Polypeptides binding to gamma delta t cells and uses thereof

Abstract

The present invention provides VHH-containing polypeptides that bind to gamma delta T cells. Uses of the VHH-containing polypeptides are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 296,774, filed on January 5, 2022, and U.S. Provisional Application No. 63 / 417,926, filed on October 20, 2022; each of which is incorporated herein by reference in its entirety for any purpose. Technical Field

[0003] The present invention relates to polypeptides that bind to γδ T cells, and methods for regulating the biological activity of γδ T cells using polypeptides that bind to γδ T cells. The methods include, but are not limited to, methods for treating cancer. In some embodiments, the polypeptide that binds to γδ T cells is a fusion polypeptide comprising a polypeptide that binds to γδ T cells and a polypeptide that binds to an antigen other than γδ T cells. Background Art

[0004] The activation of T cells is controlled by other molecules such as IL-2, IL-15, IL-7, IL-6, IL-12, IFNα, IFNβ and IFNγ. Interleukin-2 (IL-2), a cytokine synthesized and secreted by activated T cells themselves, is a pleiotropic cytokine that regulates the proliferation and lytic activity of γδ T cells. IL-2 binds to a high-affinity receptor consisting of three subunits (IL-2α, IL-2β and γc) on the surface of T cells. Signaling via the IL-2 receptor complex triggers T cells to develop through cell division, driving the clonal expansion of activated T cells.

[0005] There is a need for polypeptides that bind to γδ T cells and that can specifically target activating molecules to γδ T cells to increase the potency and selectivity of cytotoxic γδ T cell responses. SUMMARY OF THE INVENTION

[0006] Provided herein are polypeptides that bind to γδ T cells, and methods of using polypeptides that bind to γδ T cells to treat, for example, cancer. In some embodiments, the polypeptide that binds to γδ T cells comprises one or more additional binding domains and / or cytokine sequences. Certain numbered embodiments are provided below.

[0007] Embodiment 1. A polypeptide comprising at least one VHH domain that binds to a γδ TCR, wherein at least one VHH domain comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO:3, 144, 145, 146, 147, 148 or 149; a CDR2 comprising the amino acid sequence of SEQ ID NO:4, 150, 151, 152, 153, 154, 155 or 156; and a CDR3 comprising the amino acid sequence of SEQ ID NO:5.

[0008] Embodiment 2. A polypeptide as described in embodiment 1, wherein at least one VHH domain comprises CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 respectively comprise the amino acid sequence of SEQ ID NO: 3, 4 and 5; 144, 4 and 5; 145, 4 and 5; 146, 4 and 5; 147, 4 and 5; 148, 4 and 5; 149, 4 and 5; 3, 150 and 5; 3, 151 and 5; 3, 152 and 5; 3, 153 and 5; 3, 154 and 5; 3, 155 and 5; or 3, 156 and 5.

[0009] Embodiment 3. A polypeptide according to embodiment 1 or embodiment 2, wherein at least one VHH domain comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 4; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 5.

[0010] Embodiment 4. A polypeptide according to any one of embodiments 1 to 3, wherein at least one VHH domain or each VHH domain is humanized.

[0011] Embodiment 5. The polypeptide of any one of embodiments 1 to 4, wherein at least one VHH domain comprises SEQ ID NO: 180, wherein X1, X2, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 25 , X 26 and X 27 are independently selected, and where:

[0012] X1 is V or A; X 11 D or G; X 20 is T or A;

[0013] X2 is R or G; X 12 A or S; X 21 A or T;

[0014] X3 is K or T; X 13 A or T; X 22V or L;

[0015] X4 is I or F; X 14 is E or Y; X 23 N or S;

[0016] X5 is Q, G or E; X 15 V or A; X 24 is K or Y;

[0017] X6 is R or L; X 16 D, E, A, G, V, S, X 25 N, S, E, Y,

[0018] X7 is L, W or F; Y, L or Q; A, S, G, Q;

[0019] X8 is A or S; X 17 S, P, T, A, V, L, X 26 For S, T, A, L,

[0020] X9 is H or A; I, or G; V, N or G; and

[0021] X 10 is T or S; X 18 is G or D; X 27 K, R, E or D.

[0022] X 19 is S or N;

[0023] Embodiment 6. The polypeptide of any one of embodiments 1 to 4, wherein at least one VHH domain comprises SEQ ID NO: 180, wherein X3 is K, X1, X2, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 25 , X 26 and X 27 are independently selected, and where:

[0024] X1 is V or A; X 11 D or G; X20 is T or A;

[0025] X2 is R or G; X 12 A or S; X 21 A or T;

[0026] X4 is I or F; X 13 A or T; X 22 V or L;

[0027] X5 is Q, G or E; X 14 is E or Y; X 23 N or S;

[0028] X6 is R or L; X 15 V or A; X 24 is K or Y;

[0029] X7 is L, W or F; X 16 is D, E, A, L or Q; X 25 N or S;

[0030] X8 is A or S; X 17 is S, P, T, V, L or G; X 26 is S, T or G; and

[0031] X9 is H or A; X 18 is G or D; X 27 K, R, E or D.

[0032] X 10 is T or S; X 19 is S or N;

[0033] Embodiment 7. The polypeptide of any one of embodiments 1 to 4, wherein at least one VHH domain comprises SEQ ID NO: 180, wherein X2 is R; X 25 is N; and X1 and X3 are K, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 26 and X 27are independently selected, and where:

[0034] X1 is V or A; X 11 D or G; X 19 is S or N;

[0035] X4 is I or F; X 12 A or S; X 20 is T or A;

[0036] X5 is Q, G or E; X 13 A or T; X 21 A or T;

[0037] X6 is R or L; X 14 is E or Y; X 22 V or L;

[0038] X7 is L, W or F; X 15 V or A; X 23 N or S;

[0039] X8 is A or S; X 16 is D, E, A or Q; X 24 is K or Y;

[0040] X9 is H or A; X 17 is S, P or G; X 26 is S or T; and

[0041] X 10 is T or S; X 18 is G or D; X 27 K, R, E or D.

[0042] Embodiment 8. The polypeptide of any one of embodiments 1 to 4, wherein at least one VHH domain comprises SEQ ID NO: 180, wherein X2 is R; X3 is K, X4 is I; X9 is H; X 25 is N; and X1, X5, X6, X7, X8, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 26 and X27 are independently selected, and where:

[0043] X1 is V or A; X 13 A or T; X 20 is T or A;

[0044] X5 is Q, G or E; X 14 is E or Y; X 21 A or T;

[0045] X6 is R or L; X 15 V or A; X 22 V or L;

[0046] X7 is L, W or F; X 16 is D, E or A; X 23 N or S;

[0047] X8 is A or S; X 17 is S or P; X 24 is K or Y;

[0048] X 10 is T or S; X 18 is G or D; X 26 is S or T; and

[0049] X 11 D or G; X 19 S or N; X 27 K, R, E or D.

[0050] X 12 A or S;

[0051] Embodiment 9. The polypeptide of any one of embodiments 1 to 4, wherein at least one VHH domain comprises SEQ ID NO: 180, wherein X1 is V; X2 is R; X3 is K, X4 is I; X9 is H; X 10 is T; X 11 D;X 12 A; X 13 A; X 14 E; X 25 is N; and X5, X6, X7, X8, X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 26 and X27 are independently selected, and where:

[0052] X5 is Q, G or E; X 17 is S or P; X 22 V or L;

[0053] X6 is R or L; X 18 is G or D; X 23 N or S;

[0054] X7 is L or W; X 19 S or N; X 24 is K or Y;

[0055] X8 is A or S; X 20 is T or A; X 26 is S or T; and

[0056] X 15 V or A; X 21 A or T; X 27 K, R, E or D.

[0057] X 16 is D, E or A;

[0058] Embodiment 10. A polypeptide as described in any one of embodiments 1 to 9, wherein at least one VHH domain comprises an amino acid sequence that is at least 85%, 90%, 95% or at least 99% identical to the amino acid sequence of SEQ ID NO: 2, 17 to 31, 72 to 77, 80 to 143, 158 to 159, or 166 to 179.

[0059] Embodiment 11. A polypeptide as described in any one of embodiments 1 to 10, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 2, 17 to 31, 72 to 77, 80 to 143, 158 to 159, or 166 to 179.

[0060] Embodiment 12. A polypeptide as described in any one of embodiments 1 to 11, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 99, 143 or 158.

[0061] Embodiment 13. The polypeptide according to any one of embodiments 1 to 12, comprising two VHH domains.

[0062] Embodiment 14. The polypeptide of any one of embodiments 1 to 13, comprising three VHH domains.

[0063] Embodiment 15. The polypeptide of any one of embodiments 1 to 14, wherein the polypeptide comprises an immune cell activation cytokine.

[0064] Embodiment 16. The polypeptide of embodiment 15, wherein the immune cell activating cytokine is fused to the N-terminus or C-terminus of the VHH domain that binds to γδ T cells.

[0065] Embodiment 17. The polypeptide of embodiment 10 or embodiment 16, wherein the immune cell activating cytokine is IL-2, IL-15, IL-7, IL-6, IL-12, IFNα, IFNβ or IFNγ, or an attenuated or modified form thereof.

[0066] Embodiment 18. The polypeptide of any one of embodiments 1 to 17, wherein the polypeptide comprises an Fc region.

[0067] Embodiment 19. The polypeptide of embodiment 18, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NO: 32 to 70, optionally wherein the Fc region does not have a C-terminal lysine residue.

[0068] Embodiment 20. The polypeptide of embodiment 18 or embodiment 19, wherein the polypeptide comprises an immune cell activation cytokine.

[0069] Embodiment 21. The polypeptide of embodiment 20, wherein the immune cell activating cytokine is IL-2, IL-15, IL-7, IL-6, IL-12, IFNα, IFNβ or IFNγ, or an attenuated or modified form thereof.

[0070] Embodiment 22. The polypeptide of embodiment 21, wherein the immune cell activating cytokine is fused to the C-terminus of the Fc region.

[0071] Embodiment 23. The polypeptide of any one of embodiments 1 to 22, wherein the polypeptide comprises at least one antigen binding domain that binds an antigen other than γδ TCR.

[0072] Embodiment 24. The polypeptide of embodiment 23, wherein the polypeptide comprises at least one antigen binding domain that binds to: Lag3, TGFBR1, TGFBR2, Fas, TNFR2, 1-92-LFA-3, 5T4, α-4 integrin, α-V integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33 , CD38, CD39, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD 73. CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD1 38. CD166, CD172A, CD248, CDH6, CEACAM5(CEA), CEACAM6(NCA-90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FcRH5, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GPRC5D, GRP78, HAVCAR1, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MICA, MICB, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4. NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine 1 phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, T LR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, TROP-2uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, or WISP-3.

[0073] Embodiment 25. The polypeptide of embodiment 23 or 24, wherein the polypeptide comprises at least one antigen binding domain that binds to a tumor cell antigen.

[0074] Embodiment 26. A polypeptide according to any one of embodiments 23 to 25, wherein at least one antigen binding domain that binds an antigen other than a γδ TCR is a VHH domain.

[0075] Embodiment 27. A polypeptide according to embodiment 26, wherein each antigen binding domain that binds an antigen other than a γδ TCR is a VHH domain.

[0076] Embodiment 28. A polypeptide according to any one of embodiments 23 to 26, wherein at least one antigen binding domain that binds an antigen other than a γδ TCR comprises a heavy chain variable region and a light chain variable region.

[0077] Embodiment 29. A polypeptide according to embodiment 28, wherein each antigen binding domain that binds an antigen other than γδ TCR comprises a heavy chain variable region and a light chain variable region.

[0078] Embodiment 30. A complex comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is a polypeptide as in any one of embodiments 18 to 29, wherein the first polypeptide comprises a first Fc region, and wherein the second polypeptide comprises a second Fc region, and wherein the first Fc region is the same as or different from the second Fc region.

[0079] Embodiment 31. A complex as in embodiment 30, wherein the second polypeptide comprises at least one VHH domain that binds to γδ TCR, at least one immune cell activating cytokine and / or at least one antigen binding domain that binds to an antigen other than γδ TCR.

[0080] Embodiment 32. The complex of embodiment 31, wherein if the antigen binding domain that binds an antigen other than γδ TCR comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region is fused to a heavy chain constant region comprising the second Fc region.

[0081] Embodiment 33. The complex of embodiment 31 or 32, wherein at least one antigen binding domain that binds an antigen other than a γδ TCR binds Lag3, TGFBR1, TGFBR2, Fas, TNFR2, 1-92-LFA-3, 5T4, α-4 integrin, α-V integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33 , CD38, CD39, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD 73. CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD1 38. CD166, CD172A, CD248, CDH6, CEACAM5(CEA), CEACAM6(NCA-90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FcRH5, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GPRC5D, GRP78, HAVCAR1, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MICA, MICB, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4. NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine 1 phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, T LR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, TROP-2uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, or WISP-3.

[0082] Embodiment 34. A polypeptide as described in any one of embodiments 31 to 33, wherein at least one antigen binding domain that binds an antigen other than a γδ TCR binds, wherein the polypeptide comprises at least one antigen binding domain that binds a tumor cell antigen.

[0083] Embodiment 35. The complex of any one of embodiments 31 to 34, wherein at least one antigen binding domain that binds an antigen other than a γδ TCR is a VHH domain.

[0084] Embodiment 36. The complex of any one of embodiments 32 to 35, wherein the first Fc region comprises a knob mutation and the second Fc region comprises a hole mutation.

[0085] Embodiment 37. The complex of embodiment 36, wherein the first Fc region comprises a T366W mutation and the second Fc region comprises T366S, L368A and Y407V mutations.

[0086] Embodiment 38. The complex of embodiment 37, wherein the second Fc region comprises a H435R or H435K mutation.

[0087] Embodiment 39. The polypeptide or complex of any one of embodiments 18 to 38, wherein the polypeptide is a dimer under physiological conditions, or wherein the complex is formed under physiological conditions.

[0088] Embodiment 40. A polypeptide or complex according to any one of embodiments 1 to 39, wherein the γδ TCR is a human γδ TCR.

[0089] Embodiment 41. The polypeptide or complex of any one of embodiments 1 to 40, wherein the VHH domain binds to a human γδ TCR comprising human γ9 and human δ2.

[0090] Embodiment 42. An immunoconjugate comprising a polypeptide or complex according to any one of embodiments 1 to 41 and a cytotoxic agent.

[0091] Embodiment 43. The immunoconjugate of embodiment 42, wherein the cytotoxic agent is selected from calicheamicin, auristatin, dolastatin, tubulicin, maytansinoid, cryptophycin, duocarmycin, esperamicin, pyrrolobenzodiazepine and enediyne antibiotics.

[0092] Embodiment 44. A pharmaceutical composition comprising the polypeptide or complex of any one of embodiments 1 to 41 or the immunoconjugate of embodiment 42 or embodiment 43 and a pharmaceutically acceptable carrier.

[0093] Embodiment 45. An isolated nucleic acid encoding a polypeptide or complex according to any one of embodiments 1 to 41.

[0094] Embodiment 46. A vector comprising the nucleic acid of embodiment 45.

[0095] Embodiment 47. A host cell comprising the nucleic acid of embodiment 45 or the vector of embodiment 46.

[0096] Embodiment 48. A host cell expressing the polypeptide or complex of any one of embodiments 1 to 41.

[0097] Embodiment 49. A method of producing a polypeptide or complex according to any one of embodiments 1 to 40, comprising culturing a host cell according to embodiment 47 or embodiment 48 under conditions suitable for expressing the polypeptide or complex.

[0098] Embodiment 50. The method of embodiment 49, further comprising isolating the polypeptide or complex.

[0099] Embodiment 51. A method of increasing proliferation of γδ T cells, comprising contacting the T cells with a polypeptide or complex according to any one of embodiments 1 to 41.

[0100] Embodiment 52. A method according to embodiment 51, wherein the γδ T cells are in vitro.

[0101] Embodiment 53. A method according to embodiment 51, wherein the γδ T cells are in vivo.

[0102] Embodiment 54. A method of treating cancer, comprising administering a pharmaceutically effective amount of a polypeptide or complex according to any one of embodiments 1 to 41 or a pharmaceutical composition according to embodiment 44 to an individual suffering from cancer.

[0103] Embodiment 55. The method of embodiment 54, wherein the cancer is selected from basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain cancer and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer; gastrointestinal cancer; glioblastoma; hepatic carcinoma; liver tumor; intraepithelial neoplasia; kidney cancer or renal cancer; laryngeal cancer; liver cancer; lung cancer; small cell lung cancer; non-small cell lung cancer; lung adenocarcinoma; squamous carcinoma of the lung lung; melanoma; myeloma; neuroblastoma; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; colorectal cancer; respiratory cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancer; vulvar cancer; lymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; massive NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; and chronic myeloid leukemia.

[0104] Embodiment 56. The method of embodiment 54 or 55, further comprising administering an additional therapeutic agent.

[0105] Embodiment 57. The method of embodiment 56, wherein the additional therapeutic agent is an anti-cancer agent.

[0106] Embodiment 58. A method as described in Embodiment 56, wherein the anticancer agent is selected from chemotherapeutic agents, anticancer biological agents, radiation therapy, CAR-T therapy and oncolytic viruses.

[0107] Embodiment 59. The method of any one of embodiments 56 to 57, wherein the additional therapeutic agent is an anti-cancer biologic.

[0108] Embodiment 60. The method of embodiment 59, wherein the anti-cancer biologic is an agent that inhibits PD-1 and / or PD-L1.

[0109] Embodiment 61. A method as described in Embodiment 59, wherein the anti-cancer biologic is an agent that inhibits VISTA, gpNMB, B7H3, B7H4, HHLA2, CTLA4 or TIGIT.

[0110] Embodiment 62. The method of any one of embodiments 57 to 61, wherein the anticancer agent is an antibody.

[0111] Embodiment 63. The method of embodiment 59, wherein the anti-cancer biologic is a cytokine.

[0112] Embodiment 64. A method as described in Embodiment 57 or Embodiment 58, wherein the anticancer agent is CAR-T therapy.

[0113] Embodiment 65. The method of embodiment 57 or embodiment 58, wherein the anticancer agent is an oncolytic virus.

[0114] Embodiment 66. The method of any one of Embodiments 54 to 65, further comprising tumor resection and / or radiation therapy.

[0115] BRIEF DESCRIPTION OF THE DRAWINGS

[0116] Figure 1A , Figure 1C , Figure 1E and Figure 1G Shown are the median fluorescence intensity of intracellular phosphorylated STAT5 staining in γδ T cells, NK cells and αβ T cells after treatment with monovalent or bivalent single domain antibodies specific for γδ TCR linked to affinity-reduced IL-2 variants IL-2_X or IL-2_Y, as assessed by flow cytometry. Figure 1B , Figure 1D , Figure 1F and Figure 1H Shown are the % of cells with phosphorylated STAT5 staining on γδ T cells, NK cells and αβ T cells after treatment with monovalent or bivalent single domain antibodies specific for γδ TCR linked to affinity-reduced IL-2 variants IL-2_X or IL-2_Y as assessed by flow cytometry.

[0117] Figure 2A Shown are the percentages of γδ T cells that proliferated in response to treatment with low-affinity IL-2_X targeting the γδ TCR (cx11005) or non-targeting low-affinity IL-2_X (cx9452). Figure 2B Shown are the percentages of γδ T cells in all cell lines after treatment with the above test articles. Figure 2C The percentage of proliferating αβ T cells after treatment is shown, and Figure 2DThe percentage of αβ T cells after treatment is shown for all cell lines.

[0118] Figure 3A We show that Vδ2 is upregulated upon treatment with a monovalent single domain antibody specific for γδ TCR (cx11026) linked to the affinity-reduced IL-2 variant IL-2_X, as assessed by flow cytometry. + Median fluorescence intensity of intracellular phosphorylated STAT5 in γδ T cells and αβ T cells. Figure 3B We show that Vδ2 is activated upon treatment with a monovalent single domain antibody specific for the γδ TCR linked to the affinity-reduced IL-2 variant IL-2_X, as assessed by flow cytometry. + % of cells with phosphorylated STAT5 staining in γδ T cells and αβ T cells.

[0119] Figure 4A Figure 3 shows proliferation of Vδ2 cells in response to treatment with low affinity IL-2_X (cx11026) targeting γδ TCR or non-targeting low affinity IL-2_X (cx9452) as determined by flow cytometric analysis of CellTrace Violet dilution. + Percentage of γδ T cells. Figure 4B Display total CD3 after treatment + T cell line Vδ2 + Percentage of γδ T cells.

[0120] Figure 5A , Figure 5B , Figure 5C , Figure 5D , Figure 5E , Fig. 5F , Figure 5G , Figure 5H , Fig.5I , Figure 5J and Figure 5K Binding of the γδ TCR-binding VHH, 1D7, and its humanized variants formatted as monovalent VHH-hlgG1-Fc fusion proteins was demonstrated as assessed by flow cytometry on expanded human Vδ2+γδ T cells.

[0121] FIG. 6A to FIG. 6B Target cell killing curves are shown for THP-1 (6A, left), HT29 (6A, right), Daudi (6B, upper left), NCI-H460 (6B, upper right), MM1S (6B, lower left), and A375 (6B, lower right) cells after treatment with Vγ9Vδ2 T cells expanded with IL-2 molecules targeting the γδ TCR (1D7 x IL-2_X) (plotted as an overlay of caspase 3 / 7-green and cyto-ID red over time).

[0122] FIG. 7A to FIG. 7D The binding and cytotoxic activities of the γδTCR x CD20 bispecific peptides were demonstrated. Fig. 7A Demonstrated binding to Raji cells expressing CD20. Figure 7B Demonstrated binding to Vγ9Vδ2 T cells. FIG. 7C to FIG. 7D Shown are γδ T cell-mediated killing of Raji target cells by freshly isolated (7C) and expanded (7D) Vγ9Vδ2 T cells treated with γδTCRxCD20 bispecific polypeptides, rituximab-an or untreated.

[0123] FIG. 8A to FIG. 8E The binding and cytotoxic activities of the γδTCR x CD33 bispecific peptides were demonstrated. FIG. 8A to FIG. 8B Binding to CD33-expressing Molm-13 and MV-411 cells is shown, respectively. Figure 8C Demonstrated binding to Vγ9Vδ2 T cells. FIG. 8D to FIG. 8E γδ T cell-mediated killing of MOLM-13 (8D) and MV-411 (8E) target cells by expanded Vγ9Vδ2 T cells treated with γδTCR x CD33 bispecific polypeptide, non-targeting CD33 x UT polypeptide or left untreated is shown.

[0124] 9A to 9D Shown is the ability of the γδTCR x 5T4 construct to elicit antigen-dependent γδT cell-mediated cytotoxicity in the 5T4+ cell line, A375 (9A and 9C), but not in A375Δ5T4 cells, a 5T4 cell line (9B and 9D), as assessed by caspase-3 / 7 activation using a cell imaging system (3 hour time point shown) (9A and 9B) and cell survival using CellTiter-Glo analysis (9C and 9D). Fig. 9A and Fig. 9C A table of EC50 values ​​(nM) is provided in .

[0125] FIG. 10A to FIG. 10F Demonstrates cross-reactivity with cynomolgus macaque γδ TCR. Fig. 10A Display shows the binding of the γδ TCR-binding VHH, 1D7, and the humanized variant 1D7v9 formatted as a bivalent VHH-hIgG1-xELL Fc fusion protein as assessed by flow cytometry on cynomolgus macaque Vγ9+γδ T cells. Fig. 10BShown are the % of cells with phosphorylated STAT5 staining on cynomolgus macaque γδ T cells and αβ T cells from three donors following treatment with a monovalent γδ TCR linked to a reduced affinity IL-2 variant (cx11026), as assessed by flow cytometry. Fig. 10C and Fig.10E Shown are the percentages of cynomolgus macaque γδ T cells from two different cynomolgus macaque donors that proliferated in response to treatment with low-affinity IL-2_X (cx11026:1D7 x IL-2_X) or non-targeting low-affinity IL-2_X (cx9452:UT x IL-2_X) monovalently targeting the γδ TCR. Fig. 10D and Fig.10F Shown are the percentages of γδ T cells from all cell lines in these donors after treatment with the above test articles.

[0126] FIG. 11A to FIG. 11C The parent anti-γδ TCR VHH 1D7 (SEQ ID NO: 2) and the binding affinity (K) determined by flow cytometry analysis were shown. D ) is 100 nM (see Table 2). Fig.11D Shown is a sequence alignment of the parental anti-γδ TCR VHH 1D7 (SEQ ID NO: 2) and a consensus sequence representing aligned humanized 1D7 variants (SEQ ID NO: 180).

[0127] FIG. 12A to FIG. 12B Eight non-limiting exemplary formats of polypeptides that bind γδ T cells are shown. Fig. 12A, the first format shows an exemplary bispecific monovalent anti-γδ T cell × antigen construct, which is a complex comprising: a first polypeptide comprising an anti-antigen VHH domain (specific for an antigen other than γδ TCR) and an Fc region, and a second polypeptide comprising an anti-γδ TCR VHH domain and an Fc region. The second format shows an exemplary bispecific monovalent anti-γδ T cell × antigen construct with a single cytokine polypeptide (e.g., a modified IL-2 polypeptide), which is a complex comprising: a first polypeptide comprising an anti-antigen VHH domain (specific for an antigen other than γδ TCR) and an Fc region, and a second polypeptide comprising an anti-γδ TCR VHH domain, an Fc region, and a cytokine polypeptide. The third format shows an exemplary bispecific monovalent anti-γδ T cell × antigen construct, which is a complex comprising: a first polypeptide comprising an Fc region, and a second polypeptide comprising an anti-antigen VHH domain (specific for an antigen other than γδ TCR), an anti-γδ TCR VHH domain, and an Fc region. The fourth format shows an exemplary bivalent anti-γδ T cell construct, which is a single polypeptide comprising an anti-antigen VHH domain (specific for an antigen other than γδ TCR) and an anti-γδ TCR VHH. The fifth format shows an exemplary bivalent anti-γδ T cell construct with a single cytokine polypeptide (e.g., a modified IL-2 polypeptide), which is a single polypeptide comprising an anti-antigen VHH domain (specific for an antigen other than γδ TCR), an anti-γδ TCR VHH, and a cytokine polypeptide. Fig. 12B , the first format shows an exemplary trispecific construct, which is a complex comprising: a first polypeptide comprising two anti-antigen VHH domains (specific for two different antigens other than γδ TCR) and an Fc region, and a second polypeptide comprising an anti-γδ TCR VHH domain and an Fc region. The second format shows an exemplary trispecific construct, which is a complex comprising: a first polypeptide comprising an anti-antigen VHH domain (specific for an antigen other than γδ TCR) and an Fc region, and a second polypeptide comprising a different anti-antigen VHH domain (specific for a different antigen other than γδ TCR), an anti-γδ TCR VHH domain, and an Fc region. The third format shows an exemplary trispecific construct, which is a single polypeptide comprising two anti-antigen VHH domains (specific for two different antigens other than γδ TCR) and an anti-γδ TCR VHH. The order of the tandem VHH domains can be changed. Similarly, for molecules comprising two polypeptide chains, the VHH domain can be located on either chain. DETAILED DESCRIPTION

[0128] Embodiments provided herein relate to polypeptides that bind γδ T cells and their use in various methods of treating, for example, cancer.

[0129] Definition and various implementations

[0130] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0131] All references, including patent applications, patent publications, and Genbank Accession Numbers, cited herein are hereby incorporated by reference to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety.

[0132] The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are routinely employed using conventional methods, such as the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., (2003)); METHODS IN ENZYMOLOGY series (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames, and GR Taylor, eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Human Biology, 1996; Press; Cell Biology: A Laboratory Notebook (JECellis, 1998) Academic Press; Animal Cell Culture (RIFreshney), 1987); Introduction to Cell and Tissue Culture (JPMather and PERoberts, 1998) Plenum Press; Cell and TissueCulture Laboratory Procedures (A.Doyle, JBGriffiths and DGNewell, 1993-8) J.Wiley and Sons; Handbook of Experimental Immunology (edited by DMWeir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JEColigan et al., 1991); Short Protocols in Molecular Biology (Wileyand Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (edited by D. Catty., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., JS Lippincott Company, 1993); and updated versions thereof.

[0133] Unless otherwise defined, scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill. In addition, unless the context otherwise requires or otherwise explicitly indicated, singular terms shall include the plural and plural terms shall include the singular. With respect to any conflicts in definitions between various sources or references, the definitions provided herein shall prevail.

[0134] In general, the residue numbering in immunoglobulin heavy chains is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0135] It should be understood that embodiments of the invention described herein include "consisting of" embodiments and / or "consisting essentially of" embodiments. As used herein, the singular forms "a", "an", and "the" include plural references unless otherwise indicated. The use of the term "or" herein is not intended to imply that alternatives are mutually exclusive.

[0136] In this application, the use of "or" means "and / or" unless explicitly stated or understood by one skilled in the art. In the case of multiple dependent items, the use of "or" refers back to more than one of the preceding independent items or dependent items.

[0137] The words "reference sample", "reference cell" or "reference tissue" represent a sample with at least one known characteristic that can be used as a comparison with a sample with at least one unknown characteristic. In some embodiments, the reference sample can be used as a positive or negative indicator. The reference sample can be used to determine, for example, the amount of protein and / or mRNA present in healthy tissue, in contrast to the amount of protein and / or mRNA present in a sample with unknown characteristics. In some embodiments, the reference sample is from the same individual, but from a different part of the individual tested. In some embodiments, the reference sample is from a tissue area surrounding or adjacent to a cancer. In some embodiments, the reference sample is not from the individual tested, but from a sample from an individual known to have or not have the disorder in question. In some embodiments, the reference sample is from the same individual, but from a time point before the individual suffers from cancer. In some embodiments, the reference sample is a benign cancer sample from the same or different individual. When using a negative reference sample for comparison, the expression amount or amount of the molecule in question in the negative reference sample will indicate the amount that will be understood by those skilled in the art assuming that there is no such molecule and / or there is a low amount of such molecule in the present invention. When a positive reference sample is used for comparison, the expression level or amount of the molecule in question in the positive reference sample will indicate the amount that one skilled in the art would understand if a certain amount of the molecule were present in the present invention.

[0138] The terms "benefit", "clinical benefit", "response" and "therapeutic response" as used herein in the context of benefiting from or responding to the administration of a therapeutic agent can be measured by assessing various evaluation indicators, such as inhibiting disease progression to some extent, including slowing and complete arrest; reducing the number of disease attacks and / or symptoms; reducing the size of lesions; inhibiting (i.e., reducing, slowing or completely stopping) disease cell infiltration to adjacent peripheral organs and / or tissues; inhibiting (i.e., reducing, slowing or completely stopping) disease spread; alleviating to some extent one or more symptoms associated with the disorder; increasing the length of time that the patient is disease-free, such as progression-free survival, after treatment; increasing overall survival; improving response rate; and / or reducing mortality at a given time point after treatment. "Non-responsive" or "unresponsive" individuals or cancers are individuals or cancers that do not meet the above-mentioned restrictions on "response".

[0139] The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" are used interchangeably and refer to nucleotide polymers. Such nucleotide polymers may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA and PNA. "Nucleic acid sequence" refers to a linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0140] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers and polymers of amino acid residues. The definition encompasses full-length proteins and fragments thereof. The term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation and the like. In addition, for the purposes of the present invention, "polypeptide" refers to a protein that includes modifications to the native sequence, such as deletions, additions and substitutions (which are usually conservative in practice), as long as the protein maintains the desired activity. These modifications may be intentional, such as induced by site-directed mutagenesis; or may be accidental, such as by mutations in the host producing the protein or errors caused by PCR amplification.

[0141] The term "specific binding" to an antigen or epitope is a term that is well understood in the art, and methods for determining the specific binding are also well known in the art. If a molecule reacts or associates with a specific cell or substance more frequently, more rapidly, for a longer duration and / or with greater affinity than it does with an alternative cell or substance, it is said to exhibit "specific binding" or "preferential binding". If a single domain antibody (sdAb) or a VHH-containing polypeptide binds to a target with greater affinity, avidity, easier and / or longer duration than it binds to other substances, it "specifically binds" or "preferentially binds" to the target. For example, an sdAb or a VHH-containing polypeptide that specifically or preferentially binds to a γδ T cell is an sdAb or a VHH-containing polypeptide that binds to this epitope with greater affinity, greater avidity, easier and / or longer duration than it binds to other T cells or non-T cells. By reading this definition, it should also be understood that, for example, an sdAb or a VHH-containing polypeptide that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind an antigen.

[0142] The term "inhibition" refers to the reduction or cessation of any expression characteristic, or the reduction or cessation of the incidence, degree or possibility of that characteristic. "Reduction" or "inhibition" is to reduce, reduce or stagnate the activity, function and / or amount compared to a reference. In some embodiments, "reduction" or "inhibition" means the ability to reduce by 10% or more overall. In some embodiments, "reduction" or "inhibition" means the ability to reduce by 50% or more overall. In some embodiments, "reduction" or "inhibition" means the ability to reduce by 75%, 85%, 90%, 95% or more overall. In some embodiments, the amounts mentioned above are inhibited or reduced relative to the control within the same period of time over a period of time.

[0143] As used herein, the term "direct inhibition" and similar terms refer to an inhibition profile in which increasing antibody concentrations result in increased inhibition. In some embodiments, after a particular concentration, maximum inhibition is reached and the inhibition profile levels off. Maximum inhibition need not be 100% inhibition, but may be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

[0144] As used herein, the term "epitope" refers to a site where an antigen binding molecule (e.g., sdAb or VHH-containing polypeptide) binds to a target molecule (e.g., an antigen, such as a protein, nucleic acid, carbohydrate, or lipid). Epitopes often include a group of molecules with chemical activity on the surface, such as amino acids, polypeptides, or sugar side chains, and have specific three-dimensional structural characteristics and mass-to-charge ratio characteristics. The epitope can be formed by adjacent and / or juxtaposed non-adjacent residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of the target molecule. Epitopes formed by adjacent residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are usually retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost when treated with denaturing solvents. An epitope may include, but is not limited to, at least 3, at least 5, or 8 to 10 residues (e.g., amino acids or nucleotides). In some embodiments, the length of the epitope is less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues, or less than 12 residues. If two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope within the antigen. In some embodiments, epitopes can be identified by a certain minimum distance from the CDR residues on the antigen binding molecules. In some embodiments, epitopes can be identified by the above distances, and are further limited to those residues involved in the bond (e.g., hydrogen bond) between the residues of the antigen binding molecules and the antigen residues. Epitopes can also be identified by various scans, such as alanine or arginine scans that can indicate one or more residues that the antigen binding molecules can interact with. Unless explicitly stated, a group of residues as an epitope does not exclude other residues as part of the epitope of a specific antigen binding molecule. In fact, the presence of the group represents a minimum series of epitopes (or a group of substances). Therefore, in some embodiments, the residue set identified as an epitope represents the minimum relevant epitope of the antigen, rather than the exclusive residue list of the epitope on the non-antigen.

[0145] A "non-linear epitope" or "conformational epitope" comprises non-adjacent polypeptides, amino acids and / or sugars within an antigenic protein to which an antigen binding molecule specific for the epitope binds. In some embodiments, at least one residue will not be adjacent to other indicated residues of the epitope; however, one or more residues may also be adjacent to other residues.

[0146] A "linear epitope" comprises adjacent polypeptides, amino acids, and / or sugars within an antigen protein to which an antigen binding molecule specific for the epitope binds. It should be noted that in some embodiments, not every residue within a linear epitope needs to be directly bound (or associated with a bond) to an antigen binding molecule. In some embodiments, a linear epitope may be derived from immunization with a peptide effectively consisting of the sequence of a linear epitope, or from a structural portion of a protein that is relatively isolated from the rest of the protein (such that an antigen binding molecule may at least first interact with that sequence portion).

[0147] The term "antibody" is used in the broadest sense and encompasses various polypeptides comprising an antibody-like antigen binding domain, including but not limited to conventional antibodies (generally comprising at least one heavy chain and at least one light chain), single domain antibodies (sdAb, comprising at least one VHH domain and an Fc region), VHH-containing polypeptides (polypeptides comprising at least one VHH domain), and fragments of any of the foregoing, as long as they exhibit the desired antigen binding activity. In some embodiments, the antibody comprises a dimerization domain. The dimerization domain includes but is not limited to a heavy chain constant domain (comprising CH1, hinge, CH2, and CH3, wherein CH1 is generally paired with a light chain constant domain CL, and the hinge mediates dimerization) and an Fc region (comprising hinge, CH2, and CH3, wherein the hinge mediates dimerization).

[0148] The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as camel (including llama), shark, mouse, human, cynomolgus monkey, etc.

[0149] As used herein, the term "antigen binding domain" refers to a part of an antibody that is sufficient to bind to an antigen. In some embodiments, the antigen binding domain of a conventional antibody comprises three heavy chain CDRs and three light chain CDRs. Therefore, in some embodiments, the antigen binding domain comprises: a heavy chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any part of FR1 and / or FR4 required for maintaining binding to an antigen; and a light chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any part of FR1 and / or FR4 required for maintaining binding to an antigen. In some embodiments, an sdAb or an antigen binding domain containing a VHH polypeptide comprises three CDRs of a VHH domain. Therefore, in some embodiments, an sdAb or an antigen binding domain containing a VHH polypeptide comprises a VHH domain, which comprises CDR1-FR2-CDR2-FR3-CDR3 and any part of FR1 and / or FR4 required for maintaining binding to an antigen.

[0150] As used herein, the term "VHH" or "VHH domain" or "VHH antigen binding domain" refers to the antigen binding portion of a single domain antibody such as a camelid antibody or a shark antibody. In some embodiments, a VHH comprises three CDRs and four framework regions, referred to as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, a VHH may be truncated at the N-terminus or C-terminus such that it comprises only a portion of FR1 and / or FR4, or lacks one or both of those framework regions, as long as the VHH substantially maintains antigen binding and specificity.

[0151] The terms "single domain antibody" and "sdAb" are used interchangeably herein to refer to an antibody comprising at least one light chain-free monomer domain such as a VHH domain and an Fc region. In some embodiments, an sdAb is a dimer of two polypeptides, each of which comprises at least one VHH domain and an Fc region. As used herein, the terms "single domain antibody" and "sdAb" encompass polypeptides comprising multiple VHH domains, such as polypeptides having the structure VHH1-VHH2-Fc or VHH1-VHH2-VHH3-Fc, wherein VHH1, VHH2, and VHH3 may be the same or different.

[0152] The term "VHH polypeptide" refers to a polypeptide comprising at least one VHH domain. In some embodiments, the VHH polypeptide comprises two, three or four or more VHH domains, wherein each VHH domain may be the same or different. In some embodiments, the VHH polypeptide comprises an Fc region. In some such embodiments, the VHH polypeptide may be referred to as an sdAb. In addition, in some such embodiments, the VHH polypeptide may form a dimer. Non-limiting structures of VHH polypeptides, which are also sdAbs, include VHH1-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc, wherein VHH1, VHH2, and VHH3 may be the same or different. In some embodiments of the structure, one VHH may be connected to another VHH via a linker, or one VHH may be connected to Fc via a linker. In some such embodiments, the linker comprises 1 to 20 amino acids, preferably 1 to 20 amino acids that mainly comprise glycine and optionally comprise serine. In some embodiments, when the VHH-containing polypeptide comprises an Fc, it forms a dimer. Thus, if the structure VHH1-VHH2-Fc forms a dimer, it is considered to be tetravalent (i.e., the dimer has four VHH domains). Similarly, if the structure VHH1-VHH2-VHH3-Fc forms a dimer, it is considered to be hexavalent (i.e., the dimer has six VHH domains).

[0153] The term "monoclonal antibody" refers to an antibody (including sdAb or VHH polypeptide) of a substantially homogeneous group of antibodies, that is, the individual antibodies comprising this group are identical except for the possible presence of a small number of naturally occurring mutations. Monoclonal antibodies are highly specific for a single antigenic site. In addition, in contrast to polyclonal antibody preparations that typically include different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Therefore, a sample of a monoclonal antibody can bind to the same epitope on the antigen. The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies, and should not be understood as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies can be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or can be made by a recombinant DNA method such as described in U.S. Patent No. 4,816,567. For example, monoclonal antibodies can also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554.

[0154] The term "CDR" means complementarity determining region, as defined by one skilled in the art in at least one identifiable manner.

[0155] In some embodiments, the CDRs may be defined according to any of the Chothia numbering scheme, the Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, and / or the contact definition. A VHH comprises three CDRs, referred to as CDR1, CDR2, and CDR3.

[0156] As used herein, the term "heavy chain constant region" refers to a region comprising at least three heavy chain constant domains C H 1. Hinge, C H 2 and C H3. Of course, unless otherwise indicated, deletions and changes in the domain that do not change function are included in the scope of the term "heavy chain constant region". Non-limiting exemplary heavy chain constant regions include γ, δ and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody comprising a γ constant region is an IgG antibody, an antibody comprising a δ constant region is an IgD antibody, and an antibody comprising an α constant region is an IgA antibody. In addition, an antibody comprising a μ constant region is an IgM antibody, and an antibody comprising an ε constant region is an IgE antibody. Specific isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (comprising a γ1 constant region), IgG2 (comprising a γ2 constant region), IgG3 (comprising a γ3 constant region) and IgG4 (comprising a γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (comprising an α1 constant region) and IgA2 (comprising an α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0157] As used herein, "Fc region" refers to the portion of the heavy chain constant region that includes CH2 and CH3. In some embodiments, the Fc region includes a hinge, CH2, and CH3. In various embodiments, when the Fc region includes a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0158] As discussed herein, an "acceptor human framework" as used herein is an immunoglobulin framework comprising a heavy chain variable domain (V H ) framework. The acceptor human framework derived from a human immunoglobulin framework or a human common framework may comprise the same amino acid sequence therewith, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes in all human frameworks of a single antigen binding domain such as a VHH is less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3.

[0159] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody, such as an sdAb, or a VHH-containing polypeptide) and its binding partner (e.g., an antigen). The affinity or apparent affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K) and the affinity of a molecule X for its partner Y, respectively. D ) or K D-表观 Affinity can be expressed by common methods known in the art (such as ELISA K D, KinExA, flow cytometry and / or surface plasmon resonance devices), including those described herein. The methods include but are not limited to those involving or flow cytometry analysis techniques.

[0160] As used herein, the term "K D ” refers to the equilibrium dissociation constant of the antigen binding molecule / antigen interaction. When the term “K D ", it includes K D and K D-表观 .

[0161] In some embodiments, the K of the antigen binding molecule is D K is measured by flow cytometry using a cell line expressing the antigen and fitting the mean fluorescence measured at each antibody concentration to a nonlinear single-site binding equation (Prism Software graphpad). In some such embodiments, K D K D-表观 .

[0162] The term "biological activity" refers to any one or more biological properties of a molecule (whether found naturally in vivo or provided or achieved by recombinant means). Biological properties include, but are not limited to, binding to a ligand, inducing or increasing cell proliferation (such as T cell proliferation), and inducing or increasing the expression of cytokines.

[0163] "Agonist" or "activating" antibodies are antibodies that increase and / or activate the biological activity of a target antigen. In some embodiments, an agonist antibody binds to an antigen and increases its biological activity by at least about 20%, 40%, 60%, 80%, 85% or more.

[0164] An "antagonist", a "blocking" or "neutralizing" antibody, is an antibody that inhibits, reduces and / or does not activate the biological activity of a target antigen. In some embodiments, a neutralizing antibody binds to an antigen and reduces its biological activity by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%, 99% or more.

[0165] An "affinity matured" sdAb or VHH-containing polypeptide refers to a sdAb or VHH-containing polypeptide having one or more alterations in one or more CDRs that improve the affinity of the sdAb or VHH-containing polypeptide for an antigen, compared to a parent sdAb or VHH-containing polypeptide that does not have such alterations.

[0166] As used herein, "humanized VHH" refers to a VHH in which one or more framework regions have been substantially replaced by human framework regions. In some cases, certain framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized VHHs may include residues that are neither present in the original VHH nor in the human framework sequence, but include the residues to further improve and optimize the sdAb containing VHH polypeptide potency. In some embodiments, humanized sdAb or VHH containing polypeptides include human Fc regions. As will be appreciated, humanized sequences can be identified by their primary sequence and do not necessarily represent the process of producing antibodies.

[0167] An "effector-positive Fc region" has the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include Fc receptor binding; Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation, etc. The effector functions generally require an Fc region in combination with a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of assays.

[0168] A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0169] "Variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of the native sequence Fc region by at least one amino acid modification. In some embodiments, the "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of the native sequence Fc region by at least one amino acid modification, but retains at least one effector function of the native sequence Fc region. In some embodiments, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or compared to the Fc region of the parent polypeptide, for example, about one to about ten amino acid substitutions in the native sequence Fc region or in the Fc region of the parent polypeptide, and preferably about one to about five amino acid substitutions. In some embodiments, the variant Fc region herein has at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the native sequence Fc region and / or with the Fc region of the parent polypeptide.

[0170] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, FcγR is a natural human FcR. In some embodiments, FcR is an FcR (gamma receptor) that binds to an IgG antibody and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allele variants and alternative splicing forms of those receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), both of which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain, and the inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcRs are reviewed in, e.g., Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" herein encompasses other FcRs, including those identified in the future. For example, the term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulates the constant expression of immunoglobulins. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.)).

[0171] As used herein, the term "substantially similar" or "substantially the same" means that the similarity between two or more values ​​is high enough that, in the case of a biological characteristic measured by the values, a person skilled in the art would consider the difference between the two or more values ​​to have little or no biological and / or statistical significance. In some embodiments, two or more substantially similar values ​​differ by no more than approximately any of the following: 5%, 10%, 15%, 20%, 25%, or 50%.

[0172] A polypeptide "variant" means a biologically active polypeptide having at least about 80% amino acid sequence identity with a native sequence polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum sequence identity percentage, and not considering any conservative substitutions as part of the sequence identity. The variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of the polypeptide. In some embodiments, the variant will have at least about 80% amino acid sequence identity. In some embodiments, the variant will have at least about 90% amino acid sequence identity. In some embodiments, the variant will have at least about 95% amino acid sequence identity with the native sequence polypeptide.

[0173] As used herein, "percentage (%) of amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percentage of amino acid sequence identity can be achieved in various ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

[0174] Amino acid substitutions may include, but are not limited to, replacing one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 1. Amino acid substitutions may be introduced into related antibodies and products screened for desired activities, such as antigen binding retention / improvement, immunogenicity reduction, or ADCC or CDC improvement.

[0175] Table 1

[0176] Initial residue Exemplary substitutions Ala(A) Val; Leu; Ile Arg(R) Lys; Gln; Asn Asn(N) Gln; His; Asp, Lys; Arg Asp(D) Glu; Asn Cys(C) Ser; Ala Gln(Q) Asn;Glu Glu(E) Asp; Gln Gly(G) Ala His(H) Asn; Gln; Lys; Arg Ile(I) Leu; Val; Met; Ala; Phe; Norleucine Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Lys(K) Arg; Gln; Asn Met(M) Leu; Phe; Ile Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Pro(P) Ala Ser(S) Thr Thr(T) Val; Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Phe; Thr; Ser Val(V) Ile; Leu; Met; Phe; Ala; norleucine

[0177] Amino acids can be grouped according to common side chain properties:

[0178] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0179] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0180] (3) Acidic: Asp, Glu;

[0181] (4) Basic: His, Lys, Arg;

[0182] (5) Residues that affect chain orientation: Gly, Pro;

[0183] (6) Aromatic: Trp, Tyr, Phe.

[0184] Non-conservative substitutions will result in exchanging a member of one of these classes for another class.

[0185] The term "vector" is used to describe a polynucleotide that can be engineered to contain one or more cloned polynucleotides that can be propagated in a host cell. A vector may include one or more of the following components: an origin of replication, one or more regulatory sequences that regulate expression of a polypeptide of interest (such as a promoter and / or enhancer), and / or one or more selectable marker genes (such as antibiotic resistance genes and genes that can be used in colorimetric assays, e.g., β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0186] "Host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. A host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, Host cells include 293 and CHO cells, and derivatives thereof, such as 293-6E, CHO-DG44, CHO-K1, CHO-S and CHO-DS cells. Host cells include progeny of a single host cell, and progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental or deliberate mutations. Host cells include cells transfected in vivo with the polynucleotides provided herein.

[0187] As used herein, the term "isolated" refers to a molecule that has been separated from at least some of the components that are usually found or produced together in nature. For example, when a polypeptide is separated from at least some components of the cell that produces the polypeptide, the polypeptide is referred to as "isolated". If the polypeptide is secreted by a cell after expression, then physically separating the supernatant containing the polypeptide from the cell that produces it is considered to be an "isolated" polypeptide. Similarly, when a polynucleotide is not part of a larger polynucleotide that is usually found in nature (such as genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide), or is separated from at least some components of the cell that produces it (for example, in the case of an RNA polynucleotide), the polynucleotide is referred to as "isolated". Therefore, a DNA polynucleotide contained in a vector inside a host cell can be referred to as "isolated".

[0188] The terms "individual" and "subject" are used interchangeably herein to refer to an animal; for example, a mammal. In some embodiments, methods of treating mammals are provided, including but not limited to humans, rodents, monkeys, cats, dogs, horses, cattle, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sports animals, and mammalian pets. In some embodiments, an "individual" or "subject" refers to an individual or individual in need of treatment for a disease or condition. In some embodiments, the individual receiving treatment may be a patient, indicating the fact that the individual has been identified as suffering from a condition associated with the treatment, or is at great risk of developing the condition.

[0189] As used herein, "disease" or "disorder" refers to a condition for which treatment is needed and / or desired.

[0190] Unless otherwise indicated, the terms "tumor cell," "cancer cell," "cancer," "tumor," and / or "neoplasm" are used interchangeably herein and refer to cells that exhibit uncontrolled growth and / or abnormally increased cell survival and / or inhibition of apoptosis, which interfere with the normal function of body organs and systems. Included in this definition are benign and malignant cancers, polyps, hyperplasias, and latent tumors or micrometastases.

[0191] The terms "cancer" and "tumor" encompass solid cancers and hematological / lymphatic cancers, and also encompass malignant, pre-malignant, and benign growths, such as dysplasias. Exemplary cancers include, but are not limited to, basal cell carcinoma, bile duct cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer (including gastrointestinal cancer); glioblastoma; hepatoma; liver tumor; intraepithelial neoplasia; kidney cancer or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous lung cancer); melanoma; myeloma; neuroblastoma; oral cancer (lip cancer, tongue cancer, mouth cancer, and pharynx cancer); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancer; vulvar cancer; lymphoma; lymphomas, including Hodgkin's and non-Hodgkin's lymphomas and B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; massive NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with macular degeneration, edema (such as associated with brain tumors) and Meigs' syndrome.

[0192] As used herein, the term "non-tumor cell" refers to normal cells or tissues. Exemplary non-tumor cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, monocytes, macrophages, epithelial cells, fibroblasts, hepatocytes, interstitial renal cells, fibroblast-like synoviocytes, osteoblasts, and cells located in breast, skeletal muscle, pancreas, stomach, ovary, small intestine, placenta, uterus, testis, kidney, lung, heart, brain, liver, prostate, large intestine, lymphoid organs, bone and bone-derived mesenchymal stem cells. As used herein, the term "cells or tissues located in the periphery" refers to non-tumor cells that are not located near tumor cells and / or inside the tumor microenvironment.

[0193] As used herein, the term "cells or tissues within the tumor microenvironment" refers to cells, molecules, extracellular matrix and / or blood vessels that surround and / or feed tumor cells. Exemplary cells or tissues within the tumor microenvironment include, but are not limited to, tumor vascular structures; tumor infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPC); cancer-associated fibroblasts; coat cells; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T cells; regulatory T cells (Treg cells); macrophages; neutrophils; bone marrow-derived suppressor cells (MDSC) and other immune cells located near the tumor. As described below, methods for identifying tumor cells and / or cells / tissues located within the tumor microenvironment are well known in the art.

[0194] In some embodiments, "increase" or "decrease" refers to a statistically significant increase or decrease, respectively. As will be apparent to those skilled in the art, "modulation" may also include a change (which may be an increase or decrease) in affinity, avidity, specificity and / or selectivity for a ligand, binding partner, one or more of the partners binding to a homopolymer or heteropolymer form or substrate of a target or antigen, compared to the same conditions but without the test agent; a change (which may be an increase or decrease) in the sensitivity of the target or antigen to one or more conditions (such as pH, ionic strength, presence of cofactors, etc.) in the medium or environment in which the target or antigen is present; and / or cell proliferation or cytokine production. Depending on the target involved, this may be determined by any suitable means and / or using any suitable assay known per se or described herein.

[0195] As used herein, "immune response" is meant to encompass cellular and / or humoral immune responses sufficient to inhibit or prevent the onset of disease or ameliorate disease symptoms (e.g., cancer or cancer metastasis). "Immune response" can encompass aspects of both the innate and acquired immune systems.

[0196] As used herein, "treatment" is a method for obtaining a favorable or desired clinical outcome. "Treatment" as used herein covers the administration or application of any therapeutic agent to a mammal, including humans, for a disease. For purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, any one or more of the following: alleviating one or more symptoms, reducing the extent of the disease, preventing or delaying disease spread (e.g., metastasis, such as metastasis to the lungs or lymph nodes), preventing or delaying disease recurrence, delaying or slowing disease progression, improving the disease state, inhibiting disease or disease progression, inhibiting or slowing disease or its progress, curbing its development, and alleviating (whether partially or completely). "Treatment" also encompasses the alleviation of pathological results of proliferative diseases. The methods provided herein contemplate any one or more of these therapeutic aspects. According to the above, the term treatment does not require one hundred percent removal of all disease aspects.

[0197] "Relief" means that one or more symptoms are alleviated or improved compared to when no therapeutic agent is administered. "Improvement" also includes shortening or reducing the duration of symptoms.

[0198] The term "anticancer agent" is used in the broadest sense herein to refer to an agent used to treat one or more cancers. Exemplary classes of such agents include, but are not limited to, chemotherapeutic agents, anticancer biologics (such as cytokines, receptor extracellular domain-Fc fusions, and antibodies), radiation therapy, CAR-T therapy, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNA), and oncolytic viruses.

[0199] The term "biological sample" means a quantity of a substance from a living organism or a formerly living organism. Such substances include, but are not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0200] The term "control" or "reference" refers to a composition known to not contain the analyte (a "negative control") or a composition known to contain the analyte (a "positive control"). A positive control may comprise a known concentration of the analyte.

[0201] As used herein, "delaying the development of a disease" means to postpone, hinder, slow, stifle, stabilize, contain and / or delay the development of a disease (such as cancer). Depending on the history of the disease and / or the individual being treated, this delay may be of varying lengths of time. As will be apparent to one skilled in the art, sufficient or significant delay may actually encompass prevention, such that the individual does not develop the disease. For example, the development of advanced cancers, such as cancer metastases, may be delayed.

[0202] As used herein, "preventing" includes providing a preventive effect on the appearance or recurrence of a disease in an individual who may be susceptible to the disease but has not yet been diagnosed with the disease. Unless otherwise specified, the terms "reducing," "inhibiting," or "preventing" do not imply or require complete prevention at all times, but only over a measured period of time.

[0203] A "therapeutically effective amount" of a substance / molecule, agonist or antagonist may vary according to factors such as the disease state, age, sex and weight of the individual, and the ability of the substance / molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or deleterious effects of the substance / molecule, agonist or antagonist. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount that is effective to achieve the desired therapeutic and / or preventive result at the necessary dosage and time.

[0204] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to a preparation that is in a form that permits the biological activity of the active ingredient to be effective, and contains no additional components that are unacceptably toxic to the subject to which the formulation would be administered. The formulation may be sterile.

[0205] "Pharmaceutically acceptable carrier" refers to a conventional non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid or carrier in the art, which is used together with the therapeutic agent to constitute a "pharmaceutical composition" for administration to an individual. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration used and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation used.

[0206] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0207] The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents wherein at least portions of the administration overlap in time, or wherein the administration of one therapeutic agent falls within a shorter time period for the administration of the other therapeutic agent, or wherein the therapeutic effects of the two agents overlap for at least one time period.

[0208] The term "sequentially" is used herein to refer to the administration of two or more therapeutic agents without overlap in time, or wherein the therapeutic effects of the agents do not overlap.

[0209] As used herein, "in combination with" means administering one treatment modality in addition to another treatment modality. Thus, "in combination with" means administering one treatment modality before, during, or after another treatment modality is administered to an individual.

[0210] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0211] An "article of manufacture" is any article (e.g., package or container) or kit comprising at least one agent, such as a drug for treating a disease or disorder (e.g., cancer) or a probe for specifically detecting a biomarker described herein. In some embodiments, the article of manufacture or kit is marketed, distributed, or sold in unit form for performing the methods described herein.

[0212] The terms "label" and "detectable label" mean a moiety that is attached to an antibody or antigen, for example, to make the reaction (e.g., binding) between members of a specific binding pair detectable. A labeled member of a specific binding pair is referred to as "detectably labeled." Thus, the term "labeled binding protein" refers to a protein into which a tag is incorporated to facilitate identification of the binding protein. In some embodiments, the label is a detectable marker that produces a signal that can be detected visually or instrumentally, such as a polypeptide that incorporates a radiolabeled amino acid or is linked to a biotinyl moiety that can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Embodiments of labels for polypeptides include, but are not limited to, the following: a radioisotope or radionuclide (e.g., 3 H. 14 C. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I. 177 Lu, 166 Ho or 153 Sm); chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphor), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent labels; biotinyl; a predetermined polypeptide epitope recognized by a secondary reporter (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag); and a magnetic agent, such as a gadolinium chelate. Representative embodiments of labels commonly used in immunoassays include a light-generating moiety, such as an acryl compound, and a fluorescence-generating moiety, such as fluorescein. In this regard, the moiety itself may not be detectably labeled, but may become detectable after reaction with another moiety.

[0213] Exemplary γδ T cell binding polypeptides

[0214] Provided herein are polypeptides that bind to γδ T cells. In various embodiments, the polypeptide that binds to γδ T cells comprises at least one VHH domain that binds to γδ T cells. In some embodiments, the polypeptide that binds to γδ T cells provided herein comprises one, two, three, four, five, six, seven, or eight VHH domains that bind to γδ T cells. In some embodiments, the polypeptide that binds to γδ T cells provided herein comprises one, two, three, or four VHH domains that bind to γδ T cells. The polypeptide that binds to γδ T cells may comprise one or more additional VHH domains that bind to one or more target proteins other than γδ T cells, and / or may comprise one or more additional polypeptide sequences, such as cytokine sequences.

[0215] In some embodiments, the polypeptide binding to γδ T cells comprises at least one VHH domain and an Fc region that binds to γδ T cells. In some embodiments, the polypeptide binding to γδ T cells provided herein comprises one, two, three or four VHH domains and an Fc region that bind to γδ T cells. In some embodiments, the Fc region mediates dimerization of the polypeptide binding to γδ T cells under physiological conditions so as to form a dimer with double the number of γδ T cell binding sites. For example, a polypeptide binding to γδ T cells comprising three VHH domains and an Fc region that binds to γδ T cells is trivalent as a monomer, but under physiological conditions, the Fc region can mediate dimerization so that the polypeptide binding to γδ T cells exists as a hexavalent dimer under such conditions.

[0216] In some embodiments, the polypeptide that binds to γδ T cells comprises at least two VHH domains, wherein a first VHH domain binds to a first epitope of γδ T cells and a second VHH domain binds to a second epitope of γδ T cells. When the polypeptide that binds to γδ T cells comprises a VHH domain that binds to a first epitope of γδ T cells and a VHH domain that binds to a second epitope of γδ T cells, the polypeptide that binds to γδ T cells can be referred to as "bi-epitopic" or "bispecific".

[0217] In some embodiments, the polypeptide that binds to γδ T cells is a complex of a first polypeptide and a second polypeptide, the first polypeptide comprising a first VHH domain and a first Fc domain that binds to γδ T cells; the second polypeptide comprising a second VHH domain and a second Fc domain that binds to γδ T cells, optionally wherein the first or second polypeptide further comprises a cytokine polypeptide. In some embodiments, the polypeptide that binds to γδ T cells is a complex of a first polypeptide and a second polypeptide, the first polypeptide comprising a first VHH domain and a first Fc domain that binds to γδ T cells; the second polypeptide comprising an antigen binding domain that binds to an antigen other than γδ T cells and a second Fc domain, optionally wherein the first or second polypeptide further comprises a cytokine polypeptide. In some such embodiments, the first or second Fc domain comprises a "knob" mutation and the other Fc domain comprises a "hole" mutation. Thus, in some embodiments, the polypeptide that binds to γδ T cells is a complex of a first polypeptide and a second polypeptide. In some such embodiments, the complex comprises two VHH domains that bind to γδ T cells. In some such embodiments, the complex comprises one VHH domain that binds to γδ T cells, and one antigen binding domain that binds to an antigen other than γδ TCR.

[0218] Peptides that bind to γδ T cells

[0219] In various embodiments, the VHH domain that binds the γδ TCR comprises a CDR1 sequence selected from SEQ ID NOs: 3, 144, 145, 146, 147, 148, and 149; a CDR2 sequence selected from SEQ ID NOs: 4, 150, 151, 152, 153, 154, 155, and 156; and a CDR3 sequence of SEQ ID NO: 5. In various embodiments, the VHH domain that binds the γδ TCR comprises CDR1, CDR2 and CDR3, which CDR1, CDR2 and CDR3 respectively comprise the amino acid sequence of SEQ ID NO: 3, 4 and 5; 144, 4 and 5; 145, 4 and 5; 146, 4 and 5; 147, 4 and 5; 148, 4 and 5; 149, 4 and 5; 3, 150 and 5; 3, 151 and 5; 3, 152 and 5; 3, 153 and 5; 3, 154 and 5; 3, 155 and 5; or 3, 156 and 5. In various embodiments, the VHH domain that binds to a γδ TCR comprises a CDR1 sequence selected from the group consisting of SEQ ID NOs: 3, 144, 146, 147, and 148; a CDR2 sequence selected from the group consisting of SEQ ID NOs: 4, 150, 151, 152, 153, 154, 155, and 156; and a CDR3 sequence of SEQ ID NO: 5. In various embodiments, the VHH domain that binds to a γδ TCR comprises a CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 3, 4, and 5; 144, 4, and 5; 146, 4, and 5; 147, 4, and 5; 148, 4, and 5; 3, 150, and 5; 3, 151, and 5; 3, 152, and 5; 3, 153, and 5; 3, 154, and 5; 3, 155, and 5; or 3, 156, and 5, respectively. In various embodiments, the VHH domain that binds a γδ TCR comprises a CDR1 sequence of SEQ ID NO: 3; a CDR2 sequence of SEQ ID NO: 4; and a CDR3 sequence of SEQ ID NO: 5. In various embodiments, the VHH domain is humanized.

[0220] In some embodiments, the VHH domain that binds to a γδ TCR comprises SEQ ID NO: 180, wherein X1, X2, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, X27, 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X22 , X 23 , X 24 , X 25 , X 26 and X 27 are independently selected, and where:

[0221] X1 is V or A; X11 is D or G; X20 is T or A;

[0222] X2 is R or G; X12 is A or S; X21 is A or T;

[0223] X3 is K or T; X13 is A or T; X22 is V or L;

[0224] X4 is I or F; X14 is E or Y; X23 is N or S;

[0225] X5 is Q, G or E; X15 is V or A; X24 is K or Y;

[0226] X6 is R or L; X16 is D, E, A, G, V, S, X25 is N, S, E, Y, A,

[0227] X7 is L, W or F; Y, L or Q; S, G, Q;

[0228] X8 is A or S; X17 is S, P, T, A, V, L, I; X26 is S, T, A, L, V,

[0229] X9 is H or A; or G; N or G; and

[0230] X10 is T or S; X18 is G or D; X27 is K, R, E or D.

[0231] X19 is S or N;

[0232] In some embodiments, the VHH domain that binds to a γδ TCR comprises SEQ ID NO: 180, wherein X3 is K, and X1, X2, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X2 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 25, X 26 and X 27 are independently selected, and where:

[0233] X1 is V or A; X11 is D or G; X 20 is T or A;

[0234] X2 is R or G; X12 is A or S; X21 is A or T;

[0235] X4 is I or F; X13 is A or T; X22 is V or L;

[0236] X5 is Q, G or E; X14 is E or Y; X23 is N or S;

[0237] X6 is R or L; X15 is V or A; X24 is K or Y;

[0238] X7 is L, W or F; X16 is D, E, A, L or Q; X25 is N or S;

[0239] X8 is A or S; X17 is S, P, T, V, L or G; X26 is S, T or G; and

[0240] X9 is H or A; X18 is G or D; X27 is K, R, E or D.

[0241] X10 is T or S; X19 is S or N;

[0242] In some embodiments, the VHH domain that binds to a γδ TCR comprises SEQ ID NO: 180, wherein X2 is R; X 25 is N; and X1 and X3 are K, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 26 and X 27 are independently selected, and where:

[0243] X1 is V or A; X11 is D or G; X19 is S or N;

[0244] X4 is I or F; X12 is A or S; X20 is T or A;

[0245] X5 is Q, G or E; X13 is A or T; X21 is A or T;

[0246] X6 is R or L; X14 is E or Y; X22 is V or L;

[0247] X7 is L, W or F; X15 is V or A; X23 is N or S;

[0248] X8 is A or S; X16 is D, E, A or Q; X24 is K or Y;

[0249] X9 is H or A; X17 is S, P or G; X26 is S or T; and

[0250] X10 is T or S; X18 is G or D; X27 is K, R, E or D.

[0251] In some embodiments, the VHH domain that binds to a γδ TCR comprises SEQ ID NO: 180, wherein X2 is R; X3 is K, X4 is I; X9 is H; X 25 is N; and X1, X5, X6, X7, X8, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 26 and X 27 are independently selected, and where:

[0252] X1 is V or A; X13 is A or T; X20 is T or A;

[0253] X5 is Q, G or E; X14 is E or Y; X21 is A or T;

[0254] X6 is R or L; X15 is V or A; X22 is V or L;

[0255] X7 is L, W or F; X16 is D, E or A; X23 is N or S;

[0256] X8 is A or S; X17 is S or P; X24 is K or Y;

[0257] X10 is T or S; X18 is G or D; X26 is S or T; and

[0258] X11 is D or G; X19 is S or N; X27 is K, R, E or D.

[0259] X12 is A or S;

[0260] In some embodiments, the VHH domain that binds to a γδ TCR comprises SEQ ID NO: 180, wherein X1 is V; X2 is R; X3 is K, X4 is I; X9 is H; X 10 is T; X 11 D;X 12 A; X 13 A; X 14 E; X 25 is N; and X5, X6, X7, X8, X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 26 and X 27 are independently selected, and where:

[0261] X5 is Q, G or E; X17 is S or P; X22 is V or L;

[0262] X6 is R or L; X18 is G or D; X23 is N or S;

[0263] X7 is L or W; X19 is S or N; X24 is K or Y;

[0264] X8 is A or S; X20 is T or A; X26 is S or T; and

[0265] X15 is V or A; X21 is A or T; X27 is K, R, E or D.

[0266] X16 is D, E or A;

[0267] In some embodiments, the VHH domain that binds to a γδ TCR comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 2, 17 to 31, 72 to 77, 80 to 143, 158 to 159, and 166 to 179. In some embodiments, the VHH domain that binds to a γδ TCR comprises an amino acid sequence selected from SEQ ID NOs: 2, 17 to 31, 72 to 77, 80 to 143, 158 to 159, and 166 to 179, wherein position 114 of the VHH domain has been substituted with lysine (K) aspartic acid (D), glutamic acid (E), or arginine (R). In some embodiments, the VHH domain that binds the γδ TCR comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to an amino acid sequence selected from SEQ ID NO: 2, 17 to 19, 21 to 23, 25 to 31, 72 to 77, 80, 81, 84 to 86, 88 to 93, 95, 97 to 107, 109, 111 to 129, 131 to 133, 135 to 137, 139 to 143, 158 to 159, and 166 to 179. In some embodiments, the VHH domain that binds to a γδ TCR comprises an amino acid sequence selected from SEQ ID NO: 2, 17 to 19, 21 to 23, 25 to 31, 72 to 77, 80, 81, 84 to 86, 88 to 93, 95, 97 to 107, 109, 111 to 129, 131 to 133, 135 to 137, 139 to 143, 158 to 159, and 166 to 179, wherein position 114 of the VHH domain has been substituted with lysine (K) aspartic acid (D), glutamic acid (E), or arginine (R). In some embodiments, the VHH domain that binds to a γδ TCR comprises an amino acid sequence selected from SEQ ID NO: 2, 17 to 31, 72 to 77, 80 to 143, 158 to 159, and 166 to 179. In some embodiments, the VHH domain that binds a γδ TCR comprises an amino acid sequence selected from SEQ ID NO: 2, 17 to 19, 21 to 23, 25 to 31, 72 to 77, 80, 81, 84 to 86, 88 to 93, 95, 97 to 107, 109, 111 to 129, 131 to 133, 135 to 137, 139 to 143, 158 to 159, and 166 to 179.

[0268] In some embodiments, the VHH domain that binds to the γδ TCR comprises the CDR1 sequence of SEQ ID NO: 3; the CDR2 sequence of SEQ ID NO: 4; and the CDR3 of SEQ ID NO: 5. In some embodiments, the VHH domain comprises an amino acid sequence that is at least 85%, 90%, 95% or at least 99% identical to SEQ ID NO: 99, 143 or 158. In some embodiments, the VHH domain comprises an amino acid sequence selected from SEQ ID NO: 99, 143 and 158, wherein position 114 of the VHH domain has been substituted with lysine (K) aspartic acid (D), glutamic acid (E) or arginine (R). In some embodiments, the VHH domain comprises an amino acid sequence of SEQ ID NO: 99, 143 or 158.

[0269] In various embodiments, the γδ T cell binding polypeptide comprises one, two, three, or four γδ T cell binding VHH domains.

[0270] In some embodiments, the VHH domain that binds to γδ T cells can be humanized. Humanized antibodies (such as sdAb or VHH polypeptides) are suitable for use as therapeutic molecules because humanized antibodies reduce or eliminate human immune responses to non-human antibodies, which can cause immune responses to antibody therapeutics and reduce the effectiveness of therapeutics. In general, humanized antibodies include one or more variable domains, wherein CDR (or a portion thereof) is derived from non-human antibodies, and FR (or a portion thereof) is derived from human antibody sequences. Humanized antibodies will optionally also include at least a portion of a human constant region. In some embodiments, some FR residues in humanized antibodies are replaced with corresponding residues from non-human antibodies (e.g., antibodies from which CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0271] Humanized antibodies and methods for their preparation are reviewed in, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633, and further described in, e.g., Riechmann et al., (1988) Nature 332:323-329; Queen et al., (1989) Proc. Natl Acad. Sci. USA 86:10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., (2005) Methods 36:25-34; Padlan, (1991) Mol. Immunol. 28:489-498 (describing "surface remodeling"); Dall'Acqua et al., (2005) Methods 36:43-60 (describing "FR shuffling"); and Osbourn et al., (2005) Methods 36:61-68; and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (describing the "guided selection" method of FR shuffling).

[0272] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. (1993) J. Immunol. 151: 2296); framework regions derived from the consensus sequence of human antibodies having a particular subgroup of heavy chain variable regions (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4285; and Presta et al. (1993) J. Immunol, 151: 2296); 1:2623); human mature (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front.Biosci.13:1619-1633); and framework regions derived from screening FR libraries (see, e.g., Baca et al., (1997) J.Biol.Chem.272:10678-10684 and Rosok et al., (1996) J.Biol.Chem.271:22611-22618). Typically, the FR regions of a VHH are replaced with human FR regions to generate a humanized VHH. In some embodiments, certain FR residues of a human FR are replaced in order to improve one or more properties of a humanized VHH. A VHH domain having such replaced residues is still referred to herein as "humanized".

[0273] In various embodiments, the Fc region included in the polypeptide that binds to γδ T cells is a human Fc region, or is derived from a human Fc region. In some embodiments, the Fc region included in the polypeptide that binds to γδ T cells is derived from a human Fc region and lacks a C-terminal lysine residue. In some embodiments, the Fc region included in the polypeptide that binds to γδ T cells is derived from a human Fc region and comprises a C-terminal lysine residue. In some embodiments, the C-terminal amino acid of the Fc region is an amino acid other than lysine.

[0274] In some embodiments, the Fc region included in the polypeptide that binds γδ T cells is derived from a human Fc region and comprises three amino acid deletions corresponding to IgG1 E233, L234, and L235 in the lower hinge, referred to herein as "Fc xELL". Fc xELL polypeptides do not bind FcγRs and are therefore referred to as "effector silent" or "effector void", however in some embodiments, the xELL Fc region binds FcRn and therefore has an extended half-life and endocytic trafficking associated with FcRn-mediated recycling.

[0275] In some embodiments, the Fc region included in the polypeptide that binds to γδ T cells is derived from a human Fc region and comprises mutations M252Y and M428V, referred to herein as "Fc-γV". In some embodiments, the mutations enhance binding to FcRn at the acidic pH of the endosome (close to 6.5), while losing detectable binding at neutral pH (about 7.2), resulting in enhanced FcRn-mediated recycling and extended half-life.

[0276] In some embodiments, the Fc region included in the polypeptide that binds to γδ T cells is derived from a human Fc region and comprises mutations designed for heterodimerization, referred to herein as "knob" and "hole". In some embodiments, the "knob" Fc region comprises the mutation T366W. In some embodiments, the "hole" Fc region comprises the mutations T366S, L368A, and Y407V. In some embodiments, the Fc region used for heterodimerization comprises additional mutations, such as the mutation S354C located on the first member of the heterodimeric Fc pair that forms an asymmetric disulfide bond and the corresponding mutation Y349C located on the second member of the heterodimeric Fc pair. In some embodiments, one member of the heterodimeric Fc pair comprises the modification H435R or H435K to prevent protein A from binding while maintaining FcRn binding. In some embodiments, one member of the heterodimeric Fc pair comprises the modification H435R or H435K, while the second member of the heterodimeric Fc pair is not modified at H435. In various embodiments, the holding Fc region comprises the modification H435R or H435K (in some cases, when modified to H435R, referred to as "hole-R"), while the knob Fc region does not comprise a modification. In some cases, the hole-R mutation improves purification of heterodimers relative to the homodimeric hole Fc region that may be present.

[0277] Non-limiting exemplary Fc regions that can be used in polypeptides that bind to γδ T cells include Fc regions comprising an amino acid sequence of SEQ ID NOs: 32 to 70. In some embodiments, the polypeptide that binds to γδ T cells includes an Fc region comprising an amino acid sequence selected from SEQ ID NOs: 32 to 70, wherein the Fc region does not have a C-terminal lysine residue. In some embodiments, the polypeptide that binds to γδ T cells includes an Fc region comprising an amino acid sequence selected from SEQ ID NOs: 34, 35, 41 to 52, and 58 to 70. In some embodiments, the polypeptide that binds to γδ T cells includes an Fc region comprising an amino acid sequence selected from SEQ ID NOs: 34, 35, 41 to 52, 58 to 70, wherein the Fc region does not have a C-terminal lysine residue.

[0278] Exemplary Activities of Polypeptides Binding to γδ T Cells

[0279] In various embodiments, the polypeptides provided herein that bind to γδ T cells stimulate γδ T cells in vitro and / or in vivo. In some embodiments, the methods provided in the embodiments herein can be used to determine the stimulation or activity of γδ T cells in vitro and / or in vivo.

[0280] In some embodiments, the polypeptides provided herein that bind to γδ T cells include an immune cell activating cytokine or an antigen binding domain that binds to an antigen other than γδ T cells and stimulates γδ T cells. In some embodiments, the γδ T cell stimulating activity of the antigen binding domain of the immune cell activating cytokine or the antigen binding domain that binds to an antigen other than γδ T cells is increased and / or more specifically targets cytotoxic T cells when fused to a VHH that binds to γδ T cells than when used alone. In some embodiments, the toxicity of the antigen binding domain of the immune cell activating cytokine or the antigen binding domain that binds to an antigen other than γδ T cells is reduced by specifically targeting it to γδ T cells.

[0281] In some embodiments, a γδ T cell binding polypeptide provided herein comprising an immune cell activating cytokine or an antigen binding domain that binds an antigen other than a γδ T cell increases T cell proliferation in vitro and / or in vivo.

[0282] In some embodiments, the polypeptides provided herein that bind to γδ T cells include VHHs and immune cell activation cytokines provided herein that bind to γδ T cells. In some such embodiments, the immune cell activation cytokines are IL-2, IL-15, IL-7, IL-6, IL-12, IFNα, IFNβ or IFNγ. In some such embodiments, the immune cell activation cytokines are wild-type immune cell activation cytokines. In some embodiments, the immune cell activation cytokines include mutations that weaken the activity of immune cell activation cytokines relative to the activity of wild-type cytokines. In some embodiments, polypeptides that bind to γδ T cells comprising immune cell activation cytokines stimulate γδ T cell activation and proliferation in vivo. In some embodiments, polypeptides that bind to γδ T cells comprising immune cell activation cytokines are used in methods for treating cancer.

[0283] The increase in the proliferation of activated γδ T cells can be determined by any method in the art, such as the method provided in the embodiments herein. A non-limiting exemplary analysis is as follows. γδ T cells can be isolated from one or more healthy human donors. T cells are stained with CellTrace Violet (CTV) and contacted with a polypeptide comprising a modified IL-2, and then analyzed by FACS. CTV staining loss indicates proliferation. In some embodiments, such as by measuring the proliferation of γδ T cells isolated from different healthy human donors, the increase in γδ T cell proliferation is determined in the form of the average value of a group of experiments or combined T cells. In some embodiments, the increase in γδ T cell proliferation is determined in the form of the average value of experiments performed using T cells from at least five or at least ten different healthy donors or T cell pools from at least five or at least ten different healthy donors.

[0284] In some embodiments, the polypeptides provided herein that bind to γδ T cells include VHHs that bind to γδ T cells and antigen binding domains that bind to antigens other than γδ T cells. In some such embodiments, the antigen is Lag3, CTLA4, TGFBR1, TGFBR2, Fas, TNFR2, PD1, PDL1, or TIM3. In some embodiments, the antigen is 1-92-LFA-3, 5T4, α-4 integrin, α-V integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33 , CD38, CD39, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD 73. CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD1 38. CD166, CD172A, CD248, CDH6, CEACAM5(CEA), CEACAM6(NCA-90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FcRH5, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GPRC5D, GRP78, HAVCAR1, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MICA, MICB, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch1, Notch 2, Notch 3, Notch 4. NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine 1 phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, T LR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, TROP-2uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2 or WISP-3. In some embodiments, the polypeptide that binds to γδ T cells comprises a VHH that binds to γδ T cells and an antigen binding domain that binds to a tumor cell antigen. In some such embodiments, the polypeptide that binds to γδ T cells that comprises an antigen binding domain that binds to a tumor cell antigen increases γδ T cell-mediated killing of tumor cells that express the antigen.

[0285] Peptide expression and production

[0286] A nucleic acid molecule comprising a polynucleotide encoding a polypeptide that binds to γδ T cells is provided. In some embodiments, the nucleic acid molecule may also encode a leader sequence that directs secretion of the polypeptide that binds to γδ T cells, which leader sequence is typically cleaved so that it is not present in the secreted polypeptide. The leader sequence may be a native heavy chain (or VHH) leader sequence, or may be another heterologous leader sequence.

[0287] Nucleic acid molecules can be constructed using recombinant DNA techniques routine in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0288] Provided are vectors comprising nucleic acids encoding polypeptides that bind to γδ T cells as described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, vectors optimized to express polypeptides in desired cell types such as CHO or CHO-derived cells or NSO cells are selected. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20: 880-889 (2004).

[0289] In some embodiments, the polypeptide that binds to γδ T cells can be expressed in prokaryotic cells, such as bacterial cells; or eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. The expression can be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44.Lec13 CHO cells, and FUT8 CHO cells; cells (Crucell); and NSO cells. In some embodiments, polypeptides that bind γδ T cells can be expressed in yeast. See, for example, U.S. Publication No. US2006 / 0270045A1. In some embodiments, a particular eukaryotic host cell line is selected based on its ability to produce the desired post-translational modification of the polypeptide. For example, in some embodiments, CHO cells produce a polypeptide that has a higher level of sialylation than the same polypeptide produced in 293 cells.

[0290] The introduction of one or more nucleic acids (such as vectors) into the desired host cells can be accomplished by any method, including but not limited to calcium phosphate transfection, DEAE-polydextrose mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Edition Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into the desired host cells according to any suitable method.

[0291] Host cells comprising any nucleic acid or vector described herein are also provided. In some embodiments, a host cell expressing a polypeptide described herein that binds to γδ T cells is provided. The polypeptides expressed in the host cell that bind to γδ T cells can be purified by any suitable method. The method includes, but is not limited to, the use of an affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include ROR1 ECD and agents that bind to the Fc region. For example, protein A, protein G, protein A / G, or antibody affinity columns can be used to bind to the Fc region and purify polypeptides that bind to γδ T cells comprising the Fc region. Hydrophobic interaction chromatography, such as butyl or phenyl columns, may also be applicable to the purification of some polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) is also applicable to the purification of some polypeptides, such as antibodies. Mixed mode chromatography (e.g., reverse phase / anion exchange, reverse phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) may also be applicable to the purification of some polypeptides, such as antibodies. Many methods for purifying polypeptides are known in the art.

[0292] In some embodiments, the polypeptide that binds to γδ T cells is produced in a cell-free system. Non-limiting exemplary cell-free systems are described in, for example, Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0293] In some embodiments, a polypeptide that binds to a γδ T cell prepared by the above method is provided. In some embodiments, the polypeptide that binds to a γδ T cell is prepared in a host cell. In some embodiments, the polypeptide that binds to a γδ T cell is prepared in a cell-free system. In some embodiments, the polypeptide that binds to a γδ T cell is purified. In some embodiments, a cell culture medium containing the polypeptide that binds to a γδ T cell is provided.

[0294] In some embodiments, a composition comprising an antibody prepared by the methods described above is provided. In some embodiments, the composition comprises a polypeptide that binds to γδ T cells prepared in a host cell. In some embodiments, the composition comprises a polypeptide that binds to γδ T cells prepared in a cell-free system. In some embodiments, the composition comprises a purified polypeptide that binds to γδ T cells.

[0295] Exemplary methods for treating disease using polypeptides that bind γδ T cells

[0296] In some embodiments, a method of treating a disease in an individual is provided, comprising administering a polypeptide that binds to γδ T cells. The disease includes a disease that would benefit from T cells, such as γδ T cells. + Any disease in which the proliferation and activation of T cells is increased. In some embodiments, methods for treating cancer in an individual are provided. In some embodiments, the method of treating cancer comprises increasing the proliferation and / or activation of γδ T cells by administering a polypeptide that binds to γδ T cells comprising a VHH that binds to γδ T cells and an immune cell activating cytokine or an antigen binding domain that binds to a tumor cell antigen other than γδ T cells.

[0297] The method comprises administering to an individual an effective amount of a polypeptide that binds to γδ T cells as provided herein. The method of treatment may be for humans or animals. In some embodiments, a method of treating humans is provided. Non-limiting exemplary cancers that can be treated with the polypeptide that binds to γδ T cells as provided herein include basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain cancer and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer; gastrointestinal cancer; glioblastoma; hepatic carcinoma; liver tumor; intraepithelial neoplasia; kidney cancer or renal cancer; laryngeal cancer; liver cancer (liver cancer); lung cancer; small cell lung cancer; non-small cell lung cancer; adenocarcinoma of the lung; squamous cell lung cancer; melanoma; myeloma; neuroblastoma; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; colorectal cancer; respiratory cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancer; and vulvar cancer; lymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma; B-cell lymphoma; low-grade / follicular Non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; massive NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; and chronic myeloid leukemia.

[0298] The polypeptide that binds to γδ T cells may be administered to the individual as needed. The frequency of administration may be determined by a person skilled in the art, such as an attending physician, based on consideration of the condition being treated, the age of the individual being treated, the severity of the condition being treated, the general health of the individual being treated, and the like. In some embodiments, one or more effective doses of a polypeptide that binds to γδ T cells are administered to the individual. In some embodiments, an effective dose of a polypeptide that binds to γδ T cells is administered to the individual daily, semi-weekly, weekly, biweekly, monthly, etc. An effective dose of a polypeptide that binds to γδ T cells is administered to the individual at least once. In some embodiments, an effective dose of a polypeptide that binds to γδ T cells may be administered multiple times, including multiple times over the course of at least one month, at least six months, or at least one year.

[0299] In some embodiments, the pharmaceutical composition is administered in an amount effective to treat (including prevent) cancer and / or increase T cell proliferation. The therapeutically effective amount is generally determined by the body weight of the individual being treated, their physiology or health, the extension of the condition being treated, or the age of the individual being treated. In general, the antibody can be administered in an amount ranging from about 0.05 mg / kg body weight to about 100 mg / kg body weight per administration.

[0300] In some embodiments, polypeptides that bind to γδ T cells can be administered in vivo by various routes, including but not limited to intravenous, intraarterial, parenteral, intraperitoneal or subcutaneous. Appropriate formulations and routes of administration can be selected according to the intended application.

[0301] In some embodiments, therapeutic treatment using a polypeptide that binds to γδ T cells is achieved by increasing T cell proliferation and / or activation, and / or by contacting γδ T cells with cancer cells. In some embodiments, increasing T cell proliferation and / or activation inhibits cancer growth.

[0302] Pharmaceutical composition

[0303] In some embodiments, the composition comprising a polypeptide that binds to γδ T cells is provided in the form of a formulation with various pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th Edition (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd Edition, Pharmaceutical Press (2000)). Various pharmaceutically acceptable carriers including vehicles, adjuvants, and diluents can be used. In addition, various pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like can also be used. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0304] In some embodiments, the pharmaceutical composition comprises a polypeptide that binds γδ T cells at a concentration of at least 10 mg / mL.

[0305] Combination therapy

[0306] The polypeptide binding to γδ T cells can be administered alone or in combination with other treatment modes such as other anticancer agents. It can be provided before, substantially simultaneously or afterwards (that is, in parallel or sequentially) other treatment modes. In some embodiments, the treatment methods described herein may further include administration: radiotherapy, chemotherapy, vaccination, targeted tumor therapy, CAR-T therapy, oncolytic virus therapy, cancer immunotherapy, cytokine therapy, surgical resection, chromosome modification, ablation, cryotherapy, antisense agents for tumor targets, siRNA agents for tumor targets, microRNA agents for tumor targets or anticancer / tumor agents, or biological agents, such as antibodies, cytokines or receptor cell extracellular domain-Fc fusions.

[0307] In some embodiments, the γδ T cell binding polypeptides provided herein are administered concurrently with a second therapeutic agent, e.g., PD1 or PD-L1 therapy. Implementation options for PD-1 / PD-L1 therapy include nivolumab (BMS); pidilizumab (CureTech, CT-011), pembrolizumab (Merck); durvalumab (Medimmune / AstraZeneca); atezolizumab (Genentech / Roche); avelumab (Pfizer); AMP-224 (Amplimmune); BMS-936559; AMP-514 (Amplimmune); MDX-1105 (Merck); TSR-042 (Tesaro / AnaptysBio, ANB-011); STI-A1010 (Sorrento Therapeutics); STI-A1110 (Sorrento Therapeutics); and other agents targeting programmed death-1 (PD-1) or programmed death-ligand 1 (PD-L1).

[0308] In some embodiments, the polypeptides provided herein that bind to γδ T cells are administered concurrently with an immunostimulant such as an agonist of a member of the tumor necrosis factor receptor superfamily (TNFRSF) or a member of the B7 family. Non-limiting embodiments of immunostimulatory TNFRSF members include OX40, GITR, 41BB, CD27, and HVEM. Non-limiting embodiments of B7 family members include CD28 and ICOS. Therefore, in some embodiments, the polypeptides provided herein that bind to γδ T cells are administered concurrently with an agonist (such as an agonist antibody) of OX40, GITR, 41BB, CD27, HVEM, CD28, and / or ICOS.

[0309] In some embodiments, the polypeptides provided herein that bind to γδ T cells are administered concurrently with CAR-T (chimeric antigen receptor T cell) therapy, oncolytic virus therapy, cytokine therapy, and / or agents targeting other checkpoint molecules such as VISTA, gpNMB, B7H3, B7H4, HHLA2, CTLA4, TIGIT, and the like.

[0310] Non-Limiting Exemplary Diagnostic and Therapeutic Methods

[0311] In some embodiments, the methods described herein are applicable to evaluating an individual and / or a sample from an individual (eg, a cancer patient). In some embodiments, the evaluation is one or more of a diagnosis, a prognosis, and / or a response to treatment.

[0312] In some embodiments, the methods described herein comprise assessing the presence, absence, or amount of a protein. In some embodiments, the methods described herein comprise assessing the presence, absence, or expression of a nucleic acid. The compositions described herein can be used for these measurements. For example, in some embodiments, the methods described herein comprise contacting a sample of a tumor or cells cultured from a tumor with a therapeutic agent as described herein.

[0313] In some embodiments, the assessment can guide treatment (including treatment with the antibodies described herein). In some embodiments, the assessment can guide the use or retention of adjuvant therapy after resection. Adjuvant therapy, also known as adjuvant care, is a treatment given in addition to primary, primary or preliminary treatment. By means of non-limiting embodiments, adjuvant therapy can be an additional treatment given usually after surgery when all detectable diseases have been removed, but there is still a statistical risk of recurrence due to recessive diseases. In some embodiments, antibodies are used as adjuvant therapy in cancer treatment. In some embodiments, antibodies are used as the only adjuvant therapy in cancer treatment. In some embodiments, antibodies described herein are rejected as adjuvant therapy in cancer treatment. For example, if the patient is unlikely to react to the antibodies described herein or will have a minimal response, then for the sake of quality of life and to avoid unnecessary toxicity from ineffective chemotherapy, treatment may not be administered. In these cases, palliative care can be used.

[0314] In some embodiments, the molecule is administered as a neoadjuvant therapy before resection. In some embodiments, neoadjuvant therapy refers to a therapy that shrinks and / or downgrades a tumor before any surgery. In some embodiments, neoadjuvant therapy means chemotherapy administered to a cancer patient before surgery. In some embodiments, neoadjuvant therapy means an antibody administered to a cancer patient before surgery. Cancer types that are generally considered neoadjuvant chemotherapy include, for example, breast cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, and lung cancer. In some embodiments, antibodies are used as neoadjuvant therapy in cancer treatment. In some embodiments, it is used before resection.

[0315] In some embodiments, the tumor microenvironment encompassed in the methods described herein is one or more of the following: tumor vasculature; tumor infiltrating lymphocytes; fibroblastic reticular cells; endothelial precursor cells (EPC); cancer-associated fibroblasts; coat cells; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T cells; regulatory T cells; macrophages; neutrophils; and other immune cells located in close proximity to the tumor.

[0316] Reagent test kit

[0317] Also provided are articles of manufacture and kits comprising any polypeptide that binds to γδ T cells as described herein and suitable packaging. In some embodiments, the invention includes a kit having (i) a polypeptide that binds to γδ T cells, and (ii) instructions for using the kit to administer the polypeptide that binds to γδ T cells to an individual.

[0318] Suitable packaging for the compositions described herein is known in the art and includes, for example, vials (e.g., sealed vials), containers, ampoules, bottles, cans, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles may be further sterilized and / or sealed. The present invention also provides unit dosage forms comprising the compositions described herein. The unit dosage forms may be stored in suitable packaging in single or multiple unit doses and may also be further sterilized and sealed. The instructions supplied in the kits of the present invention are typically written instructions on a label or drug instructions (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. Instructions for use of the antibody generally include information on dosage, dosing schedule, and route of administration for intended treatment or industrial use. The kit may further include a description of selecting a suitable individual or treatment.

[0319] The container may be a unit dose, bulk (e.g., multi-dose packaging), or a subunit dose. For example, a kit containing a sufficient dose of the molecules disclosed herein to provide an extended period of effective treatment to an individual may also be provided, such as about 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or more. The kit may also include multiple unit doses of molecules and instructions for use and be packaged in sufficient amounts for storage and use in pharmacies (e.g., hospital pharmacies and dispensing pharmacies). In some embodiments, the kit includes a dry (e.g., lyophilized) composition that may be reconstituted, resuspended, or rehydrated to form a generally stable aqueous suspension of the antibody.

[0320] Example

[0321] The embodiments discussed below are intended only to illustrate the present invention and should not be considered to limit the present invention in any way. The embodiments are not intended to represent that the following experiments are all experiments or the only experiments performed. Efforts have been made to ensure the accuracy of the quantities used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0322] Example 1: IL-2 targeting γδ TCR activates STAT5 signaling pathway in γδ T cells

[0323] T cell activation by IL-2 signaling leads to phosphorylation of the transcription factor STAT5. PBMCs were separated from healthy donor leukopak by lymphocyte separation agent density gradient centrifugation. The cells were labeled with the following fluorescent conjugated antibodies for 20 minutes at room temperature: non-competitive anti-γδTCR-FITC, anti-CD3-BV785 and anti-CD56-BV421. After washing, 200,000 PBMCs / well were seeded in a 96-well plate. The cells were treated with a titration of a fusion protein comprising IL-2_X or IL-2_Y (both of which are attenuated IL-2 polypeptides) fused to the C-terminus of anti-γδTCR-5C8 or anti-γδTCR-6C4, starting with a starting concentration of 100nM, and the entire plate was titrated in duplicate at 1:5. The plate was incubated at 37°C / 5% CO2 for 20 minutes. With BD Cytofix / Cytoperm TM (BD Biosciences), fixed cells, permeabilized in 90% ice-cold methanol, and the levels of phosphorylated STAT5 ("pSTAT5") were measured by flow cytometry using a phospho-specific anti-pSTAT5-PE antibody (1:70) and gated on CD3-BV785 + γδTCR-FITC +To identify γδ T cells, circle CD56-BV421 + CD3-BV785 - To identify NK cells and select CD3-BV785 + γδTCR-FITC - To identify αβ T cells.

[0324] like Figures 1A to 1H As shown in Figure 2, treatment with bivalent or monovalent anti-γδ TCR-5C8 or anti-γδ TCR-6C4 targeting IL-2_X or IL-2_Y resulted in pSTAT5 + Dose-dependent increase in the percentage of γδ T cells and median fluorescence intensity of pSTAT5 on γδ T cells. In contrast, there was little pSTAT5 activation on NK or αβ T cells below 100 nM.

[0325] Example 2: Increased proliferation and accumulation of γδ T cells upon treatment with low-affinity IL-2_X targeting γδTCR

[0326] IL-2 promotes the activation and proliferation of T cell populations. In order to assess the effect of IL-2_X targeting γδTCR on proliferation, PBMCs were separated from healthy donor leukocytes using lymphocyte separation agent density gradient medium. Cells were labeled with CellTrace Violet proliferative dye for 10 minutes at 37°C. After washing, the cells were resuspended in RPMI+10% FBS, and 300,000 cells per well were added to a 96-well plate. Cells were treated with titration of fusion proteins containing IL-2_X fused to the C-terminus of anti-γδTCR-5C8 or non-targeted control VHH, starting with a starting concentration of 100nM, and the entire plate was titrated in duplicate at 1:5. The plate was cultivated for 7 days at 37°C / 5% CO2. Cells were labeled with the following fluorescent conjugated antibodies for 30 minutes at 4°C: CD3-BV785, γδTCR-FITC, and viability dye iodide propidium. Cells were washed and analyzed by flow cytometry.

[0327] like FIG. 2A to FIG. 2D As shown in Figure 3, treatment with anti-γδTCR-5C8-targeted IL-2_X resulted in a dose-dependent increase in γδ T cell proliferation. + The percentage of γδ T cells in the population also increased with anti-γδTCR-5C8 targeted IL-2_X treatment, and αβ T cells decreased simultaneously. This effect was specific for γδ T cells, as the αβ T cell population did not proliferate in response to treatment with anti-γδTCR-5C8 targeted IL-2_X. Non-targeting VHH fused to IL-2_X did not promote the proliferation of γδ or αβ T cells, demonstrating target specificity.

[0328] Example 3: IL-2 Activates Vδ2 by Targeting γδTCR + STAT5 signaling in γδ T cells

[0329] IL-2 signaling leads to phosphorylation of the transcription factor STAT5 and activation of downstream gene expression. To evaluate STAT5 phosphorylation, PBMCs were isolated from whole blood of healthy human donors by lymphocyte separation agent density gradient centrifugation. The cells were labeled with the following fluorescent conjugated antibodies at room temperature for 20 minutes: non-competitive anti-γδTCR-FITC, anti-CD3-BV785 and anti-Vδ2-BV421. After washing, 400,000 PBMCs / well were seeded in a 96-well plate. The cells were treated with a titration of a fusion protein containing IL-2_X fused to the C-terminus of anti-γδTCR-1D7, starting with a starting concentration of 50nM, and the entire plate was titrated in duplicate at 1:3. The plate was incubated at 37°C / 5% CO2 for 20 minutes. BD Cytofix / Cytoperm TM (BD Biosciences) fixed cells and incubated in 90% ice-cold BD Phosflow TM The cells were permeabilized in Perm Buffer III (BD Biosciences), and Vδ2 was measured by flow cytometry using a phosphorylation-specific anti-pSTAT5-PE antibody (1:70). + γδ T cells or Vδ2 - γδ - Levels of phosphorylated STAT5 ("pSTAT5") on αβ T cells.

[0330] like FIG. 3A to FIG. 3B As shown in Figure 2, treatment with monovalent anti-γδTCR-1D7 targeting IL-2_X resulted in pSTAT5 + Vδ2 + The percentage of γδ T cells ( Figure 3A ) and Vδ2 + Median fluorescence intensity of pSTAT5 on γδ T cells ( Figure 3B ). In contrast, no detectable pSTAT5 was present on αβ T cells at any concentration tested.

[0331] Example 4: Treatment with low-affinity IL-2_X targeting γδTCR potently expands Vδ2 + γδ T cells

[0332] IL-2 promotes the proliferation and effector function of γδ T cells. To determine the effect of IL-2_X targeting γδTCR on proliferation, PBMCs were separated as described in Example 2 and labeled with CellTrace Violet and added at 300,000 per well. Cells were treated with titration of fusion proteins comprising IL-2_X fused to the C-terminus of anti-γδTCR-1D7 or non-targeted control VHH, starting with a starting concentration of 100nM, and the entire dish was titrated in duplicate at 1:5. The dish was cultivated for 7 days at 37°C / 5% CO2. The cells were labeled with the following fluorescent conjugated antibodies at 4°C for 30 minutes: CD3-BV785, γδTCR-FITC, Vδ2-PE, and viability dye iodine propidium. The cells were washed and analyzed by flow cytometry.

[0333] like FIG. 4A to FIG. 4B As shown in Figure 2, treatment with anti-γδTCR-1D7 (cx11026) targeting IL-2_X resulted in a dose-dependent increase in the proliferation of Vδ2 γδT cells ( Figure 4A ). In addition, total CD3 + The percentage of γδ T cells in the T cell population was substantially increased with anti-γδTCR-1D7-targeted IL-2_X treatment ( Figure 4B ). Non-targeting VHH (cx9452) fused with IL-2_X did not promote the proliferation of any Vδ2γδ T cells, demonstrating target specificity.

[0334] Example 5: Development of VHH domains that bind γδ TCR

[0335] Single domain antibodies targeting human γδ TCR were generated by immunizing llamas with enriched γδ T cells and heterodimeric knob-hole constructs consisting of human γ9 extracellular domain cloned with reduced effector knob Fc paired with human δ2 extracellular domain containing reduced effector hole Fc. After specific anti-γδ TCR antibody titers were generated, llama peripheral blood mononuclear cells (PBMCs) were isolated from 500 mL of blood from immunized animals and total mRNA was isolated using Qiagen RNeasy Maxi Kit and subsequently converted to first strand cDNA using Thermo Superscript IV reverse transcriptase and oligo-dT priming. VHH sequences were specifically amplified via PCR using cDNA as template and cloned into a yeast surface display vector as VHH-Fc-AGA2 fusion protein. The Fc is human IgG1 Fc (SEQ ID NO:32), or in some cases, a variant IgG1 Fc with reduced effector function (eg, Fc xELL; SEQ ID NO:33).

[0336] Yeast libraries displaying VHH-Fc-AGA2 fusion proteins were enriched using a recombinant form of the γδ TCR ECD, separated by magnetic beads, followed by fluorescence activated cell sorting (FACS). The sorted yeast were precipitated, and the isolated colonies were selected and placed in 96-well plates and grown in culture medium, converting surface displayed VHH-Fc expression to secretion into the culture medium. Supernatants from 96-well yeast secretion cultures were applied to 293F cells transiently transfected with γδ TCR (γδ TCR positive) or untransfected 293F cells (γδ TCR negative), washed, treated with a fluorophore-labeled anti-human IgG1 Fc secondary antibody, and analyzed by 96-well flow cytometry.

[0337] The nucleic acid sequence encoding VHHs that bind to γδ TCR-positive cells and do not bind to γδ TCR-negative cells was cloned into a mammalian expression vector in frame with the human Fc xELL coding region and expressed by transient transfection using polyethyleneimine in HEK293 Freestyle cells (293F cells) or CHO cells. The supernatant was collected after 3-7 days, the secreted recombinant protein was purified by protein A chromatography, and the concentration was calculated by the absorbance at 280 nm and the extinction coefficient.

[0338] Binding of γδ TCR-binding VHH, 1D7 and its humanized version formatted as a monomeric VHH-hIgG1-Fc fusion protein using a non-dimeric human IgG1 Fc variant region lacking the hinge Fc NNT was evaluated by flow cytometry on fresh or frozen expanded human Vδ2+γδ T cells. + γδ T cells. Freshly isolated PBMCs were resuspended at 1.0×10^6 cells / mL in complete RPMI medium supplemented with 10% FBS, 5uM zoledronate and 500IU / ml IL-2. On day 3, half of the medium was removed and IL-2 was added at a final concentration of 500IU / mL. For the remainder of the expansion, the medium was replaced and IL-2 was added at a final concentration of 500IU / ml every 2-3 days for a total of two weeks. Purity was assessed by flow cytometry and was typically greater than 80% Vδ2 +γδ T cells. Freshly expanded γδ T cells were plated at 30,000 cells / well in a 96-well plate in FACS buffer (PBS, 1% BSA, 0.1% NaN3, pH 7.4), or thawed expanded γδ T cells were plated at 50,000 cells / well in a 96-well plate. Untransfected HEK293F cells were used as γδ TCR negative controls and plated at 30,000 cells / well in a separate plate. The test polypeptide was then diluted to 2× final concentration 1000 nM, and 3, 4, and 5-fold serial dilutions were performed. FACS buffer without polypeptide was used as a control for secondary antibody only. The polypeptide dilutions were added to an equal volume of cells, and the analysis plate was incubated at 4°C for 30 minutes. After washing twice with 150 μL FACS buffer / well, cells were resuspended in FACS buffer with fluorescently labeled anti-human Fc antibody diluted 1:1000 or 1:2000 to detect binding. The assay plates were incubated at 4°C for 20 minutes and washed once with 150 μL FACS buffer / well. Binding to γδ T cells was determined by flow cytometry using Intllicyt iQue Plus, and anti-human A647 median fluorescence intensity was calculated using onboard software. Data were plotted and analyzed using GraphPad Prism analysis software. Binding curves of the initial humanized 1D7 VHH fusion protein were FIG. 5A to FIG. 5I The binding curve of the improved humanized variant 1D7v158 is shown in Figure 5J This variant has a more favorable pI profile and has comparable binding affinity to the parent VHH. Other humanized variants based on 1D7v158 were also generated, and the binding curves of the parent 1D7v158 and other variants were Figure 5K Displayed in.

[0339] The affinity (K) of 1D7 ("1D7-p") and its humanized version was determined by combining data from flow cytometry using a nonlinear fitting model. D ), and are shown in Table 2.

[0340] The amino acid sequences of 1D7 VHH and humanized versions thereof are provided in the table of certain sequences provided below. It is specified that the amino acid at residue 114 in any of the disclosed 1D7 VHH domains may be lysine (K) aspartic acid (D), glutamic acid (E) or arginine (R). For example, the parent 1D7 VHH (SEQ ID NO: 2) comprises lysine (K) at residue 114, hu1D7v39 VHH (SEQ ID NO: 97) comprises arginine (R) at residue, and hu1D7v158 (SEQ ID NO: 158) comprises glutamic acid (E) at residue 114. Thus, the residue at position 114 may be substituted with lysine, aspartic acid, glutamic acid or arginine.

[0341] Table 2

[0342]

[0343]

[0344] *Determined from data in Figure 6J

[0345] ^Determined from the data in Figure 6K

[0346] Example 6: PBMCs expanded with IL-2 targeting γδTCR exhibit potent cytotoxicity in a wide range of tumor cell lines

[0347] The ability of PBMCs expanded with IL-2 molecules targeting γδTCR (cx11026) to kill target cell lines derived from a variety of blood and solid tumors was evaluated. Briefly, PBMCs isolated from Leuko75 were expanded with 1 nM cx11026 essentially as described in Example 7, and after a two-week expansion process, the cells were resuspended to 2.0×10^6 cells / ml for killing analysis. 1-2×10^6 target cells were washed with HBSS, labeled with Cyto-ID red for 6 minutes following the kit instructions, and then the cells were resuspended in complete RPMI medium and plated at 100 microliters / well (10,000 cells). Control wells using untreated PBMCs or containing only target cells were included. Adherent cells were plated on a flat bottom, and non-adherent cells were plated on a 96-well plate coated with Poly-L, and the plate was incubated at room temperature for 20 minutes, followed by incubation at 37°C / 5% CO2 for 2 hours before adding effector cells. Effector cells were then added at 100,000 / well in a 50 μL volume at a 10:1 ratio to target cells. Apoptosis protease 3 / 7-green was added at a final concentration of 1.25 μM in 50 μL / well. Cells were allowed to settle for 20 minutes at room temperature and analyzed for cell killing using an Incucyte cell imager. After equilibration at temperature for 30 minutes, target cell killing was assessed by the overlap of apoptosis protease 3 / 7-green and cyto-ID red every 1.5 hours. After making masks for target cells and dead cells, the overlap data was plotted and analyzed using GraphPad Prism analysis software.

[0348] like FIG. 6A to FIG. 6B As shown in , treatment of PMBCs with an IL-2 molecule targeting the γδTCR (1D7 x IL-2_X) resulted in the expansion of Vγ9Vδ2 T cells and extensive anti-tumor killing activity in a wide variety of cell lines, including THP-1 acute monocytic leukemia, HT29 colorectal adenocarcinoma, Daudi Burkitt's lymphoma, NCI-H460 lung cancer, MM1S multiple myeloma, and A375 malignant melanoma cell lines.

[0349] Example 7: Activity of γδTCR x CD20 bispecific molecules

[0350] The binding activity of a bispecific γδ TCR molecule (cx11498) comprising an anti-γδ VHH domain 1D7v9, an anti-CD20 VHH domain and a heterodimeric knob-hole Fc region binding to a γδ TCR and a representative target antigen CD20 (γδ TCR x CD20) was evaluated by flow cytometry on CD20-expressing Raji target cells and Vγ9Vδ2 T cells. PBMCs and Raji cells were thawed and resuspended in FACS buffer to 1.0×10^6 cells / ml. 100,000 PBMCs or Raji cells were seeded in a 96-well plate. Test articles were added at a final starting concentration of 50 nM and the entire plate was titrated at 1:4. Cells were incubated with antibodies for 30 minutes at 4°C, centrifuged briefly, and washed four times with FACS buffer. Cells were labeled with anti-human Fcγ-A647 (Jackson ImmunoResearch) and PBMCs were also labeled with anti-human γδ TCR-FITC (Biolegend) in 50uL FACS at 4°C for 30 minutes. Plates were washed twice with FACS buffer, resuspended in FACS buffer and read on an IQue flow cytometer. The mean fluorescence intensity of anti-human Fcγ-A647 was calculated using the onboard software and graphically plotted using GraphPad Prism. γδ TCR x antigen molecules bind to Raji cells expressing antigen (CD20) in a titration-dependent manner ( Fig. 7A ) and Vγ9Vδ2 T cells ( Figure 7B ).

[0351] Raji cells and freshly isolated Vγ9Vδ2 T cells or expanded Vγ9Vδ2 T cells were used to evaluate the killing activity of Vγ9Vδ2 T cells mediated by γδ TCR x CD20 molecules (cx11498) and sequence analogs of the anti-CD20 antibody rituximab (Invivogen, hcd20-mab164-43-01) in a FACS-based killing assay. Freshly isolated Vγ9Vδ2 T cells were isolated from thawed PBMCs washed in complete medium with CTL thawing buffer using the Stemcell EasySep Human Vγ9Vδ2 T Cell Isolation Kit, the cells were resuspended in 2% FBS / PBS and stained with Vδ2-PE antibody for 20 minutes, washed and resuspended in 2% FBS / PBS and run on a cell sorter to selectively sort Vδ2+ T cells, the sorted cells were resuspended to 2×10^6 cells / mL in complete assay medium before use. Expanded Vγ9Vδ2 T cells were prepared from thawed PBMCs. Briefly, PBMCs were washed with CTL thawing buffer in complete medium and resuspended in a culture flask to 1.0×10^6 cells / mL, and 1 nM of the monovalent IL-2 molecule 1D7 x IL-2_X (cx11026) targeting γδTCR was added. The culture flask was incubated at 37°C / 5% CO2 for 7 days. On the third day, the cells were briefly centrifuged, resuspended and re-added with 1 nM 1D7 xIL-2_X. After 7 days of expansion, the cells were resuspended to 20×10^6 cells / ml and stained for Vδ2-PE in complete medium for 30 minutes, washed and resuspended in 2% FBS / PBS+1 mM EDTA, and run on a cell sorter to isolate pure Vγ9Vδ2 T cells. Raji cells were labeled with Far Red cell tracer (ThermoFisher), then plated at 10,000 cells / well in 50 μL, sorted Vγ9Vδ2 T cells (freshly isolated or expanded) were added at a ratio of 4:1 (effector: target) cells in 50 μL, and the plate was titrated down at 1:2. 25 μL of test article (at 8× final concentration 5nM concentration in RPMI) or culture medium alone was added to the cells, and culture medium was added to a final volume of 200 μL, and cells were incubated overnight at 37°C / 5% CO2. The next day, cells were labeled with the following components in FACS buffer at 4°C for 30 minutes: anti-human CD3-BV785, anti-human Vδ2-PE, Apotracker-green, and PI. Subsequently, the plates were washed and analyzed by flow cytometry (Quanteon). The percentage of apoptotic cells was determined for labeled Raji target cells using Apotracker green.

[0352] like FIG. 7C to FIG. 7DAs shown in Figure 2, treatment with γδTCR x CD20 peptide (cx11498) resulted in γδ T cell-mediated activation of newly isolated ( Figure 7C ) and Vγ9Vδ2T cells expanded by anti-Vγ9xIL2-X ( Fig.7D ) increased killing of Raji target cells. Compared with freshly isolated Vγ9Vδ2 T cells, Vγ9Vδ2 T cells amplified by 1D7 x IL-2_X exhibited excellent killing activity in the presence and absence of anti-CD20xVγ9-1D7v9. In addition, treatment with rituximab had little effect on Raji cell killing in the case of freshly isolated Vγ9Vδ2 T cells, but increased the killing activity of Vγ9Vδ2 T cells amplified by 1D7 x IL-2_X. This indicates that treatment with anti-1D7 x IL-2_X enhances tumor cell killing activity through multiple mechanisms including improved ADCC activity. These data indicate that additional targeting of bispecific or conventional ADCC antibodies improves Vγ9Vδ2 T cell target cell killing activity.

[0353] Example 8: Activity of γδTCR x CD33 bispecific molecules

[0354] The binding activity of the bispecific γδ TCR molecule (cx12083) comprising the binding γδTCR and the representative target antigen CD33 (γδTCR x CD33) of the anti-γδ VHH domain 1D7v9, anti-CD33 VHH domain and heterodimeric knob-hole Fc region was evaluated on MOLM-13 and MV-411 cell line target cells and Vγ9Vδ2 T cells expressing CD33 by flow cytometry analysis. The monospecific CD33 construct (CD33 x UT; cx12056) comprising an unrelated second VHH domain rather than a γδTCR VHH domain was also tested. In brief, frozen PCMCs (previously treated with zoledronate to amplify γδ T cells) were thawed and washed twice in the assay medium using 1x CTL thawing agent. The cells were resuspended in FACs buffer to 1×10^6 cells / mL and plated on a 96-well plate (100,000 cells / well). 50uL test article is added to each well with 4× final starting concentration 200nM and titrated throughout at 1:4. 50uL FACS buffer is added to reach a final total volume of 200uL. Cells are cultivated with test article at 4°C for 30 minutes, washed three times, and stained with Vd2-PE, CD3-BV785 and anti-human-A647 antibodies in FACS buffer at 4°C for 30 minutes. Cells are then washed 2 times with FACS buffer, resuspended in 70ul FACS buffer and read on Novocyte. All molecules bind to MOLM-13 ( expressing CD33) in a titration-dependent manner. Fig. 8A ) and MV4-11( Figure 8B ) cells. However, only the bispecific γδTCR x CD33 molecules bound to Vγ9Vδ2 T cells ( Figure 8C ).

[0355] Vγ9Vδ2 T cell-mediated killing activity of γδTCR x CD33 molecules (cx12083) and CD33 x UT control molecules was evaluated in a FACS-based killing assay using MOLM-13 and MV-411 target cells and expanded Vγ9Vδ2 T cells. Vδ2 was expanded from frozen PBMCs isolated from peripheral blood leukocyte concentrates of healthy human donors. + γδ T cells were cultured and resuspended at 1.0×10^6 cells / mL in complete RPMI medium with 10% FBS. On the first day, 1 nM anti-Vγ9xIL2-X was added to the medium. On the 4th day, the cells were briefly centrifuged and 1 nM anti-Vγ9xIL2-X was added again. The cells were expanded for a total of 8 days. The expanded cells were labeled with the viability dye PI and anti-Vδ2-PE in 2% FBS / PBS. Vδ2 was selected by using a Sony SH800 cell sorter + PI - Cell isolation of live Vγ9Vδ2 T cells. Purity was assessed by flow cytometry and was greater than 95% Vδ2 + γδ T cells. MOLM-13 and MV-411 cells were removed from the culture, washed once with complete RPMI, then washed with 0.1% BSA / PBS buffer, and labeled with CellTrace Violet at a dilution of 1:1000 for 10 minutes at 37°C. Target cells were resuspended in complete RPMI culture medium and seeded on a Corning 96-well U bottom plate at 100uL / well (10,000 cells). Sorted Vγ9Vδ2T cells were added at a ratio of 5:1 effector cells: target cells in a 50uL volume. Treatment groups were plated in duplicate. Test article CD33 x 1D7v9 or non-targeted control CD33 x UT was added to each plate at a final concentration of 100nM at 4× in 50uL complete RPMI. Cells were cultured overnight at 37°C / 5% CO2. On the second day, cells were centrifuged briefly and stained with the following components at 4°C for 30 minutes in 50uL FACS buffer: anti-human CD3-BV785, anti-human Vδ2-PE, Apotracker-green and PI. Subsequently, the dish was washed 2 times with FACS buffer, resuspended in 70uL FACS buffer and read on Quanteon flow cytometer. Apotracker green and PI were used to measure the percentage of dead cells for labeled MOLM-13 or MV-411 target cells.

[0356] like FIG. 8D to FIG. 8E As shown in Figure 2, treatment with CD33 x 1D7v9 resulted in expanded Vγ9Vδ2 T cells responding to MOLM-13 ( Fig.8D ) and MV-411( Fig. 8E ) increased killing of target cells. Killing activity requires the participation of CD33 and Vγ9, as CD33 x UT molecules lacking the γδ TCR binding domain did not lead to increased killing compared to the no antibody control.

[0357] Example 9: Activity of γδTCR x 5T4 bispecific molecules of different affinities

[0358] exist The Vγ9Vδ2 T cell-mediated killing activity of bispecific γδ TCR molecules comprising an anti-5T4 VHH domain, a low-affinity humanized anti-γδ VHH domain 1D7v9 (1D7v9 x 5T4) or a high-affinity humanized anti-γδ VHH domain 1D7v158 (1D7v158 x 5T4) and a heterodimeric knob-hole Fc region binding to γδ TCR and the representative target antigen 5T4 (γδTCR x 5T4) was evaluated in a cytotoxicity assay. Briefly, 5T4-positive (A375) cells (and 5T4-negative cells (A375Δ5T4) were labeled with CYTO-ID red, washed and plated in 96-well plates (10,000 cells / well) in complete RPMI and allowed to adhere for 2 hours at 37°C. Frozen PBMCs (previously treated with zoledronate to expand γδ T cells) were thawed in complete RPMI medium and added to target cells at a ratio of 5:1 (effector:target cells). Treatment groups were plated in duplicate. Test articles were added at a starting concentration of 50 nM and titrated across the plate at 1:3, as well as green apoptosis protease-3 / 7 reagent, which fluorescently labels nuclear DNA of cells undergoing apoptosis. The plates were incubated at 37°C for 22 hours. The assay plates were imaged periodically. Target cell death was determined by measuring the total red / green overlap object area. After 22 hours, the supernatant was removed and the remaining attached (viable target) cells were gently washed with PBS and then used 2.0, a luciferase-based reagent that results in bioluminescence in the presence of ATP (living cells).

[0359] like Fig. 9A and Fig. 9B As shown in , two bispecific γδTCR x 5T4 molecules induced γδ T cell-mediated caspase-3 / 7 activation in A549 cells but not A375Δ5T4 cells. Fig. 9C and Fig.9DAs shown in , target-dependent apoptotic protease activation induced by γδTCRx5T4 molecules correlates with target-dependent γδT cell-mediated cytotoxicity (assessed by cell survival). Fig. 9A and Fig. 9B As can be seen from the EC50 values ​​presented in , the potency of each molecule is affected by the affinity of the anti-γδTCR VHH domain, with the molecule comprising 1D7v158 exhibiting approximately 2-fold higher potency than the molecule comprising 1D7v9. Thus, the potency of a molecule can be modulated using anti-γδTCR VHH domains of varying affinity.

[0360] Example 10: Anti-γδ TCR binding VHH 1D7 binds to cynomolgus macaque γδ T cells and effectively targets fusion molecules

[0361] The ability of bivalent molecules containing anti-γδ TCR binding VHH (1D7 or 1D7v9) fused to human IgG1 Fc xELL (SEQ ID NO: 33) to specifically bind to the Vγ9Vδ2 subset from cynomolgus macaque PBMCs was tested. Briefly, expanded γδ T cells from cynomolgus macaque donors were thawed, washed twice with CTL thaw agent, and resuspended in complete assay medium at 0.5×10^6 cells / mL. 200uL of resuspended cells or 100,000 cells / well were added to a non-sterile 96-well U-bottom plate. The cells were briefly centrifuged and 100uL of FACs buffer was added to the wells. 100uL of antibody was added to the U-bottom plate at 2× final starting concentration 100nM, and the plate was titrated down at 1:5 in FACs buffer. For wells where test article was not added, 100uL / well FACs buffer was added instead. The plate was incubated at 4°C for 30 minutes. Cells were washed twice with FACS buffer and stained with CD3-BV421, anti-Vγ9-FITC and anti-human Fcγ-A647 in 50 uL FACS buffer for 30 minutes at 4° C. After incubation, the plates were washed twice with FACS buffer, resuspended in 70 uL FACS buffer, and binding to γδ T cells was determined by flow cytometry on Novocyte.

[0362] like Fig. 10A As shown in , both bivalent molecules specifically bind to the Vγ9+γδ T cell subset in a dose-dependent manner. The bivalent comprising 1D7v9 VHH exhibits lower binding affinity to cynomolgus macaque γδ T cells, and lower affinity of this humanized variant to human γδ T cells was also observed (see Example 5, above).

[0363] The IL-2_X molecule (cx11026) containing the monovalent targeting γδTCR of anti-γδTCR binding VHH 1D7 was tested in several cynomolgus macaque PBMC donors to enhance the ability of IL2 signal transduction via STAT5 phosphorylation. In short, PBMCs from three donors were separated and left to stand overnight in complete medium. The cells were labeled with the following fluorescent binding antibodies at room temperature for 20 minutes: non-competitive anti-Vγ9-FITC, anti-CD3-B421. After washing, 400,000 PBMC / wells were inoculated in 96-well plates. The cells were treated with titration of the fusion protein (cx11026) of IL-2_X fused to the C-terminus of anti-γδTCR-1D7, which was added with 4×final starting concentration 100nM, and the whole plate was titrated with 1:5 in the assay medium (50uL). The final total volume was 200uL / well. The plates were incubated at 37°C / 5% CO2 for 20 minutes. Cells were fixed in 100uL / well BD Fixation / Permeabilization buffer for 45 minutes, then permeabilized with 100uL / well BD Permeabilization buffer III for 1 hour, and washed 3 times with FACS buffer. Cells were then stained with 50uL / well FACS diluted pSTAT5 antibody overnight at 4°C. The next day, cells were washed twice with FACS buffer, resuspended in 70ul FACS buffer, and Vγ9 was measured by flow cytometry on a Quanteon flow cytometer. + γδ T cells or Vγ9 - γδ - Levels of phosphorylated STAT5 ("pSTAT5") on αβ T cells.

[0364] like Fig. 10B As shown in Figure 2, treatment with monovalent anti-γδTCR-1D7 targeting IL-2_X resulted in a decrease in pSTAT5 in all three cynomolgus macaque PBMC donor samples. + Vγ9 + There was a dose-dependent increase in the percentage of γδ T. There was no detectable pSTAT5 on αβ T cells from either donor.

[0365] A monovalent γδTCR-targeting IL-2_X molecule (cx11026) containing anti-γδ TCR binding VHH 1D7 was tested in several cynomolgus macaque PBMC donors to enhance Vγ9 +The ability of γδ T cells to proliferate. In brief, PBMCs were freshly isolated from fresh cynomolgus macaque blood samples as described in Example 2 and labeled with CellTrace Violet and added at 300,000 per well. 50uL of fusion protein containing IL-2_X fused to the C-terminus of anti-γδTCR-1D7 (cx11026) or non-targeting control VHH (cx9452) was added at 4× final starting concentration 100nM, and titrated throughout at 1:5 in the assay medium. 50uL of culture medium was added to a final total volume of 200uL / well, and the assay plate was incubated at 37°C / 5% CO2 for 7 days. The cells were labeled with the following fluorescent conjugated antibodies at 4°C for 30 minutes: CD3-APC, Vγ9-FITC, DNAM1-PE, NKGD-APC / Cy7, and viability dye propidium iodide. The cells were washed and analyzed on a Quanteon flow cytometer by flow cytometry.

[0366] FIG. 10C to FIG. 10F Data from two representative donors are presented in Figure 1 and show that treatment with anti-γδTCR-1D7 (cx11026)-targeted IL-2_X resulted in a dose-dependent increase in the proliferation of Vγ9 γδ T cells ( Fig. 10C and Fig.10E In addition, total CD3 + The percentage of Vγ9γδ T cells in the T cell population was substantially increased with anti-γδTCR-1D7-targeted IL-2_X treatment ( Fig. 10D and Fig.10F ). Non-targeting VHH (cx9452) fused with IL-2_X did not promote the proliferation of Vγ9 γδ T cells, showing target specificity.

[0367] Together, these studies demonstrate that VHHs that bind to human γδ TCRs, 1D7 and the humanized version, specifically cross-react with cynomolgus macaque γδ T cells, and that these VHH domains effectively target fusion molecules (e.g., γδTCR x IL-2_X) to cynomolgus macaque γδ T cells.

[0368] The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics of the present invention. The foregoing embodiments are therefore intended to be illustrative in all respects and not restrictive of the present invention. Therefore, the scope of the present invention is indicated by the appended claims rather than the foregoing description, and therefore it is intended herein to cover all changes that occur within the meaning and scope of the equivalents of the claims.

[0369] Tables of some compounds

[0370]

[0371]

[0372] OprI18v13 VHH, 4GFTv1 VHH and 401A9v1 VHH domains bind to irrelevant antigens and serve as non-binding control VHH domains

[0373] Tables of some sequences

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

Claims

1. A polypeptide comprising at least one VHH domain that binds to a γδ TCR, wherein at least one VHH domain comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 3, 144, 145, 146, 147, 148 or 149; a CDR2 comprising the amino acid sequence of SEQ ID NO: 4, 150, 151, 152, 153, 154, 155 or 156; and a CDR3 comprising the amino acid sequence of SEQ ID NO:

5.

2. The polypeptide of claim 1 , wherein at least one VHH domain comprises CDR1 , CDR2 and CDR3, wherein the CDR1 , CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO: 3, 4 and 5; 144, 4 and 5; 145, 4 and 5; 146, 4 and 5; 147, 4 and 5; 148, 4 and 5; 149, 4 and 5; 3, 150 and 5; 3, 151 and 5; 3, 152 and 5; 3, 153 and 5; 3, 154 and 5; 3, 155 and 5; or 3, 156 and 5, respectively.

3. The polypeptide of claim 1 or claim 2, wherein at least one VHH domain comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 4; and a CDR3 comprising the amino acid sequence of SEQ ID NO:

5.

4. A polypeptide according to any one of claims 1 to 3, wherein at least one VHH domain or each VHH domain is humanized.

5. The polypeptide according to any one of claims 1 to 4, wherein at least one VHH domain comprises SEQ ID NO: 180, wherein X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 25 , X 26 and X 27 are independently selected, and wherein X1 is V or A; X2 is R or G; X3 is K or T; X4 is I or F; X5 is Q, G or E; X6 is R or L; X7 is L, W or F; X8 is A or S; X9 is H or A; X 10 T or S; X 11 D or G; X 12 A or S; X 13 A or T; X 14 is E or Y; X 15 V or A; X 16 D, E, A, G, V, S, Y, L or Q; X 17 is S, P, T, A, V, L, I or G; X 18 G or D; X 19 S or N; X 20 is T or A; X 21 A or T; X 22 V or L; X 23 N or S; X 24 K or Y; X 25 N, S, E, Y, A, S, G, Q; X 26 is S, T, A, L, V, N or G; and X 27 K, R, E or D.

6. The polypeptide according to any one of claims 1 to 5, wherein at least one VHH domain comprises: a) an amino acid sequence that is at least 85%, 90%, 95% or at least 99% identical to the amino acid sequence of SEQ ID NO: 2, 17 to 31, 72 to 77, 80 to 143, 158 to 159 or 166 to 179; or b) the amino acid sequence of SEQ ID NO: 2, 17 to 31, 72 to 77, 80 to 143, 158 to 159 or 166 to 179.

7. The polypeptide according to any one of claims 1 to 6, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 99, 143 or 158.

8. The polypeptide according to any one of claims 1 to 7, comprising two VHH domains.

9. The polypeptide according to any one of claims 1 to 7, comprising three VHH domains.

10. The polypeptide according to any one of claims 1 to 9, wherein the polypeptide comprises an immune cell activation cytokine.

11. The polypeptide according to claim 10, wherein the immune cell activating cytokine is fused to the N-terminus or C-terminus of the VHH domain that binds to γδ T cells.

12. The polypeptide according to claim 10 or claim 11, wherein the immune cell activating cytokine is IL-2, IL-15, IL-7, IL-6, IL-12, IFNα, IFNβ or IFNγ, or an attenuated or modified form thereof.

13. The polypeptide according to any one of claims 1 to 12, wherein the polypeptide comprises an Fc region.

14. The polypeptide of claim 13, wherein the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32 to 70, optionally wherein the Fc region lacks a C-terminal lysine residue.

15. A polypeptide according to claim 13 or claim 14, wherein the polypeptide comprises an immune cell activation cytokine.

16. The polypeptide of claim 15, wherein the immune cell activating cytokine is IL-2, IL-15, IL-7, IL-6, IL-12, IFNα, IFNβ or IFNγ, or an attenuated or modified form thereof.

17. The polypeptide of claim 16, wherein the immune cell activating cytokine is fused to the C-terminus of the Fc region.

18. The polypeptide of any one of claims 1 to 17, wherein the polypeptide comprises at least one antigen binding domain that binds an antigen other than a γδ TCR.

19. The polypeptide of claim 18, wherein the polypeptide comprises at least one antigen binding domain that binds to: Lag3, TGFBR1, TGFBR2, Fas, TNFR2, 1-92-LFA-3, 5T4, alpha-4 integrin, alpha-V integrin, alpha4beta1 integrin, alpha4beta7 integrin, AGR2, anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33 , CD38, CD39, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD 73. CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD1 38. CD166, CD172A, CD248, CDH6, CEACAM5(CEA), CEACAM6(NCA-90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FcRH5, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GPRC5D, GRP78, HAVCAR1, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MICA, MICB, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4. NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine 1 phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, T LR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, TROP-2uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, or WISP-3.

20. The polypeptide of claim 18 or 19, wherein the polypeptide comprises at least one antigen binding domain that binds a tumor cell antigen.

21. The polypeptide of any one of claims 18 to 20, wherein at least one antigen binding domain that binds an antigen other than a γδ TCR is a VHH domain.

22. The polypeptide of claim 21, wherein each antigen binding domain that binds an antigen other than a γδ TCR is a VHH domain.

23. The polypeptide of any one of claims 18 to 21, wherein at least one antigen binding domain that binds an antigen other than γδ TCR comprises a heavy chain variable region and a light chain variable region.

24. The polypeptide of claim 23, wherein each antigen binding domain that binds an antigen other than γδ TCR comprises a heavy chain variable region and a light chain variable region.

25. A complex comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is the polypeptide according to any one of claims 13 to 24, wherein the first polypeptide comprises a first Fc region, and wherein the second polypeptide comprises a second Fc region, and wherein the first Fc region is the same as or different from the second Fc region.

26. The complex of claim 25, wherein the second polypeptide comprises at least one VHH domain that binds to a γδ TCR, at least one immune cell activating cytokine and / or at least one antigen binding domain that binds to an antigen other than a γδ TCR.

27. The complex of claim 26, wherein if the antigen binding domain that binds an antigen other than γδ TCR comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region is fused to a heavy chain constant region comprising the second Fc region.

28. The complex of claim 26 or 27, wherein at least one antigen binding domain that binds an antigen other than a γδ TCR binds Lag3, TGFBR1, TGFBR2, Fas, TNFR2, PD1, PDL1, TIM3, 1-92-LFA-3, 5T4, α-4 integrin, α-V integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33 , CD38, CD39, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD 73. CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD1 38. CD166, CD172A, CD248, CDH6, CEACAM5(CEA), CEACAM6(NCA-90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FcRH5, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GPRC5D, GRP78, HAVCAR1, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MICA, MICB, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch1, Notch 2, Notch 3, Notch 4. NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine 1 phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, T LR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, TROP-2uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, or WISP-3.

29. The polypeptide of any one of claims 26 to 28, wherein at least one antigen binding domain that binds an antigen other than a γδ TCR binds, wherein the polypeptide comprises at least one antigen binding domain that binds a tumor cell antigen.

30. The complex of any one of claims 26 to 29, wherein at least one antigen binding domain that binds an antigen other than a γδ TCR is a VHH domain.

31. The complex of any one of claims 27 to 30, wherein the first Fc region comprises a knob mutation and the second Fc region comprises a hole mutation.

32. The complex of claim 31, wherein the first Fc region comprises a T366W mutation and the second Fc region comprises T366S, L368A, and Y407V mutations.

33. The complex of claim 32, wherein the second Fc region comprises a H435R or H435K mutation.

34. The polypeptide or complex according to any one of claims 13 to 33, wherein the polypeptide is a dimer under physiological conditions, or wherein the complex is formed under physiological conditions.

35. The polypeptide or complex of any one of claims 1 to 34, wherein the γδ TCR is a human γδ TCR.

36. The polypeptide or complex of any one of claims 1 to 35, wherein the VHH domain binds a human γδ TCR comprising human γ9 and human δ2.

37. An immunoconjugate comprising the polypeptide or complex according to any one of claims 1 to 36 and a cytotoxic agent.

38. The immunoconjugate of claim 37, wherein the cytotoxic agent is selected from the group consisting of calicheamicin, auristatin, dolastatin, tubulysin, maytansine, nostoc, duocarmycin, esperamicin, pyrrolobenzodiazepine and enediyne antibiotics.

39. A pharmaceutical composition comprising the polypeptide or complex according to any one of claims 1 to 36 or the immunoconjugate according to claim 37 or claim 38 and a pharmaceutically acceptable carrier.

40. An isolated nucleic acid encoding the polypeptide or complex according to any one of claims 1 to 36.

41. A vector comprising the nucleic acid according to claim 40.

42. A host cell comprising the nucleic acid of claim 40 or the vector of claim 41.

43. A host cell expressing the polypeptide or complex according to any one of claims 1 to 36.

44. A method of producing a polypeptide or complex according to any one of claims 1 to 36, comprising incubating a host cell according to claim 42 or claim 43 under conditions suitable for expression of the polypeptide or complex.

45. The method of claim 44, further comprising isolating the polypeptide or complex.

46. ​​A method of increasing the proliferation of γδ T cells, comprising contacting the T cells with a polypeptide or complex according to any one of claims 1 to 36.

47. The method of claim 46, wherein the γδ T cells are in vitro.

48. The method of claim 46, wherein the γδ T cells are in vivo.

49. A method for treating cancer, comprising administering a pharmaceutically effective amount of the polypeptide or complex according to any one of claims 1 to 36 or the pharmaceutical composition according to claim 39 to an individual suffering from cancer.

50. The method of claim 49, wherein the cancer is selected from the group consisting of basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain cancer and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, gastrointestinal cancer, glioblastoma, liver cancer, liver tumors, intraepithelial neoplasia, kidney cancer or renal cancer, laryngeal cancer, liver cancer, lung cancer, small cell lung cancer ; non-small cell lung cancer; lung adenocarcinoma; squamous cell lung cancer; melanoma; myeloma; neuroblastoma; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; colorectal cancer; respiratory cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancer; vulvar cancer; lymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; massive NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; and chronic myeloid leukemia.

51. The method of claim 49 or 50, further comprising administering an additional therapeutic agent.

52. The method of claim 51, wherein the additional therapeutic agent is an anti-cancer agent.

53. The method of claim 52, wherein the anticancer agent is selected from a chemotherapeutic agent, an anticancer biological agent, radiation therapy, CAR-T therapy, and an oncolytic virus.

54. The method of any one of claims 51 to 53, wherein the additional therapeutic agent is an anti-cancer biologic.

55. The method of claim 54, wherein the anti-cancer biologic is an agent that inhibits PD-1 and / or PD-L1.

56. The method of claim 54, wherein the anticancer biologic is an agent that inhibits VISTA, gpNMB, B7H3, B7H4, HHLA2, CTLA4, or TIGIT.

57. The method of any one of claims 52 to 56, wherein the anti-cancer agent is an antibody.

58. The method of claim 54, wherein the anti-cancer biologic is a cytokine.

59. The method of claim 52 or claim 53, wherein the anticancer agent is CAR-T therapy.

60. The method of claim 52 or claim 53, wherein the anticancer agent is an oncolytic virus.

61. The method of any one of claims 49 to 60, further comprising tumor resection and / or radiation therapy.

Images