Gamma delta T cell binding polypeptides and uses thereof

Abstract

Provided herein are VHH-containing polypeptides that bind to γδ T cells. Uses of the VHH-containing polypeptides are also provided.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 296,774, filed January 5, 2022, and U.S. Provisional Application No. 63 / 417,926, filed October 20, 2022, each of which is incorporated by reference in its entirety for all purposes.

[0002] The present invention relates to γδ T cell-binding polypeptides and methods of using γδ T cell-binding polypeptides to modulate the biological activity of γδ T cells, including, but not limited to, methods of treating cancer. In some embodiments, the γδ T cell-binding polypeptide is a fusion polypeptide comprising a γδ T cell-binding polypeptide and a polypeptide that binds to an antigen other than γδ T cells. [Background technology]

[0003] T cell activation is also controlled by other molecules such as IL-2, IL-15, IL-7, IL-6, IL-12, IFNα, IFNβ, and IFNγ. The cytokine interleukin-2 (IL-2), synthesized and secreted by activated T cells themselves, is a pleiotropic cytokine that regulates the proliferation and cytolytic activity of γδ T cells. IL-2 binds to a high-affinity receptor composed of three subunits (IL-2α, IL-2β, and γc) on the T cell surface. Signaling through the IL-2 receptor complex drives T cell division and clonal expansion of activated T cells. Summary of the Invention [Problem to be solved by the invention]

[0004] There is a need for γδ T cell-binding polypeptides that can specifically target activating molecules to γδ T cells, increasing the potency and selectivity of cytotoxic γδ T cell responses. [Means for solving the problem]

[0005] Provided herein are γδ T cell-binding polypeptides and methods of using γδ T cell-binding polypeptides to treat, for example, cancer. In some embodiments, the γδ T cell-binding polypeptide comprises one or more additional binding domains and / or cytokine sequences. Certain numbered embodiments are provided below.

[0006] Embodiment 1. A polypeptide comprising at least one VHH domain that binds to a γδ TCR, wherein the at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, or SEQ ID NO:149, a CDR2 comprising the amino acid sequence of SEQ ID NO:4, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, or SEQ ID NO:156, and a CDR3 comprising the amino acid sequence of SEQ ID NO:5.

[0007] Embodiment 2. A polypeptide according to embodiment 1, wherein at least one VHH domain comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:144, SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:145, SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:146, SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:147, SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:148, SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:149, SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:150 and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:151 and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:152 and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:153 and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:154 and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:155 and SEQ ID NO:5; or SEQ ID NO:3, SEQ ID NO:156 and SEQ ID NO:5.

[0008] Embodiment 3. A polypeptide described in embodiment 1 or 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.

[0009] Embodiment 4. A polypeptide according to any one of embodiments 1 to 3, wherein at least one or each VHH domain is humanised.

[0010] Embodiment 5. 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, 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 is T or S, X 11 is D or G, X 12 is A or S, X 13 is A or T, X 14is E or Y, X 15 is V or A, X 16 is D, E, A, G, V, S, Y, L or Q, X 17 is S, P, T, A, V, L, I, or G; X 18 is G or D, X 19 is S or N, X 20 is T or A, X 21 is A or T, X 22 is V or L; X 23 is N or S, X 24 is K or Y, X 25 are N, S, E, Y, A, S, G, and Q, X 26 is S, T, A, L, V, N or G, X 27 is K, R, E, or D; A polypeptide according to any one of embodiments 1 to 4.

[0011] Embodiment 6. At least one VHH domain comprises SEQ ID NO: 180, wherein X3 is K and 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, X1 is V or A; X2 is R or G; 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 is T or S, X 11 is D or G, X 12 is A or S, X 13 is A or T, X 14 is E or Y, X 15 is V or A, X 16 is D, E, A, L or Q, X 17 is S, P, T, V, L, or G; X 18 is G or D, X 19 is S or N, X 20 is T or A, X 21 is A or T, X 22 is V or L; X 23 is N or S, X 24 is K or Y, X 25 is N or S, X 26 is S, T, or G, X 27 is K, R, E, or D; A polypeptide according to any one of embodiments 1 to 4.

[0012] Embodiment 7. At least one VHH domain comprises SEQ ID NO: 180, where X2 is R and X 25is N, X1, 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, X1 is V or A; 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 is T or S, X 11 is D or G, X 12 is A or S, X 13 is A or T, X 14 is E or Y, X 15 is V or A, X 16 is D, E, A or Q, X 17 is S, P or G, X 18 is G or D, X 19 is S or N, X 20 is T or A, X 21 is A or T, X 22 is V or L; X 23 is N or S, X24 is K or Y, X 26 is S or T, X 27 is K, R, E, or D; A polypeptide according to any one of embodiments 1 to 4.

[0013] Embodiment 8. At least one VHH domain comprises SEQ ID NO: 180, wherein X2 is R, X3 is K, X4 is I, X9 is H, and 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, X1 is V or A; X5 is Q, G or E; X6 is R or L; X7 is L, W or F; X8 is A or S; X 10 is T or S, X 11 is D or G, X 12 is A or S, X 13 is A or T, X 14 is E or Y, X 15 is V or A, X 16 is D, E or A, X 17 is S or P, X 18 is G or D, X 19 is S or N, X 20 is T or A, X 21 is A or T, X 22 is V or L; X 23 is N or S, X 24 is K or Y, X 26 is S or T, X 27 is K, R, E, or D; A polypeptide according to any one of embodiments 1 to 4.

[0014] Embodiment 9. At least one VHH domain comprises SEQ ID NO: 180, wherein Xi is V, X2 is R, X3 is K, X4 is I, X9 is H, and X 10 is T and X 11 is D and X 12 is A and X 13 is A and X 14 is E and 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, X5 is Q, G or E; X6 is R or L; X7 is L or W; X8 is A or S; X 15 is V or A, X 16 is D, E, or A, X 17 is S or P, X18 is G or D, X 19 is S or N, X 20 is T or A, X 21 is A or T, X 22 is V or L; X 23 is N or S, X 24 is K or Y, X 26 is S or T, X 27 is K, R, E, or D; A polypeptide according to any one of embodiments 1 to 4.

[0015] Embodiment 10. A polypeptide 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 99% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:17 to SEQ ID NO:31, SEQ ID NO:72 to SEQ ID NO:77, SEQ ID NO:80 to SEQ ID NO:143, SEQ ID NO:158 to SEQ ID NO:159, or SEQ ID NO:166 to SEQ ID NO:179.

[0016] Embodiment 11. A polypeptide according to any one of embodiments 1 to 10, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 17 to SEQ ID NO: 31, SEQ ID NO: 72 to SEQ ID NO: 77, SEQ ID NO: 80 to SEQ ID NO: 143, SEQ ID NO: 158 to SEQ ID NO: 159, or SEQ ID NO: 166 to SEQ ID NO: 179.

[0017] Embodiment 12. A polypeptide according to any one of embodiments 1 to 11, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 99, SEQ ID NO: 143 or SEQ ID NO: 158.

[0018] Embodiment 13. A polypeptide according to any one of embodiments 1 to 12, comprising two VHH domains.

[0019] Embodiment 14. A polypeptide according to any one of embodiments 1 to 13, comprising three VHH domains.

[0020] Embodiment 15. A polypeptide according to any one of embodiments 1 to 14, comprising an immune cell-activating cytokine.

[0021] 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.

[0022] Embodiment 17. The polypeptide of embodiment 10 or 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 version thereof.

[0023] Embodiment 18. A polypeptide according to any one of embodiments 1 to 17, comprising an Fc region.

[0024] Embodiment 19. The polypeptide of embodiment 18, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NO: 32 to SEQ ID NO: 70, and optionally, the Fc region lacks a C-terminal lysine residue.

[0025] Embodiment 20 The polypeptide of embodiment 18 or 19, comprising an immune cell activating cytokine.

[0026] 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 version thereof.

[0027] Embodiment 22 The polypeptide of embodiment 21, wherein the immune cell-activating cytokine is fused to the C-terminus of the Fc region.

[0028] Embodiment 23. A polypeptide according to any one of embodiments 1 to 22, comprising at least one antigen-binding domain that binds to an antigen other than γδ TCR.

[0029] 24. Lag3, TGFBR1, TGFBR2, Fas, TNFR2, 1-92-LFA-3, 5T4, α4 integrin, αV integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis Y, apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, complement C5, 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, CD5 1, CD52, CD56, CD64, CD70, CD71, CD73, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD 137, CD138, 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, ED B, 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 α (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, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine-1-phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM3 1. The polypeptide of embodiment 23, comprising at least one antigen-binding domain that binds to TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, TROP-2, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, or WISP-3.

[0030] Embodiment 25. A polypeptide according to embodiment 23 or 24, comprising at least one antigen-binding domain that binds to a tumor cell antigen.

[0031] Embodiment 26. A polypeptide according to any one of embodiments 23 to 25, wherein at least one antigen-binding domain that binds to an antigen other than γδ TCR is a VHH domain.

[0032] Embodiment 27. A polypeptide according to embodiment 26, wherein each antigen-binding domain that binds to an antigen other than γδ TCR is a VHH domain.

[0033] Embodiment 28. A polypeptide according to any one of embodiments 23 to 26, wherein at least one antigen-binding domain that binds to an antigen other than γδ TCR comprises a heavy chain variable region and a light chain variable region.

[0034] Embodiment 29. A polypeptide according to embodiment 28, wherein each antigen-binding domain that binds to an antigen other than γδ TCR comprises a heavy chain variable region and a light chain variable region.

[0035] Embodiment 30. A complex comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is a polypeptide described in any one of embodiments 18 to 29, the first polypeptide comprises a first Fc region, and the second polypeptide comprises a second Fc region, and the first Fc region and the second Fc region are the same or different.

[0036] Embodiment 31. A complex as described 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.

[0037] Embodiment 32. The conjugate of embodiment 31, wherein when the antigen-binding domain that binds to 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 that comprises a second Fc region.

[0038] Embodiment 33. At least one antigen-binding domain that binds to an antigen other than γδ TCR is selected from the group consisting of Lag3, TGFBR1, TGFBR2, Fas, TNFR2, 1-92-LFA-3, 5T4, α4 integrin, αV integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis Y, apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, complement C5, 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, CD73, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, 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, DSG 1, 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 α (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, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine-1-phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, T 33. A complex according to embodiment 31 or 32, which binds to LR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, TROP-2, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, or WISP-3.

[0039] Embodiment 34. A polypeptide described in any one of embodiments 31 to 33, comprising at least one antigen-binding domain that binds to an antigen other than γδ TCR, and the polypeptide comprises at least one antigen-binding domain that binds to a tumor cell antigen.

[0040] Embodiment 35. A complex according to any one of embodiments 31 to 34, wherein at least one antigen-binding domain that binds to an antigen other than γδ TCR is a VHH domain.

[0041] Embodiment 36. A conjugate according to 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.

[0042] Embodiment 37. The conjugate of embodiment 36, wherein the first Fc region comprises a T366W mutation and the second Fc region comprises T366S, L368A, and Y407V mutations.

[0043] Embodiment 38. The conjugate of embodiment 37, wherein the second Fc region comprises a H435R or H435K mutation.

[0044] Embodiment 39. A polypeptide or complex described in any one of embodiments 18 to 38, wherein the polypeptide is a dimer under physiological conditions or the complex is formed under physiological conditions.

[0045] Embodiment 40. The polypeptide or complex according to any one of embodiments 1 to 39, wherein the γδTCR is a human γδTCR.

[0046] Embodiment 41. A polypeptide or complex according to any one of embodiments 1 to 40, wherein the VHH domain binds to human γδ TCR, including human γ9 and human δ2.

[0047] Embodiment 42. An immune complex comprising the polypeptide or complex according to any one of embodiments 1 to 41 and a cytotoxic substance.

[0048] Embodiment 43. The immunoconjugate of embodiment 42, wherein the cytotoxic agent is selected from a calicheamicin, an auristatin, a dolastatin, a tubulysin, a maytansinoid, a cryptophycin, a duocarmycin, an esperamicin, a pyrrolobenzodiazepine, and an enediyne antibiotic.

[0049] Embodiment 44. A pharmaceutical composition comprising a polypeptide or complex according to any one of embodiments 1 to 41, or an immune complex according to embodiment 42 or 43, and a pharma- ceutical acceptable carrier.

[0050] Embodiment 45. An isolated nucleic acid encoding a polypeptide or complex according to any one of embodiments 1 to 41.

[0051] Embodiment 46. A vector comprising the nucleic acid described in embodiment 45.

[0052] Embodiment 47. A host cell comprising the nucleic acid of embodiment 45 or the vector of embodiment 46.

[0053] Embodiment 48. A host cell expressing a polypeptide or complex according to any one of embodiments 1 to 41.

[0054] Embodiment 49. A method for producing a polypeptide or complex described in any one of embodiments 1 to 40, comprising incubating a host cell described in embodiment 47 or 48 under conditions suitable for expression of the polypeptide or complex.

[0055] Embodiment 50. The method of embodiment 49, further comprising isolating the polypeptide or complex.

[0056] Embodiment 51 A method for increasing proliferation of γδ T cells, comprising contacting T cells with a polypeptide or complex according to any one of embodiments 1 to 41.

[0057] Embodiment 52 The method of embodiment 51, wherein the γδ T cells are present in vitro.

[0058] Embodiment 53 The method of embodiment 51, wherein the γδ T cells are present in vivo.

[0059] Embodiment 54. A method for treating cancer, comprising administering to a subject with cancer a pharma- ceutical effective amount of a polypeptide or complex described in any one of embodiments 1 to 41, or a pharmaceutical composition described in embodiment 44.

[0060] Embodiment 55. The cancer is basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, gastrointestinal cancer, glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer or renal cancer, laryngeal cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, myeloma, neuroblastoma, oral 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, 55. The method of embodiment 54, wherein the tumor is selected from 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-dividing cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloblastic leukemia.

[0061] Embodiment 56 The method of embodiment 54 or 55, further comprising administering an additional therapeutic agent.

[0062] Embodiment 57. The method of embodiment 56, wherein the additional therapeutic agent is an anti-cancer agent.

[0063] Embodiment 58. The method of embodiment 56, wherein the anticancer agent is selected from a chemotherapeutic agent, an anticancer biologic, a radiation therapy agent, a CAR-T therapy agent, and an oncolytic virus.

[0064] Embodiment 59. The method of embodiment 56 or 57, wherein the additional therapeutic agent is an anti-cancer biologic.

[0065] Embodiment 60. The method of embodiment 59, wherein the anti-cancer biologic is an agent that inhibits PD-1 and / or PD-L1.

[0066] Embodiment 61. The method of embodiment 59, wherein the anti-cancer biologic is an agent that inhibits VISTA, gpNMB, B7H3, B7H4, HHLA2, CTLA4, or TIGIT.

[0067] Embodiment 62. The method of any one of embodiments 57 to 61, wherein the anticancer agent is an antibody.

[0068] Embodiment 63 The method of embodiment 59, wherein the anticancer biologic is a cytokine.

[0069] Embodiment 64. The method of embodiment 57 or 58, wherein the anticancer agent is a CAR-T therapeutic agent.

[0070] Embodiment 65. The method of embodiment 57 or 58, wherein the anticancer agent is an oncolytic virus.

[0071] Embodiment 66 The method of any one of embodiments 54-65, further comprising tumor resection and / or radiation therapy. [Brief description of the drawings]

[0072] [Figure 1]Figures 1A, 1C, 1E, and 1G show 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 reduced affinity IL-2 mutants IL-2_X or IL-2_Y, as assessed by flow cytometry. Figures 1B, 1D, 1F, and 1H show the percentage of cells with phosphorylated STAT5 staining for γδ T cells, NK cells, and αβ T cells after treatment with monovalent or bivalent single domain antibodies specific for γδ TCR linked to reduced affinity IL-2 mutants IL-2_X or IL-2_Y, as assessed by flow cytometry. [Diagram 2] Figure 2A shows the percentage of proliferating γδ T cells in response to treatment with γδ TCR targeted low affinity IL-2_X (cx11005) or non-targeted low affinity IL-2_X (cx9452). Figure 2B shows the percentage of γδ T cells relative to total cells after treatment with the test substances. Figure 2C shows the percentage of proliferating αβ T cells after treatment, and Figure 2D shows the percentage of αβ T cells relative to total cells after treatment. [Diagram 3] Figure 3A shows the median fluorescence intensity of intracellular phosphorylated STAT5 in V52+ γδ T cells and αβ T cells after treatment with a monovalent single domain antibody specific for the γδ TCR linked to the affinity reduced IL-2 mutant IL-2_X (cx11026) as assessed by flow cytometry. Figure 3B shows the percentage of cells with phosphorylated STAT5 staining in V52+ γδ T cells and αβ T cells after treatment with a monovalent single domain antibody specific for the γδ TCR linked to the affinity reduced IL-2 mutant IL-2_X as assessed by flow cytometry. [Figure 4]Figure 4A shows the percentage of V52+ γδ T cells that expanded in response to treatment with γδ TCR targeted low affinity IL-2_X (cx11026) or non-targeted low affinity IL-2_X (cx9452) as determined by flow cytometric analysis of CellTrace Violet dilutions. Figure 4B shows the percentage of V52+ γδ T cells relative to total CD3+ T cells following treatment. [Diagram 5] Figures 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, 5I, 5J, and 5K show binding of γδ TCR-binding VHH 1D7 and its humanized variants formatted as monovalent VHH-hIgG1-Fc fusion proteins assessed by flow cytometry to expanded human Vδ2+ γδ T cells. [Figure 6] Figures 6A and 6B show target cell killing curves (plotted as overlap of caspase 3 / 7-green and cyto-ID red over time) of THP-1 (6A, left), HT29 (6A, right), Daudi (6B, top left), NCI-H460 (6B, top right), MM1S (6B, bottom left), and A375 (6B, bottom right) cells following treatment with Vy9V52 T cells expanded with a γδ TCR-targeted IL-2 molecule (1D7xIL-2_X). [Figure 7] Figures 7A-7D show the binding and cell killing activity of γδTCRxCD20 bispecific polypeptides. Figure 7A shows binding to CD20-expressing Raji cells. Figure 7B shows binding to Vγ9V52 T cells. Figures 7C and 7D show γδ T cell mediated killing of Raji target cells by freshly isolated (7C) and expanded (7D) Vγ9V52 T cells treated or not with γδTCRxCD20 bispecific polypeptides, rituximab analogue (rituximab-an). [Figure 8]Figures 8A-8E show the binding and cell killing activity of γδTCRxCD33 bispecific polypeptides. Figures 8A and 8B show binding to CD33-expressing Molm-13 and MV-411 cells, respectively. Figure 8C shows binding to Vγ9Vδ2 T cells. Figures 8D and 8E show γδ T cell mediated killing of MOLM-13 (8D) and MV-411 (8E) target cells by increased Vγ9Vδ2 T cells treated or not with γδTCRxCD33 bispecific polypeptides, non-targeting CD33xUT polypeptides. [Figure 9] Figures 9A-D show the ability of the γδTCRx5T4 construct to induce antigen-dependent γδT cell-mediated cytotoxicity in A375, a 5T4+ cell line (9A and 9C), but not in A375Δ5T4 cells, a 5T4− cell line (9B and 9D), as assessed by caspase-3 / 7 activation (3 h time point shown) using a cell imaging system (9A and 9B) and cell viability using the CellTiter-Glo assay (9C and 9D). Tables of EC50 values ​​(nM) are presented in Figures 9A and 9C. [Figure 10] Figures 10A-F show cross-reactivity with cynomolgus γδ TCR. Figure 10A shows binding of γδ TCR binding VHH 1D7 and humanized variant 1D7v9 formatted as a bivalent VHH-hIgG1-xELL Fc fusion protein assessed by flow cytometry on cynomolgus Vγ9+ γδ T cells. Figure 10B shows the percentage of cells with phosphorylated STAT5 staining on cynomolgus γδ T cells and αβ T cells from three donors after treatment with monovalent γδ TCR linked to reduced affinity IL-2 variant (cx11026) assessed by flow cytometry. Figures 10C and 10E show the percentage of proliferating cynomolgus γδ T cells in response to treatment with monovalent γδ TCR targeted low affinity IL-2_X (cx11026:1D7xIL-2_X) or non-targeted low affinity IL-2_X (cx9452:UTxIL-2_X) from two different cynomolgus donors. Figures 10D and 10F show the percentage of γδ T cells relative to total cells after treatment with the above test articles in these donors. [Figure 11] Figures 11A-C show sequence alignment of parental anti-γδ TCR VHH 1D7 (SEQ ID NO: 2) with humanized 1D7 variants (see Table 2) with binding affinity (KD) of 100 nM as determined by flow cytometry. Figure 11D shows sequence alignment of parental anti-γδ TCR VHH 1D7 (SEQ ID NO: 2) with a consensus sequence (SEQ ID NO: 180) representing the aligned humanized 1D7 variants. [Figure 12]Figures 12A and 12B show eight non-limiting exemplary formats of γδ T cell binding polypeptides. Figure 12A shows an exemplary bispecific monovalent anti-γδ T cell x antigen construct, which is a complex comprising a first polypeptide comprising an anti-antigen VHH domain (having specificity 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 x 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 (having specificity 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 x antigen construct, which is a complex comprising a first polypeptide comprising an Fc region and a second polypeptide comprising an anti-antigen VHH domain (having specificity 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 (having specificity 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 (having specificity 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 (having specificity 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 (having specificity for an antigen other than γδ TCR) and an Fc region, and a second polypeptide comprising a different anti-antigen VHH domain (having specificity 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 (having specificity for two different antigens other than γδ TCR) and an anti-γδ TCR VHH. The order of the tandem VHH domains may vary. Similarly, for molecules comprising two polypeptide chains, the VHH domain may be located on either chain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] The embodiments presented herein relate to γδ T cell-binding polypeptides and their uses in various methods of treating, for example, cancer.

[0074] Definitions and Various Embodiments The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0075] All references cited in this specification, including patent applications, patent publications, and Genbank accession numbers, are incorporated herein by reference to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety.

[0076] The techniques and procedures described or referenced herein are generally well understood and generally described in, for example, 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)), the series METHODS IN ENZYMOLOGY (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, Humana Press, Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press, Animal Cell Culture (RI Freshney, ed., 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell eds., 1993-8) J. Wiley and Sons, Handbook of Experimental Immunology (DM Weir and C.C. Blackwell, eds.), Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994), Current Protocols in Immunology (JE Coligan et al. eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (CA Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989), Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using The antibodies are used using routine methodologies by those skilled in the art, such as the widely used methodologies described in Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995), and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993) and their latest editions.

[0077] Unless otherwise specified, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context or expressly indicated otherwise, singular terms shall include the plural and plural terms shall include the singular. In the event of discrepancies in definitions among various sources or references, the definitions set forth herein shall prevail.

[0078] Generally, the numbering of residues in an immunoglobulin heavy chain 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). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0079] Embodiments of the invention described herein are understood to include "consisting of" and / or "consisting essentially of." As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise indicated. Use of the term "or" herein is not to be construed as implying that options are mutually exclusive.

[0080] In this application, the use of "or" means "and / or" unless expressly stated otherwise or understood by a person skilled in the art. In the context of a multiple dependent claim, the use of "or" refers to more than one preceding independent or dependent claim.

[0081] The phrases "reference sample", "reference cell" or "reference tissue" refer to 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, a reference sample can be used as a positive or negative indicator. A reference sample can be used to establish, for example, the level of protein and / or mRNA present in a healthy tissue relative to the level of protein and / or mRNA present in a sample with unknown characteristics. In some embodiments, the reference sample is a sample from the same subject, but from a part of the subject different from the part being tested. In some embodiments, the reference sample is a sample from a tissue area surrounding or adjacent to the cancer. In some embodiments, the reference sample is not from the subject being tested, but from a subject known to have or not have the disorder of interest. In some embodiments, the reference sample is from the same subject, but at a time before the subject developed cancer. In some embodiments, the reference sample is a sample from a benign cancer sample from the same subject or a different subject. When a negative reference sample is used for comparison, the expression level or amount of the molecule of interest in the negative reference sample indicates a level at which one skilled in the art would recognize, in view of the present disclosure, that the molecule is absent and / or present at a low level.When a positive reference sample is used for comparison, the expression level or amount of the molecule of interest in the positive reference sample indicates a level at which one skilled in the art would recognize, in view of the present disclosure, that the molecule is present at a certain level.

[0082] The terms "benefit", "clinical benefit", "responsiveness" 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 endpoints, such as a degree of inhibition of disease progression, including slowing and complete halt, a reduction in the number of disease episodes and / or symptoms, a reduction in lesion size, an inhibition (i.e., reduction, slowing, or complete halt) of disease cell invasion into adjacent peripheral organs and / or tissues, an inhibition (i.e., reduction, slowing, or complete halt) of disease spread, a degree of relief of one or more symptoms associated with the disorder, a disease-free presentation after treatment, such as an increase in the length of progression-free survival, an increase in overall survival, a higher response rate, and / or a reduction in mortality at a given time point after treatment. A "non-responsive" or "non-responsive" subject or cancer is one that does not meet the above criteria of "responding".

[0083] The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably and may refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides, including, but not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0084] 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 may include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Included in this definition are both full-length proteins and fragments thereof. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of this disclosure, "polypeptide" refers to proteins that contain modifications (generally conservative in nature), such as deletions, additions, and substitutions to the native sequence, so long as the protein maintains a desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutations of the host that produces the protein or by errors due to PCR amplification.

[0085] The term "specifically binds" to an antigen or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, with a longer duration and / or with a higher affinity with a particular cell or substance than it reacts or associates with another cell or substance. A single domain antibody (sdAb) or VHH-containing polypeptide "specifically binds" or "preferentially binds" to a target if it binds with higher affinity, avidity, more readily and / or with a longer duration than it binds to other substances. For example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to γδ T cells is an sdAb or VHH-containing polypeptide that binds to this epitope with higher affinity, avidity, more readily and / or with a longer duration than it binds to other γδ T cells or non-γδ T cells. It is also understood by interpreting this definition that, for example, an sdAb or 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 may 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 to an antigen.

[0086] The term "inhibition" or "inhibiting" refers to the reduction or cessation of any phenotypic characteristic, or the reduction or cessation of the incidence, extent, or likelihood of that characteristic. "Reduce" or "inhibit" refers to the decrease, reduction, or cessation of an activity, function, and / or amount compared to a reference. In some embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 10% or more. In some embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 50% or more. In some embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more. In some embodiments, the amount is inhibited or reduced over a period of time relative to a control over the same period of time.

[0087] As used herein, the term "direct inhibition" and similar terms refer to an inhibition profile in which increasing antibody concentration results in increasing inhibition. In some embodiments, after a certain concentration, maximum inhibition is reached and the inhibition profile plateaus. Maximum inhibition does not have to be 100% inhibition, but may be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

[0088] As used herein, the term "epitope" refers to a site on a target molecule (e.g., an antigen such as a protein, nucleic acid, carbohydrate, or lipid) to which an antigen-binding molecule (e.g., an sdAb or VHH-containing polypeptide) binds. Epitopes often comprise chemically active surface arrangements of molecules such as amino acids, polypeptides, or sugar side chains, and have specific three-dimensional structural features and specific charge characteristics. Epitopes can be formed from both contiguous and / or juxtaposed non-contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of a target molecule. Epitopes formed from contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. Epitopes can include, but are not limited to, at least 3, at least 5, or 8-10 residues (e.g., amino acids or nucleotides). In some embodiments, an epitope is less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues, or less than 12 residues in length. Two antibodies may bind to the same epitope in an antigen if they exhibit competitive binding to the antigen. In some embodiments, an epitope may be specified by a certain minimum distance to the CDR residues on the antigen-binding molecule. In some embodiments, an epitope may be specified by the above distance and further limited to those residues involved in binding (e.g., hydrogen bonding) between the antigen-binding molecule residues and the antigen residues. An epitope may also be specified by various scans. For example, an alanine scan or an arginine scan may indicate one or more residues with which an antigen-binding molecule may interact. Unless explicitly indicated, a set of residues as an epitope does not exclude other residues from being part of the epitope for a particular antigen-binding molecule. Rather, the existence of such a set indicates a minimum epitope string (or set of types). Thus, in some embodiments, the set of residues identified as an epitope is not an exclusive list of residues for the epitope on the antigen, but rather represents a minimal epitope associated with the antigen.

[0089] A "non-linear epitope" or "conformational epitope" comprises non-contiguous polypeptides, amino acids and / or sugars within an antigenic protein to which an epitope-specific antigen-binding molecule binds. In some embodiments, at least one residue is non-contiguous with other represented residues of the epitope, although one or more residues may be contiguous with other residues.

[0090] A "linear epitope" comprises a continuous polypeptide, amino acid and / or sugar in an antigenic protein to which an antigen-binding molecule specific for the epitope binds. Note that in some embodiments, not all of the residues in a linear epitope need to be directly bound (or involved in binding) by an antigen-binding molecule. In some embodiments, a linear epitope can be derived from immunization with a peptide that essentially consists of the sequence of the linear epitope, or from a structural section of a protein that is relatively isolated from the rest of the protein (so that the antigen-binding molecule can interact, at least primarily, with just that sequence section).

[0091] The term "antibody" is used in the broadest sense and includes various polypeptides, including but not limited to conventional antibodies (typically comprising at least one heavy chain and at least one light chain), single domain antibodies (sdAbs, comprising at least one VHH domain and an Fc region), VHH-containing polypeptides (polypeptides comprising at least one VHH domain), and antibody-like antigen-binding domains, including fragments of any of the above, so long as they exhibit the desired antigen-binding activity. In some embodiments, the antibody comprises a dimerization domain. Such dimerization domains include but are not limited to a heavy chain constant domain (comprising CH1, hinge, CH2, and CH3, where CH1 is typically paired with a light chain constant domain CL, while the hinge mediates dimerization) and an Fc region (comprising hinge, CH2, and CH3, where the hinge mediates dimerization).

[0092] 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.

[0093] The term "antigen-binding domain" as used herein refers to a portion of an antibody 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. Thus, in some embodiments, the antigen-binding domain comprises a heavy chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen, and a light chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen. In some embodiments, the antigen-binding domain of an sdAb or VHH-containing polypeptide comprises the three CDRs of a VHH domain. Thus, in some embodiments, the antigen-binding domain of an sdAb or VHH-containing polypeptide comprises a VHH domain comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen.

[0094] The term "VHH" or "VHH domain" or "VHH antigen-binding domain" as used herein 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, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, a VHH may be truncated at the N-terminus or C-terminus to include only a partial FR1 and / or FR4, or to lack one or both of these framework regions, so long as the VHH substantially retains antigen binding and specificity.

[0095] The terms "single domain antibody" and "sdAb" are used interchangeably herein to refer to an antibody comprising at least one monomer domain, such as a VHH domain, without a light chain and an Fc region. In some embodiments, an sdAb is a dimer of two polypeptides, each polypeptide comprising 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, for example, polypeptides having the structure VHH1-VHH2-Fc or VHH1-VHH2-VHH3-Fc, where VHH1, VHH2, and VHH3 may be the same or different.

[0096] The term "VHH-containing polypeptide" refers to a polypeptide that comprises at least one VHH domain. In some embodiments, a VHH polypeptide comprises two, three, or four or more VHH domains, where each VHH domain may be the same or different. In some embodiments, a VHH-containing polypeptide comprises an Fc region. In some such embodiments, a VHH-containing polypeptide may be referred to as an sdAb. Furthermore, in some such embodiments, a VHH polypeptide may form a dimer. Non-limiting structures of a VHH-containing polypeptide, also referred to as an sdAb, include VHH1-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc, where VHH1, VHH2, and VHH3 may be the same or different. In some embodiments of such structures, a VHH may be linked to another VHH by a linker, or a VHH may be linked to an Fc by a linker. In some such embodiments, the linker comprises 1-20 amino acids, preferably 1-20 amino acids consisting mainly of glycine and optionally serine. In some embodiments, when a VHH-containing polypeptide comprises an Fc, it forms a dimer. Thus, the structure VHH1-VHH2-Fc is considered to be tetravalent when it forms a dimer (i.e., the dimer has four VHH domains). Similarly, the structure VHH1-VHH2-VHH3-Fc is considered to be hexavalent when it forms a dimer (i.e., the dimer has six VHH domains).

[0097] The term "monoclonal antibody" refers to an antibody (including sdAb or VHH-containing polypeptide) of a substantially homogeneous antibody population. That is, the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Thus, a sample of monoclonal antibodies is capable of binding to the same epitope on the antigen. The modifier "monoclonal" indicates the nature of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567. The monoclonal antibodies can also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example.

[0098] The term "CDR" refers to a complementarity determining region defined by at least one specific manner to one skilled in the art. 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 designated CDR1, CDR2, and CDR3.

[0099] As used herein, the term "heavy chain constant region" refers to a region that comprises at least three heavy chain constant domains, i.e., H 1, hinge, C H 2, and C H3. Of course, deletions and modifications that do not change the function within the domain are included within the scope of the term "heavy chain constant region" unless otherwise specified. Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting exemplary heavy chain constant regions also include epsilon and mu. Each heavy chain constant region corresponds to one antibody isotype. For example, an antibody containing a gamma constant region is an IgG antibody, an antibody containing a delta constant region is an IgD antibody, and an antibody containing an alpha constant region is an IgA antibody. Furthermore, an antibody containing a mu constant region is an IgM antibody, and an antibody containing an epsilon constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing a γ1 constant region) antibodies, IgG2 (containing a γ2 constant region) antibodies, IgG3 (containing a γ3 constant region) antibodies, and IgG4 (containing a γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (containing an α1 constant region) antibodies and IgA2 (containing an α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0100] As used herein, "Fc region" refers to a portion of a heavy chain constant region comprising CH2 and CH3. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. In various embodiments, when the Fc region comprises a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0101] As used herein, an "acceptor human framework" refers to a heavy chain variable domain (V) derived from a human immunoglobulin framework or a human consensus framework, as discussed herein. H) framework amino acid sequence. The acceptor human framework derived from a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence or can comprise amino acid sequence changes. In some embodiments, the number of amino acid changes 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 across all human frameworks within a single antigen-binding domain such as a VHH.

[0102] "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 is generally expressed as the dissociation constant (K D ) or K D(見かけ) Affinity can be expressed by any of the conventional methods known in the art, including those described herein (e.g., ELISA K D , KinExA, flow cytometry, and / or surface plasmon resonance devices, etc. Such methods include, but are not limited to, BIAcore™, Octet™, or methods involving flow cytometry.

[0103] As used herein, "K D As used herein, the term "K" refers to the equilibrium dissociation constant of an antigen-binding molecule / antigen interaction. D When the term "K" is used, it includes D and K D(見かけ) Includes:

[0104] In some embodiments, the K of the antigen binding molecule D is measured by flow cytometry using antigen-expressing cell lines and fitting the mean fluorescence measured at each antibody concentration to a nonlinear one-site binding equation (Graphpad's Prism Software). In some such embodiments, KD is K D(見かけ) It is.

[0105] The term "biological activity" refers to any one or more biological properties of a molecule (whether naturally occurring as found in vivo or provided or made possible by recombinant means). Biological properties include, but are not limited to, binding of a ligand, induction or increase in cell proliferation (such as T cell proliferation), and induction or increase in expression of a cytokine.

[0106] An "agonist" or "activating" antibody is an antibody that increases and / or activates 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.

[0107] An "antagonist," "blocking," or "neutralizing" antibody is an antibody that inhibits, reduces and / or inactivates 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.

[0108] An "affinity matured" sdAb or VHH containing polypeptide refers to an sdAb or VHH containing polypeptide which has one or more modifications in one or more CDRs which result in an improvement in the affinity of the sdAb or VHH containing polypeptide for its antigen compared to a parent sdAb or VHH containing polypeptide which does not possess such modifications.

[0109] "Humanized VHH" as used herein refers to a VHH in which one or more framework regions have been substantially replaced with human framework regions. In some cases, certain framework region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized VHH may contain residues that are not found in the original VHH or in the human framework sequence, but are included to further improve and optimize the performance of the sdAb or VHH-containing polypeptide. In some embodiments, the humanized sdAb or VHH-containing polypeptide comprises a human Fc region. As will be appreciated, a humanized sequence may be identified by its primary sequence and does not necessarily indicate the process by which the antibody was made.

[0110] An "effector positive Fc region" has an "effector function" 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, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Such effector functions generally require combining the Fc region with a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of assays.

[0111] A "native sequence Fc region" comprises an amino acid sequence identical to that 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.

[0112] A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification. In some embodiments, a "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification, but that retains at least one effector function of the native sequence Fc region. In some embodiments, a variant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the native sequence Fc region or the Fc region of the parent polypeptide compared to the native sequence Fc region or the Fc region of the parent polypeptide. In some embodiments, a 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 the Fc region of the parent polypeptide.

[0113] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcγR is a native human FcR. In some embodiments, the FcR binds an IgG antibody (gamma receptor), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences but differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, e.g., in 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). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. For example, the term "Fc receptor" or "FcR" also includes the fetal receptor FcRn, which plays a role in 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 regulating immunoglobulin homeostasis.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.)).

[0114] As used herein, the terms "substantially similar" or "substantially the same" refer to a sufficiently high degree of similarity between two or more numerical values ​​such that one of skill in the art would consider the difference between the two or more values ​​to have little or no biological and / or statistical significance within the context of the biological characteristic measured by the values. In some embodiments, two or more substantially similar values ​​differ by no more than the approximate value of any one of 5%, 10%, 15%, 20%, 25%, or 50%.

[0115] A polypeptide "variant" refers to 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 percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Such 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, a variant has at least about 80% amino acid sequence identity. In some embodiments, a variant has at least about 90% amino acid sequence identity. In some embodiments, a variant has at least about 95% amino acid sequence identity with a native sequence polypeptide.

[0116] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide, or antibody sequence 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, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of one of ordinary skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR, Inc.) software. One of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0117] Amino acid substitutions can 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 can be introduced into a subject antibody and the products screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0118] [Table 1]

[0119] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0120] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0121] The term "vector" is used to describe a polynucleotide that can be manipulated to contain a cloned polynucleotide or polynucleotides that can be propagated in a host cell. A vector can contain one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., promoters and / or enhancers, etc.) that control the expression of a polypeptide of interest, and / or one or more selectable marker genes (e.g., antibiotic resistance genes and genes that can be used in colorimetric assays, e.g., β-galactosidase, etc.). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0122] "Host cell" refers to a cell that can be or has been the recipient of a vector or an isolated polynucleotide. A host cell can be a prokaryotic or 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, PER.C6™ cells (Crucell), and 293 and CHO cells, and their derivatives, such as 293-6E cells, CHO-DG44 cells, CHO-K1 cells, CHO-S cells, and CHO-DS cells. A host cell includes the progeny of a single host cell, although the 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. A host cell also includes a cell transfected in vivo with a polynucleotide(s) provided herein.

[0123] The term "isolated" as used herein refers to a molecule that is separated from at least some of the components that it is typically found or produced with in nature. For example, a polypeptide is referred to as "isolated" when it is separated from at least some of the components of the cell that produced it. If the polypeptide is secreted by the cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" when it is not part of a larger polynucleotide that it is typically found in nature (e.g., in the case of a DNA polynucleotide, genomic DNA or mitochondrial DNA, etc.) or when it is separated from at least some of the components of the cell that produced it, for example, in the case of an RNA polynucleotide. Thus, a DNA polynucleotide contained in a vector within a host cell can be referred to as "isolated".

[0124] The terms "individual" and "subject" are used interchangeably herein to refer to animals, e.g., mammals. In some embodiments, methods are provided for treating mammals, including, but not limited to, humans, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets. In some instances, "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject to be treated may be a patient, which means the subject has been identified as having or being at sufficient risk of suffering from a disorder relevant to the treatment.

[0125] As used herein, "disease" or "disorder" refers to a condition for which treatment is needed and / or desired.

[0126] The terms "tumor cell," "cancer cell," "cancer," "tumor," and / or "neoplasm" are used interchangeably herein, unless otherwise specified, to refer to a cell (or cells) that exhibit uncontrolled proliferation and / or abnormally increased cell survival and / or inhibited apoptosis that interferes with the normal functioning of bodily organs and systems. This definition includes benign and malignant cancers, polyps, hyperplasias, and occult tumors or micrometastases.

[0127] The terms "cancer" and "tumor" include solid cancers and hematological / lymphatic cancers, as well as malignant tumors such as dysplasia, premalignant tumors, and benign tumors. Exemplary cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney or renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), melanoma, myeloma, neuroblastoma, oral cancer (lips, tongue, mouth, and pharynx), 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, Hodgkin's lymphoma, and non-Hodgkin's lymphoma lymphomas, including non-Hodgkin's lymphoma, as well as 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-dividing cell NHL, bulky disease 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, as well as post-transplant lymphoproliferative disorders (PTLD), and abnormal blood vessel growth associated with nematoses, edema (such as edema associated with brain tumors), and Meigs syndrome.

[0128] The term "non-tumor cells" as used herein 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, stromal kidney cells, fibroblast-like synoviocytes, osteoblasts, and cells located in breast, skeletal muscle, pancreas, stomach, ovary, small intestine, placenta, uterus, testes, kidney, lung, heart, brain, liver, prostate, colon, lymphoid organs, bone, and bone-derived mesenchymal stem cells. The term "peripherally located cells or tissues" as used herein refers to non-tumor cells that are not located near tumor cells and / or within the tumor microenvironment.

[0129] The term "cells or tissues in the tumor microenvironment" as used herein refers to cells, molecules, extracellular matrix and / or blood vessels that surround and / or nourish tumor cells. Exemplary cells or tissues in the tumor microenvironment include, but are not limited to, tumor vasculature, tumor-infiltrating lymphocytes, fibroblastic reticular cells, endothelial progenitor cells (EPCs), cancer-associated fibroblasts, pericytes, other stromal cells, components of the extracellular matrix (ECM), dendritic cells, antigen-presenting cells, T cells, regulatory T cells (Treg cells), macrophages, neutrophils, myeloid-derived suppressor cells (MDSCs) and other immune cells located in the vicinity of the tumor. Methods for identifying tumor cells and / or cells / tissues located in the tumor microenvironment are well known in the art, as described herein below.

[0130] In some embodiments, "increase" or "decrease" refers to a statistically significant increase or decrease, respectively. As will be clear to the skilled artisan, "modulation" can also include causing a change (which can be either an increase or a decrease) in the affinity, avidity, specificity and / or selectivity of a target or antigen to one or more of its ligands, binding partners, partners that associate into homo- or heteromultimeric forms or substrates, compared to the same conditions except for the presence of the test agent, causing a change (which can be either an increase or a decrease) in the sensitivity of the target or antigen to one or more conditions (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. This can be determined in any suitable manner and / or using any suitable assay known per se or described herein, depending on the target involved.

[0131] As used herein, an "immune response" is intended to encompass a cellular and / or humoral immune response that is sufficient to inhibit or forestall the onset of or ameliorate a symptom of a disease (e.g., cancer or cancer metastasis). An "immune response" can encompass aspects of both the innate and adaptive immune systems.

[0132] As used herein, "treatment" is an approach to obtain beneficial or desired clinical results. As used herein, "treatment" covers any administration or application of a therapeutic agent for a disease in a mammal, including a human. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, reduction in the extent of the disease, prevention or delay of disease progression (e.g., metastasis, e.g., to the lungs or lymph nodes), prevention or delay of disease recurrence, delay or slowing of disease progression, amelioration of disease state, inhibition of disease or disease progression, inhibition or slowing of disease or its progression, arrest of its development, and remission (whether partial or total). "Treatment" also includes reduction of pathological consequences of proliferative diseases. The methods provided herein contemplate any one or more of these aspects of treatment. In accordance with the above, the term treatment does not require 100 percent elimination of all aspects of the disorder.

[0133] "Amelioration" means that one or more symptoms are lessened or improved compared to when the therapeutic agent is not administered. "Amelioration" also includes a shortening or reduction in the duration of the symptoms.

[0134] The term "anti-cancer agent" is used herein in its broadest sense 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, anti-cancer biologics (such as cytokines, receptor extracellular domain-Fc fusions, and antibodies), radiotherapy agents, CAR-T therapy agents, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNAs), and oncolytic viruses.

[0135] The term "biological sample" refers to a quantity of material from a living or formerly living organism, including, but 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.

[0136] The term "control" or "reference" refers to a composition known to be free of the analyte (a "negative control") or to a composition known to contain the analyte (a "positive control"). A positive control may contain a known concentration of the analyte.

[0137] As used herein, "delaying the onset of disease" means delaying, preventing, slowing, retarding, stabilizing, inhibiting, and / or prolonging the onset of a disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and / or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, late-stage cancer, such as the onset of metastases, can be delayed.

[0138] "Prevention" as used herein includes providing prevention against the occurrence or recurrence of a disease in a subject who may have a predisposition to the disease, but has not yet been diagnosed with the disease. Unless otherwise indicated, the terms "reduce," "inhibit," or "prevent" do not indicate or require complete prevention over the entire period of time, but only prevention over the period being measured.

[0139] A "therapeutically effective amount" of a substance / molecule, agonist or antagonist may vary depending on 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 any toxic or detrimental effects of the substance / molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective to achieve a desired therapeutic and / or prophylactic result at the required dosages for the required time.

[0140] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to a preparation that is in a form that allows the biological activity of the active ingredient(s) to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered. Such formulations may be sterile.

[0141] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventional in the art used with therapeutic agents that together comprise a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to a recipient at the dosage and concentration used and is compatible with other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation in which it is used.

[0142] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0143] The term "in combination" is used herein to refer to the administration of two or more therapeutic agents where at least a portion of the administration overlaps in time, or where the administration of one therapeutic agent is brief relative to the administration of the other, or where the therapeutic effects of both therapeutic agents overlap for at least some period of time.

[0144] The term "sequentially" is used herein to refer to the administration of two or more therapeutic agents that do not overlap in time or where the therapeutic effects of the therapeutic agents do not overlap.

[0145] As used herein, "in combination with" refers to the administration of one therapy in addition to another. Thus, "in combination with" refers to the administration of one therapy before, during, or after the administration of another therapy to an individual.

[0146] The term "package insert" is used to refer to instructions typically included in commercial packaging for a therapeutic product, which contain information regarding directions, usage, dosage, administration, concomitant therapy, contraindications and / or warnings regarding the use of such therapeutic product.

[0147] An "article of manufacture" is any manufacture (e.g., package or container) or kit that contains at least one reagent, e.g., a pharmaceutical agent for treating a disease or disorder (e.g., cancer), or a probe that specifically detects a biomarker described herein. In some embodiments, the article of manufacture or kit is advertised, delivered, or sold as a unit for performing a method described herein.

[0148] The terms "label" and "detectable label" refer to a moiety that, for example, is attached to an antibody or an antigen to render the reaction (e.g., binding) between members of a specific binding pair detectable. A labeled member of a specific binding pair is said to be "detectably labeled." Thus, the term "labeled binding protein" refers to a protein that incorporates a label that provides for the identification of the binding protein. In some embodiments, the label is a detectable marker that can generate a signal that is detectable visually or by instrumental means, such as the incorporation of a radiolabeled amino acid or the attachment of a biotinyl moiety to the polypeptide that can be detected by marked avidin (e.g., streptavidin that contains a fluorescent marker or an enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C. 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153Sm), chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), and magnetic agents such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include moieties that emit light, e.g., acridinium compounds, and moieties that emit fluorescence, e.g., fluorescein. In this regard, the moiety itself may not be detectably labeled, but may become detectable upon reaction with yet another moiety.

[0149] Exemplary γδ T Cell Binding Polypeptides Provided herein are γδ T cell binding polypeptides. In various embodiments, a γδ T cell binding polypeptide comprises at least one VHH domain that binds to a γδ T cell. In some embodiments, a γδ T cell binding polypeptide provided herein comprises one, two, three, four, five, six, seven, or eight VHH domains that bind to a γδ T cell. In some embodiments, a γδ T cell binding polypeptide provided herein comprises one, two, three, or four VHH domains that bind to a γδ T cell. Such a γδ T cell binding polypeptide may comprise one or more additional VHH domains that bind to one or more target proteins other than a γδ T cell, and / or may comprise one or more additional polypeptide sequences, such as cytokine sequences.

[0150] In some embodiments, a γδ T cell-binding polypeptide comprises at least one VHH domain that binds to a γδ T cell and an Fc region. In some embodiments, a γδ T cell-binding polypeptide provided herein comprises one, two, three, or four VHH domains that bind to a γδ T cell and an Fc region. In some embodiments, the Fc region mediates dimerization of the γδ T cell-binding polypeptide under physiological conditions, such that the formation of dimers doubles the number of γδ T cell binding sites. For example, a γδ T cell-binding polypeptide comprising three VHH domains that bind to a γδ T cell and an Fc region is trivalent as a monomer, but under physiological conditions, the Fc region can mediate dimerization such that the γδ T cell-binding polypeptide exists as a hexavalent dimer under such conditions.

[0151] In some embodiments, a γδ T cell binding polypeptide comprises at least two VHH domains, where 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 a γδ T cell binding polypeptide 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 γδ T cell binding polypeptide may be referred to as "dual epitopic" or "dual specific".

[0152] In some embodiments, the γδ T cell binding polypeptide is a complex of a first polypeptide comprising a first VHH domain and a first Fc domain that binds to γδ T cells, and a second polypeptide comprising a second VHH domain and a second Fc domain that binds to γδ T cells, optionally, either the first or the second polypeptide further comprising a cytokine polypeptide. In some embodiments, the γδ T cell binding polypeptide is a complex of a first polypeptide comprising a first VHH domain and a first Fc domain that binds to γδ T cells, and a second polypeptide comprising an antigen binding domain that binds to an antigen other than γδ T cells and a second Fc domain, optionally, either the first or the second polypeptide further comprising a cytokine polypeptide. In some such embodiments, the first Fc domain or the second Fc domain comprises a "knob" mutation(s) and the other Fc domain comprises a "hole" mutation(s). Thus, in some embodiments, the γδ T cell binding polypeptide is a complex of a first polypeptide and a second polypeptide. In some such embodiments, the complex comprises two γδ T cell binding VHH domains, hi some such embodiments, the complex comprises one γδ T cell binding VHH domain and one antigen binding domain that binds to an antigen other than a γδ TCR.

[0153] γδ T cell-binding polypeptide In various embodiments, the VHH domain that binds to γδ TCR comprises a CDR1 sequence selected from SEQ ID NO:3, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, and SEQ ID NO:149, a CDR2 sequence selected from SEQ ID NO:4, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, and SEQ ID NO:156, and a CDR3 sequence of SEQ ID NO:5. In various embodiments, the VHH domain that binds to γδ TCR comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:144, SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:145, SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:146, SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:147, SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:148, SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:149, SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:150 and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:151 and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:152 and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:153 and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:154 and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:155 and SEQ ID NO:5; or SEQ ID NO:3, SEQ ID NO:156 and SEQ ID NO:5. In various embodiments, the VHH domain that binds to γδ TCR comprises a CDR1 sequence selected from SEQ ID NO:3, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:147, and SEQ ID NO:148, a CDR2 sequence selected from SEQ ID NO:4, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, and SEQ ID NO:156, and a CDR3 sequence of SEQ ID NO:5.In various embodiments, the VHH domain that binds to γδ TCR comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; SEQ ID NO:144, SEQ ID NO:4, and SEQ ID NO:5; SEQ ID NO:146, SEQ ID NO:4, and SEQ ID NO:5; SEQ ID NO:147, SEQ ID NO:4, and SEQ ID NO:5; SEQ ID NO:148, SEQ ID NO:4, and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:150, and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:151, and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:152, and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:153, and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:154, and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:155, and SEQ ID NO:5; or SEQ ID NO:3, SEQ ID NO:156, and SEQ ID NO:5. In various embodiments, the VHH domain that binds to γδ TCR comprises the CDR1 sequence of SEQ ID NO:3, the CDR2 sequence of SEQ ID NO:4, and the CDR3 sequence of SEQ ID NO:5. In various embodiments, the VHH domain is humanized.

[0154] In some embodiments, the VHH domain that binds to γδ TCR 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, 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 is T or S, X 11 is D or G, X 12 is A or S, X 13 is A or T, X 14 is E or Y, X 15 is V or A, X 16 is D, E, A, G, V, S, Y, L or Q, X 17 is S, P, T, A, V, L, I, or G; X 18 is G or D, X 19 is S or N, X 20 is T or A, X 21 is A or T, X 22 is V or L; X 23 is N or S, X 24 is K or Y, X 25 are N, S, E, Y, A, S, G, and Q, X 26 is S, T, A, L, V, N or G, X 27 is K, R, E, or D.

[0155] In some embodiments, the VHH domain that binds to γδ TCR comprises SEQ ID NO: 180, where X3 is K and 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 , X19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 25 , X 26 , and X 27 are independently selected, X1 is V or A; X2 is R or G; 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 is T or S, X 11 is D or G, X 12 is A or S, X 13 is A or T, X 14 is E or Y, X 15 is V or A, X 16 is D, E, A, L or Q, X 17 is S, P, T, V, L, or G; X 18 is G or D, X 19 is S or N, X 20 is T or A, X 21 is A or T, X 22 is V or L; X 23 is N or S, X 24 is K or Y, X 25 is N or S, X 26 is S, T, or G, X 27is K, R, E, or D.

[0156] In some embodiments, the VHH domain that binds to γδ TCR comprises SEQ ID NO: 180, where X2 is R and X 25 is N, X1, 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, X1 is V or A; 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 is T or S, X 11 is D or G, X 12 is A or S, X 13 is A or T, X 14 is E or Y, X 15 is V or A, X 16 is D, E, A or Q, X 17 is S, P or G, X 18 is G or D, X 19 is S or N, X 20 is T or A, X 21 is A or T, X 22 is V or L; X 23 is N or S, X 24 is K or Y, X 26 is S or T, X 27 is K, R, E, or D.

[0157] In some embodiments, the VHH domain that binds to γδ TCR comprises SEQ ID NO: 180, where X2 is R, X3 is K, X4 is I, X9 is H, and 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, X1 is V or A; X5 is Q, G or E; X6 is R or L; X7 is L, W or F; X8 is A or S; X 10 is T or S, X 11 is D or G, X 12 is A or S, X 13 is A or T, X 14 is E or Y, X 15 is V or A, X16 is D, E or A, X 17 is S or P, X 18 is G or D, X 19 is S or N, X 20 is T or A, X 21 is A or T, X 22 is V or L; X 23 is N or S, X 24 is K or Y, X 26 is S or T, X 27 is K, R, E, or D.

[0158] In some embodiments, the VHH domain that binds to γδ TCR comprises SEQ ID NO: 180, where X1 is V, X2 is R, X3 is K, X4 is I, X9 is H, and X 10 is T and X 11 is D and X 12 is A and X 13 is A and X 14 is E and 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, X5 is Q, G or E; X6 is R or L; X7 is L or W; X8 is A or S; X 15 is V or A, X 16is D, E, or A, X 17 is S or P, X 18 is G or D, X 19 is S or N, X 20 is T or A, X 21 is A or T, X 22 is V or L; X 23 is N or S, X 24 is K or Y, X 26 is S or T, X 27 is K, R, E, or D.

[0159] In some embodiments, the VHH domain that binds to 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%, or at least 99% identical to an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 17 to SEQ ID NO: 31, SEQ ID NO: 72 to SEQ ID NO: 77, SEQ ID NO: 80 to SEQ ID NO: 143, SEQ ID NO: 158 to SEQ ID NO: 159, and SEQ ID NO: 166 to SEQ ID NO: 179. In some embodiments, the VHH domain that binds to γδ TCR comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 17 to SEQ ID NO: 31, SEQ ID NO: 72 to SEQ ID NO: 77, SEQ ID NO: 80 to SEQ ID NO: 143, SEQ ID NO: 158 to SEQ ID NO: 159, and SEQ ID NO: 166 to SEQ ID NO: 179, and position 114 of the VHH domain is substituted with lysine (K), aspartic acid (D), glutamic acid (E), or arginine (R). In some embodiments, the VHH domain that binds to 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%, or at least 99% identical to an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:17 to SEQ ID NO:19, SEQ ID NO:21 to SEQ ID NO:23, SEQ ID NO:25 to SEQ ID NO:31, SEQ ID NO:72 to SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:84 to SEQ ID NO:86, SEQ ID NO:88 to SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97 to SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111 to SEQ ID NO:129, SEQ ID NO:131 to SEQ ID NO:133, SEQ ID NO:135 to SEQ ID NO:137, SEQ ID NO:139 to SEQ ID NO:143, SEQ ID NO:158 to SEQ ID NO:159, and SEQ ID NO:166 to SEQ ID NO:179.In some embodiments, the VHH domain that binds to γδ TCR comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 17 to SEQ ID NO: 19, SEQ ID NO: 21 to SEQ ID NO: 23, SEQ ID NO: 25 to SEQ ID NO: 31, SEQ ID NO: 72 to SEQ ID NO: 77, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84 to SEQ ID NO: 86, SEQ ID NO: 88 to SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97 to SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111 to SEQ ID NO: 129, SEQ ID NO: 131 to SEQ ID NO: 133, SEQ ID NO: 135 to SEQ ID NO: 137, SEQ ID NO: 139 to SEQ ID NO: 143, SEQ ID NO: 158 to SEQ ID NO: 159, and SEQ ID NO: 166 to SEQ ID NO: 179, and is substituted at position 114 of the VHH domain with lysine (K), aspartic acid (D), glutamic acid (E), or arginine (R). In some embodiments, the VHH domain that binds to γδ TCR comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 17 to SEQ ID NO: 31, SEQ ID NO: 72 to SEQ ID NO: 77, SEQ ID NO: 80 to SEQ ID NO: 143, SEQ ID NO: 158 to SEQ ID NO: 159, and SEQ ID NO: 166 to SEQ ID NO: 179. In some embodiments, the VHH domain that binds to γδ TCR comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 17 to SEQ ID NO: 19, SEQ ID NO: 21 to SEQ ID NO: 23, SEQ ID NO: 25 to SEQ ID NO: 31, SEQ ID NO: 72 to SEQ ID NO: 77, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84 to SEQ ID NO: 86, SEQ ID NO: 88 to SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97 to SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111 to SEQ ID NO: 129, SEQ ID NO: 131 to SEQ ID NO: 133, SEQ ID NO: 135 to SEQ ID NO: 137, SEQ ID NO: 139 to SEQ ID NO: 143, SEQ ID NO: 158 to SEQ ID NO: 159, and SEQ ID NO: 166 to SEQ ID NO: 179.

[0160] In some embodiments, the VHH domain that binds to 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 at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 99, SEQ ID NO: 143 or SEQ ID NO: 158. In some embodiments, the VHH domain comprises an amino acid sequence selected from SEQ ID NO: 99, SEQ ID NO: 143, and SEQ ID NO: 158, wherein position 114 of the VHH domain is substituted with lysine (K), aspartic acid (D), glutamic acid (E), or arginine (R). In some embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO: 99, SEQ ID NO: 143, or SEQ ID NO: 158.

[0161] In various embodiments, the γδ T cell binding polypeptide comprises one, two, three or four VHH domains that bind to γδ T cells.

[0162] In some embodiments, the VHH domain that binds to γδ T cells may be humanized. Humanized antibodies (such as sdAbs or VHH-containing polypeptides) are useful as therapeutic molecules because they reduce or eliminate human immune responses to non-human antibodies that may result in an immune response to antibody therapeutics and reduce the efficacy of the therapeutics. Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0163] Humanized antibodies and methods for making them are reviewed, e.g., in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633, and described, e.g., in Riechmann et al., (1988) Nature 332:323-329, Queen et al., (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033, U.S. Pat. No. 5,821,337, U.S. Pat. No. 7,527,791, U.S. Pat. No. 6,982,321, and U.S. Pat. No. 7,087,409, Kashmiri et al., (2005) Methods 36:25-34, Padlan, (1991) Mol. Immunol. 28:489-498 (describing "resurfacing"), Dall'Acqua, et al. et al., (2005) Methods 36:43-60 (describing "FR shuffling"), as well as Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (describing a "guided selection" approach to FR shuffling).

[0164] Human framework regions that may 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 consensus sequences of human antibodies of particular subgroups 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, 151:2623), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633), and framework regions obtained from screening of 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 the human FR are replaced to improve one or more properties of the humanized VHH. A VHH domain having such replaced residues is also referred to herein as "humanized".

[0165] In various embodiments, the Fc region comprised in the γδ T cell binding polypeptide is a human Fc region or is derived from a human Fc region. In some embodiments, the Fc region comprised in the γδ T cell binding polypeptide is derived from a human Fc region and lacks a C-terminal lysine residue. In some embodiments, the Fc region comprised in the γδ T cell binding polypeptide is derived from a human Fc region and includes a C-terminal lysine residue. In some embodiments, the C-terminal amino acid of the Fc region is an amino acid other than lysine.

[0166] In some embodiments, the Fc region comprised in the γδ T cell-binding polypeptide is derived from a human Fc region and comprises a three amino acid deletion in the lower hinge corresponding to IgG1 E233, L234, and L235, and is referred to herein as "Fc xELL". Fc xELL polypeptides do not bind FcγR and are therefore referred to as "effector silent" or "effector null", however, in some embodiments, the xELL Fc region binds to FcRn, with associated transcytosis associated with extended half-life and FcRn mediated recycling.

[0167] In some embodiments, the Fc region comprised in a γδ T cell-binding polypeptide is derived from a human Fc region and comprises the mutations M252Y and M428V, referred to herein as "Fc-YV". In some embodiments, such mutations enhance binding to FcRn at the acidic pH of the endosome (near 6.5) while losing detectable binding at neutral pH (about 7.2), allowing for enhanced FcRn-mediated recycling and extended half-life.

[0168] In some embodiments, the Fc region comprised in the γδ T cell-binding polypeptide is derived from a human Fc region and contains mutations designed for heterodimerization, referred to herein as "knob" and "hole". In some embodiments, the "knob" Fc region contains the mutation T366W. In some embodiments, the "hole" Fc region contains the mutations T366S, L368A, and Y407V. In some embodiments, the Fc region used for heterodimerization contains an additional mutation, such as the mutation S354C on a first member of the heterodimeric Fc pair, which forms an asymmetric disulfide with the corresponding mutation Y349C on the second member of the heterodimeric Fc pair. In some embodiments, one member of the heterodimeric Fc pair contains the modification H435R or H435K to prevent Protein A 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 hole Fc region comprises the modification H435R or H435K (sometimes referred to as "hole-R" when the modification is H435R), while the knob Fc region does not. In some cases, the hole-R mutation improves purification of the heterodimer relative to a homodimeric hole Fc region that may exist.

[0169] Non-limiting exemplary Fc regions that may be used in γδ T cell-binding polypeptides include Fc regions comprising the amino acid sequences of SEQ ID NO:32-70. In some embodiments, a γδ T cell-binding polypeptide comprises an Fc region comprising an amino acid sequence selected from SEQ ID NO:32-70, wherein the Fc region lacks a C-terminal lysine residue. In some embodiments, a γδ T cell-binding polypeptide comprises an Fc region comprising an amino acid sequence selected from SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:41-52, and SEQ ID NO:58-70. In some embodiments, a γδ T cell-binding polypeptide comprises an Fc region comprising an amino acid sequence selected from SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:41-52, and SEQ ID NO:58-70, wherein the Fc region lacks a C-terminal lysine residue.

[0170] Exemplary Activities of γδ T Cell Binding Polypeptides In various embodiments, the γδ T cell binding polypeptides provided herein stimulate γδ T cells in vitro and / or in vivo. In vitro and / or in vivo stimulation or activity of γδ T cells may in some embodiments be determined using the methods provided in the Examples herein.

[0171] In some embodiments, a γδ T cell binding polypeptide provided herein comprises an antigen binding domain that binds to an immune cell activating cytokine or 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 that binds to an immune cell activating cytokine or an antigen other than γδ T cells is increased compared to when used alone and / or is more specifically targeted to cytotoxic T cells when fused to a γδ T cell binding VHH. In some embodiments, the toxicity of the antigen binding domain that binds to an immune cell activating cytokine or an antigen other than γδ T cells is reduced by specifically targeting γδ T cells.

[0172] In some embodiments, the immune cell activating cytokines provided herein, or γδ T cell binding polypeptides comprising an antigen binding domain that binds to an antigen other than γδ T cells, increase T cell proliferation in vitro and / or in vivo.

[0173] In some embodiments, a γδ T cell binding polypeptide provided herein comprises a γδ T cell binding VHH provided herein and an immune cell activating cytokine. In some such embodiments, the immune cell activating cytokine is IL-2, IL-15, IL-7, IL-6, IL-12, IFNα, IFNβ, or IFNγ. In some such embodiments, the immune cell activating cytokine is a wild-type immune cell activating cytokine. In some embodiments, the immune cell activating cytokine comprises a mutation that attenuates the activity of the immune cell activating cytokine compared to the activity of the wild-type cytokine. In some embodiments, a γδ T cell binding polypeptide comprising an immune cell activating cytokine stimulates the activation and proliferation of γδ T cells in vivo. In some embodiments, a γδ T cell binding polypeptide comprising an immune cell activating cytokine is used in a method of treating cancer.

[0174] The increase in proliferation of activated γδ T cells can be determined by any method in the art, such as, for example, the methods shown in the Examples herein. A non-limiting exemplary assay 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 analyzed by FACS after contacting with a polypeptide comprising modified IL-2. Loss of CTV staining indicates proliferation. In some embodiments, the increase in proliferation of γδ T cells is determined as an average from a series of experiments or pooled T cells, for example, by measuring the proliferation of γδ T cells isolated from different healthy human donors. In some embodiments, the increase in proliferation of γδ T cells is determined as an average from experiments performed using T cells from at least 5 or at least 10 different healthy donors, or from a pool of T cells from at least 5 or at least 10 different healthy donors.

[0175] In some embodiments, a γδ T cell-binding polypeptide provided herein comprises a γδ T cell-binding VHH and an antigen-binding domain that binds to an antigen other than a γδ T cell. 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, α4 integrin, αV integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis Y, apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, complement C5, C-242, CA9, CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD 19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD39, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD73, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD 138, 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, E GFR, 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, Notch4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine-1-phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TLR6, T LR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, TROP-2, uPAR, 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 γδ T cell binding polypeptide comprises a γδ T cell binding VHH and an antigen binding domain that binds to a tumor cell antigen. In some such embodiments, the γδ T cell binding polypeptide comprising an antigen binding domain that binds to a tumor cell antigen increases γδ T cell mediated killing of tumor cells expressing the antigen.

[0176] Polypeptide Expression and Production Nucleic acid molecules are provided that comprise a polynucleotide encoding a γδ T cell-binding polypeptide. In some embodiments, the nucleic acid molecule may also encode a leader sequence that directs secretion of the γδ T cell-binding polypeptide, the leader sequence typically being cleaved such that it is not present in the secreted polypeptide. The leader sequence may be the native heavy chain (or VHH) leader sequence or may be another heterologous leader sequence.

[0177] The nucleic acid molecule 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.

[0178] Vectors are provided that include a nucleic acid encoding a γδ T cell binding polypeptide 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, a vector is selected that is optimized for expression of the polypeptide in a desired cell type, such as a CHO cell or a CHO-derived cell, or an NSO cell. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0179] In some embodiments, γδ T cell binding polypeptides may be expressed in prokaryotic cells, such as bacterial cells, or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that may be used for expression of the polypeptide include, but are not limited to, COS cells, including COS7 cells, 293 cells, including 293-6E cells, CHO cells, including CHO-S, DG44, Lec13 CHO cells, and FUT8 CHO cells, PER.C6™ cells (Crucell), and NSO cells. In some embodiments, γδ T cell binding polypeptides may be expressed in yeast. See, for example, US Patent Application Publication No. 2006 / 0270045. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the polypeptide. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.

[0180] Introduction of one or more nucleic acids (e.g., vectors) into a desired host cell can be accomplished by any method, including, but not limited to, calcium phosphate transfection, DEAE-dextran 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, 3 rd ed. Cold Spring Harbor Laboratory Press (2001). The nucleic acid may be transiently or stably transfected into the desired host cell according to any suitable method.

[0181] Also provided are host cells comprising any of the nucleic acids or vectors described herein. In some embodiments, host cells are provided that express a γδ T cell-binding polypeptide described herein. The γδ T cell-binding polypeptide expressed in the host cell can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include agents that bind to the ROR1 ECD and Fc region. For example, Protein A, Protein G, Protein A / G, or antibody affinity columns can be used to purify γδ T cell-binding polypeptides comprising an Fc region by binding to the Fc region. Hydrophobic interaction chromatography, e.g., butyl or phenyl columns, may also be suitable for purifying some polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) may also be suitable for purifying some polypeptides, such as antibodies. Mixed-mode chromatography (e.g., reversed-phase / anion exchange, reversed-phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) may also be suitable for purifying some polypeptides, such as antibodies. Many methods of purifying polypeptides are known in the art.

[0182] In some embodiments, the γδ T cell-binding polypeptides are produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009), Spirin, Trends Biotechnol. 22: 538-45 (2004), and Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0183] In some embodiments, a γδ T cell binding polypeptide produced by the above method is provided. In some embodiments, the γδ T cell binding polypeptide is produced in a host cell. In some embodiments, the γδ T cell binding polypeptide is produced in a cell-free system. In some embodiments, the γδ T cell binding polypeptide is purified. In some embodiments, a cell culture medium comprising the γδ T cell binding polypeptide is provided.

[0184] In some embodiments, a composition is provided comprising an antibody produced by the above method. In some embodiments, the composition comprises a γδ T cell binding polypeptide produced in a host cell. In some embodiments, the composition comprises a γδ T cell binding polypeptide produced in a cell-free system. In some embodiments, the composition comprises a purified γδ T cell binding polypeptide.

[0185] Exemplary Methods of Treating Disease Using γδ T Cell Binding Polypeptides In some embodiments, a method is provided for treating a disease in an individual comprising administering a γδ T cell binding polypeptide. Such diseases include diseases that involve T cells, e.g., γδ T cells. +The present invention includes any disease that would benefit from increased proliferation and activation of T cells. In some embodiments, a method for treating cancer in an individual is provided. In some embodiments, the method for treating cancer comprises increasing proliferation and / or activation of γδ T cells by administering a γδ T cell-binding polypeptide comprising a γδ T cell-binding VHH and an antigen-binding domain that binds to an immune cell-activating cytokine or a tumor cell antigen other than γδ T cells.

[0186] The method comprises administering to an individual an effective amount of a γδ T cell-binding polypeptide as provided herein. Such a method of treatment can be a method of treatment in a human or an animal. In some embodiments, a method of treatment in a human is provided. Non-limiting exemplary cancers that can be treated using the γδ T cell-binding polypeptides provided herein include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, glioblastoma, hepatic cancer, hepatocellular carcinoma, intraepithelial neoplasia, renal or kidney cancer, laryngeal cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric 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-dividing cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloblastic leukemia.

[0187] The γδ T cell-binding polypeptide may be administered to the subject as needed. The frequency of administration may be determined by a person skilled in the art, such as the attending physician, based on considerations such as the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, etc. In some embodiments, an effective dose of the γδ T cell-binding polypeptide is administered to the subject one or more times. In some embodiments, an effective dose of the γδ T cell-binding polypeptide is administered to the subject daily, twice weekly, weekly, biweekly, monthly, etc. An effective dose of the γδ T cell-binding polypeptide is administered to the subject at least once. In some embodiments, an effective dose of the γδ T cell-binding polypeptide may be administered multiple times, including multiple times over at least one month, at least six months, or at least one year.

[0188] In some embodiments, the pharmaceutical composition is administered in an amount effective to treat cancer (including cancer prevention) and / or increase T cell proliferation. The therapeutically effective amount typically depends on the weight of the subject being treated, the physical or health condition of the subject, the extent of the condition being treated, or the age of the subject being treated. In general, the antibody may be administered in an amount ranging from about 0.05 mg / kg (body weight) to about 100 mg / kg (body weight) per dose.

[0189] In some embodiments, γδ T cell-binding polypeptides may be administered in vivo by a variety of routes, including but not limited to, intravenous, intraarterial, parenteral, intraperitoneal, or subcutaneous. Depending on the intended use, appropriate formulations and administration routes can be selected.

[0190] In some embodiments, therapeutic treatment using a γδ T cell binding polypeptide 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.

[0191] Pharmaceutical Compositions In some embodiments, compositions comprising γδ T cell-binding polypeptides are provided in formulations that include a wide variety of pharma- ceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 th ed., Lippencott Williams and Wilkins (2004), Kibbe et al., Handbook of Pharmaceutical Excipients, 3 rd ed., Pharmaceutical Press (2000). A variety of pharma- ceutically acceptable carriers, including excipients, adjuvants, and diluents, are available. In addition, a variety of pharma- ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like, are also available. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0192] In some embodiments, the pharmaceutical composition comprises a γδ T cell-binding polypeptide at a concentration of at least 10 mg / mL.

[0193] Combination therapy The γδ T cell-binding polypeptides may be administered alone or in combination with other therapeutic modalities, such as other anti-cancer agents. The γδ T cell-binding polypeptides may be provided prior to, substantially simultaneously with, or after (i.e., concomitantly or sequentially with) other therapeutic modalities. In some embodiments, the therapeutic methods described herein may further comprise administering radiotherapy, chemotherapy, vaccination, targeted tumor therapy, CAR-T therapy, oncolytic virus therapy, cancer immunotherapy, cytokine therapy, surgical resection, chromatin modification, resection, cryotherapy, antisense agents against tumor targets, siRNA agents against tumor targets, microRNA agents against tumor targets, or anti-cancer / anti-tumor agents, or biologics such as antibodies, cytokines, or receptor extracellular domain-Fc fusions.

[0194] In some embodiments, a γδ T cell-binding polypeptide provided herein is given in combination with a second therapeutic agent, for example a PD-1 or PD-L1 therapeutic agent. Examples of PD-1 / PD-L1 therapies 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 directed against programmed death-1 (PD-1) or programmed death-ligand 1 (PD-L1).

[0195] In some embodiments, the γδ T cell binding polypeptides provided herein are administered in combination with an immunostimulatory agent, such as an agonist of a member of the tumor necrosis factor receptor superfamily (TNFRSF) or a member of the B7 family. Non-limiting examples of immunostimulatory TNFRSF members include OX40, GITR, 41BB, CD27, and HVEM. Non-limiting examples of B7 family members include CD28 and ICOS. Thus, in some embodiments, the γδ T cell binding polypeptides provided herein are administered in combination with an agonist of OX40, GITR, 41BB, CD27, HVEM, CD28, and / or ICOS, such as an agonistic antibody.

[0196] In some embodiments, the γδ T cell binding polypeptides provided herein are given in combination 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, etc.

[0197] Non-Limiting Exemplary Methods of Diagnosis and Treatment In some embodiments, the methods described herein are useful for evaluating a subject and / or a sample from a subject (e.g., a cancer patient). In some embodiments, the evaluation is one or more of diagnosis, prognosis, and / or response to treatment.

[0198] In some embodiments, the methods described herein include assessing the presence, absence, or level of a protein. In some embodiments, the methods described herein include assessing the presence, absence, or level of expression of a nucleic acid. The compositions described herein can be used for these measurements. For example, in some embodiments, the methods described herein include contacting a tumor specimen or cells cultured from the tumor with a therapeutic agent described herein.

[0199] In some embodiments, the evaluation may prescribe treatment, including treatment with the antibodies described herein. In some embodiments, the evaluation may prescribe the use or withholding of adjuvant therapy after resection. Adjuvant therapy, also called adjuvant therapy, is a treatment given in addition to the primary, main, or initial treatment. As a non-limiting example, adjuvant therapy may be an additional treatment, usually given after surgery, when all detectable disease has been removed but there remains a statistical risk of relapse due to latent disease. In some embodiments, the antibody is used as an adjuvant therapy in the treatment of cancer. In some embodiments, the antibody is used as the sole adjuvant therapy in the treatment of cancer. In some embodiments, the antibody described herein is withheld as an adjuvant therapy in the treatment of cancer. For example, when a patient is unlikely to respond or has a minimal response to the antibodies described herein, treatment may not be administered for quality of life and to avoid unnecessary toxicity from ineffective chemotherapy. In such cases, palliative care may be used.

[0200] 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 the tumor before any surgery. In some embodiments, neoadjuvant therapy refers to a chemotherapy drug administered to a cancer patient before surgery. In some embodiments, neoadjuvant therapy refers to an antibody administered to a cancer patient before surgery. Cancer types for which neoadjuvant chemotherapy is usually considered include, for example, breast cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, and lung cancer. In some embodiments, the antibody is used as a neoadjuvant therapy in the treatment of cancer. In some embodiments, the use is before resection.

[0201] In some embodiments, the tumor microenvironment contemplated by the methods described herein is one or more of tumor vasculature, tumor-infiltrating lymphocytes, fibroblastic reticular cells, endothelial progenitor cells (EPCs), cancer-associated fibroblasts, pericytes, 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 proximity to the tumor.

[0202] kit Also provided are articles of manufacture and kits comprising any of the γδ T cell binding polypeptides described herein and suitable packaging. In some embodiments, the invention includes a kit comprising (i) a γδ T cell binding polypeptide and (ii) instructions for using the kit to administer the γδ T cell binding polypeptide to an individual.

[0203] Suitable packaging for the compositions described herein are known in the art and include, for example, vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may be further sterilized and / or sealed. Unit dosage forms comprising the compositions described herein are also provided. These unit dosage forms may be stored in suitable packaging in single or multiple unit dosage forms, and may also be further sterilized and sealed. Instructions provided in the kits of the invention are typically written instructions on a label or insert (e.g., a paper sheet included in the kit), although 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 regarding dosage, administration schedule, and route of administration for the intended therapeutic or industrial use. The kit may further include instructions for selecting an appropriate individual treatment.

[0204] The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Kits may also be provided that contain a sufficient dose of the molecules disclosed herein to provide effective treatment to an individual for an extended period of time, such as any approximate period of 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. Kits may also contain multiple unit doses of the molecules and instructions for use, and may be packaged in an amount sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies. In some embodiments, the kits include a dried (e.g., lyophilized) composition that can be reconstituted, resuspended, or rehydrated to form a generally stable aqueous suspension of the antibody. EXAMPLES

[0205] The examples discussed below are intended to be purely illustrative of the present invention and should not be considered as limiting the present invention in any way. These examples are not intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0206] Example 1: γδ TCR-targeted IL-2 activates the STAT5 signaling pathway in γδ T cells T cell activation via IL-2 signaling results in phosphorylation of the transcription factor STAT5. PBMCs were isolated from leukopaks of healthy donors by lymphoprep density gradient centrifugation. Cells were labeled for 20 min at room temperature with the following fluorescently conjugated antibodies: non-competitive anti-γδTCR-FITC, anti-CD3-BV785, and anti-CD56-BV421. After washing, 200000 PBMCs per well were seeded in 96-well plates. Cells were treated with titrations of fusion proteins containing either IL-2_X or IL-2_Y, both attenuated IL-2 polypeptides, fused to the C-terminus of either anti-γδTCR-5C8 or anti-γδTCR-6C4, starting from an initial concentration of 100 nM and titrated 1:5 across the plate in duplicate. Plates were incubated for 20 min at 37°C / 5% CO2. Cells were fixed with BD Cytofix / Cytoperm™ (BD Biosciences), permeabilized in 90% ice-cold methanol, and levels of phosphorylated STAT5 ("pSTAT5") were measured by flow cytometry using phospho-specific anti-pSTAT5-PE antibody (1:70) and CD3-BV785 to identify γδ T cells. + γδTCR-FITC + , CD56-BV421 to identify NK cells + CD3-BV785 - , CD3-BV785 to identify αβ T cells + γδTCR-FITC - Gated against

[0207] As shown in Figures 1A-1H, treatment with bivalent or monovalent anti-γδ TCR-5C8 or anti-γδ TCR-6C4 targeting IL-2_X or IL-2_Y reduced pSTAT5 + It resulted in a dose-dependent increase in the percentage of γδ T cells and in the median pSTAT5 fluorescence intensity on γδ T cells. In contrast, below 100 nM, little pSTAT5 activation was observed on NK cells or αβ T cells.

[0208] Example 2: Enhanced proliferation and accumulation of γδ T cells following treatment with γδ TCR-targeted low affinity IL-2_X IL-2 promotes activation and proliferation of T cell populations. To evaluate the effect of γδTCR-targeted IL-2_X on proliferation, PBMCs were isolated from leukopaks of healthy donors using lymphoprep density gradient medium. Cells were labeled with CellTrace Violet proliferation dye for 10 min at 37°C. After washing, cells were resuspended in RPMI+10% FBS and 300000 cells per well were added to a 96-well plate. Cells were treated with a titration of fusion proteins containing IL-2_X fused to the C-terminus of either anti-γδTCR-5C8 or a non-targeted control VHH, titrated 1:5 across the plate in duplicate, starting with an initial concentration of 100 nM. Plates were incubated for 7 days at 37°C / 5% CO2. Cells were labeled with the following fluorescently conjugated antibodies: CD3-BV785, γδTCR-FITC, and the viability dye propidium iodide for 30 min at 4°C. Cells were washed and analyzed by flow cytometry.

[0209] As shown in Figures 2A-D, treatment with anti-γδ TCR-5C8-targeted IL-2_X resulted in a dose-dependent increase in the proliferation of γδ T cells. + The proportion of γδ T cells in the population was also increased by anti-γδ TCR-5C8-targeted IL-2_X treatment with a concomitant decrease in αβ T cells. This effect was specific to γδ T cells, as the αβ T cell population did not proliferate in response to treatment with anti-γδ TCR-5C8-targeted IL-2_X. Non-targeted VHH fused to IL-2_X did not promote proliferation of either γδ or αβ T cells, demonstrating target specificity.

[0210] Example 3: γδ TCR-targeted IL-2 inhibits Vδ2 + Activating STAT5 signaling in γδ T cells IL-2 signaling leads to phosphorylation of the transcription factor STAT5 and activation of downstream gene expression. To assess STAT5 phosphorylation, PBMCs were isolated from whole blood of healthy human donors by lymphoprep density gradient centrifugation. Cells were labeled for 20 min at room temperature with the following fluorescently conjugated antibodies: non-competitive anti-γδTCR-FITC, anti-CD3-BV785, and anti-Vδ2-BV421. After washing, 400000 PBMCs per well were seeded in 96-well plates. Cells were treated with titrations of fusion proteins containing either IL-2_X fused to the C-terminus of anti-γδTCR-1D7, starting from an initial concentration of 50 nM and titrated 1:3 across the plate in duplicate. Plates were incubated for 20 min at 37° C. / 5% CO2. Cells were fixed with BD Cytofix / Cytoperm™ (BD Biosciences), permeabilized in 90% ice-cold BD Phosflow™ Perm Buffer III (BD Biosciences), and Vδ2 + γδ T cells or Vδ2 - γδ - Levels of phosphorylated STAT5 ("pSTAT5") on αβT cells were measured by flow cytometry using a phospho-specific anti-pSTAT5-PE antibody (1:70).

[0211] As shown in Figures 3A and 3B, treatment with monovalent anti-γδTCR-1D7-targeted IL-2_X reduced pSTAT5 + Vδ2 + Proportion of γδ T cells (Figure 3A) and Vδ2 + This resulted in a dose-dependent increase in pSTAT5 median fluorescence intensity on γδ T cells (Figure 3B). In contrast, there was no detectable pSTAT5 on αβ T cells at any concentration tested.

[0212] Example 4: Treatment with γδ TCR-targeted low affinity IL-2_X inhibits Vδ2 + Strongly increases γδ T cells IL-2 enhances proliferation and effector function of γδ T cells. To determine the effect of γδ TCR-targeted IL-2_X on proliferation, PBMCs were isolated as described in Example 2, labeled with CellTrace Violet, and added at 300000 per well. Cells were treated with titrations of fusion proteins containing IL-2_X fused to the C-terminus of either anti-γδ TCR-1D7 or a non-targeted control VHH, starting with an initial concentration of 100 nM and titrated 1:5 across the plate in duplicate. Plates were incubated for 7 days at 37° C. / 5% CO2. Cells were labeled with the following fluorescently conjugated antibodies: CD3-BV785, γδ TCR-FITC, Vδ2-PE, and the viability dye propidium iodide for 30 min at 4° C. Cells were washed and analyzed by flow cytometry.

[0213] As shown in Figure 4A and Figure 4B, treatment with anti-γδ TCR-1D7 (cx11026)-targeted IL-2_X resulted in a dose-dependent increase in the proliferation of Vδ2 γδ T cells (Figure 4A). Furthermore, total CD3 + The percentage of γδ T cells in the T cell population was substantially increased by anti-γδ TCR-1D7-targeted IL-2_X treatment (Figure 4B). A non-targeted VHH (cx9452) fused to IL-2_X did not promote the proliferation of any Vδ2 γδ T cells, demonstrating target specificity.

[0214] Example 5: Development of γδ TCR binding VHH domains Single domain antibodies targeting human γδ TCR were generated by immunizing llamas with enriched γδ T cells and a heterodimeric knob-in-hole construct consisting of a human γ9 ectodomain cloned with a truncated effector knob Fc paired with a human δ2 ectodomain containing a truncated effector hole Fc. After the development of specific anti-γδ TCR antibody titers, llama peripheral blood mononuclear cells (PBMCs) were isolated from 500 mL of blood of 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 by PCR using cDNA as template and cloned into yeast surface display vectors as VHH-Fc-AGA2 fusion proteins. Fc was human IgG1 Fc (SEQ ID NO: 32) or, in some cases, a mutant IgG1 Fc with reduced effector function (e.g., Fc xELL; SEQ ID NO: 33).

[0215] A yeast library displaying VHH-Fc-AGA2 fusion proteins was enriched with a recombinant form of the γδTCR ECD by magnetic bead isolation followed by fluorescence-activated cell sorting (FACS). Sorted yeast were plated out and isolated colonies were picked into 96-well blocks and grown in medium to switch expression from surface-displayed VHH-Fc to secretion into the medium. Supernatants from 96-well yeast secretion cultures were applied to 293F cells transiently transfected with γδTCR (γδTCR positive) or non-transfected 293F cells (γδTCR negative), washed, treated with a fluorophore-labeled anti-human IgG1 Fc secondary antibody, and analyzed by 96-well flow cytometry.

[0216] A nucleic acid sequence encoding a VHH that binds to γδTCR positive cells but not to γδTCR negative cells was cloned in frame with the human Fc xELL coding region into a mammalian expression vector and expressed by transient transfection in HEK293 Freestyle cells (293F cells) or CHO cells using polyethyleneimine. After 3 to 7 days, the supernatant was collected, and the secreted recombinant protein was purified by protein A chromatography, and the concentration was calculated from the absorbance at 280 nm and the extinction coefficient.

[0217] The binding of 1D7, a γδ TCR-binding VHH, and its humanized form formatted as a monomeric VHH-hIgG1-Fc fusion protein using a non-dimerized human IgG1 Fc mutated region lacking the hinge Fc NNT, was assessed using fresh or frozen increased human Vδ2 + was assessed by flow cytometry on γδ T cells. + γδ T cells were expanded from PBMCs derived from healthy human donor peripheral blood leukopacks. Freshly isolated PBMCs were cultured at 1.0 × 10 in complete RPMI medium supplemented with 10% FBS, 5 μM zoledronate, and 500 IU / ml IL-2. 6 On day 3, half of the medium was removed and IL-2 was added at a final concentration of 500 IU / mL. For the remainder of the expansion, medium was replaced and IL-2 was added at a final concentration of 500 IU / ml every 2-3 days for a total of 2 weeks. Purity was assessed by flow cytometry and typically showed >80% Vδ2 +The γδ T cells were. Freshly expanded γδ T cells were plated in 96-well plates at 30000 cells per well or thawed expanded γδ T cells were plated in 96-well plates at 50000 cells per well in FACS buffer (PBS, 1% BSA, 0.1% NaN3, pH 7.4). Non-transfected HEK293F cells were used as γδ TCR negative control and plated at 30000 cells per well in a separate plate. Test polypeptides were then diluted 2-fold to a final concentration of 1000 nM and serial dilutions of 3-fold, 4-fold, and 5-fold were performed. FACS buffer without polypeptide was used as a secondary antibody only control. Polypeptide dilutions were added to an equal volume of cells and the assay plate was incubated at 4° C. for 30 minutes. After washing twice with 150 μL of FACS buffer per well, cells were resuspended in FACS buffer containing fluorescently labeled anti-human Fc antibodies diluted 1000-fold or 2000-fold to detect binding. The assay plate was incubated for 20 min at 4°C and washed once with 150 μL of FACS buffer per well. Binding to γδ T cells was determined by flow cytometry using an Intellicyt iQue Plus and anti-human A647 median fluorescence intensity was calculated using the on-board software. Data were plotted and analyzed using GraphPad Prism analysis software. Binding curves for the initial humanized 1D7 VHH fusion protein are shown in Figure 5A-5I. The binding curve for the improved humanized variant 1D7v158 is shown in Figure 5J, which has a more favorable pI profile and comparable binding affinity to the parent VHH. Additional humanized variants based on 1D7v158 were also generated. The binding curves for the parental 1D7v158 and additional mutants are shown in FIG. 5K.

[0218] Affinity (K D ) was determined from the flow cytometry binding data using a nonlinear fitting model and is shown in Table 2.

[0219] The amino acid sequences of 1D7 VHH and its humanized forms are provided in the table of certain sequences shown below. It is provided that the amino acid at residue 114 in any of the disclosed 1D7 VHH domains can be lysine (K), aspartic acid (D), glutamic acid (E), or arginine (R). For example, the parent 1D7 VHH (SEQ ID NO:2) contains lysine (K) at residue 114, hu1D7v39 VHH (SEQ ID NO:97) contains arginine (R) at residue 114, and hu1D7v158 (SEQ ID NO:158) contains glutamic acid (E) at residue 114. Thus, the residue at position 114 can be substituted with lysine, aspartic acid, glutamic acid, or arginine.

[0220] [Table 2]

[0221] Example 6: PBMCs expanded with γδ TCR-targeted IL-2 exhibit potent cytotoxicity across a broad panel of tumor cell lines The ability of PBMCs expanded with a γδ TCR-targeted IL-2 molecule (cx11026) to kill target cell lines derived from a variety of hematological and solid tumors was assessed. Briefly, PBMCs isolated from Leuko 75 were expanded with 1 nM cx11026 essentially as described in Example 7, and after a 2-week expansion protocol, 2.0×10 cells were cultured for killing assays. 6 Resuspended to 1 x 10 cells / ml. 6 pieces~2×10 6Target cells were washed with HBSS and labeled with Cyto-ID red for 6 min according to the kit instructions, after which the cells were resuspended in complete RPMI medium and plated at 100 μl / well (10,000 cells). Control wells were included with untreated PBMCs or with target cells only. Adherent cells were plated on flat bottoms and non-adherent cells were plated on Poly-L coated 96-well plates and the plates were incubated for 20 min at room temperature followed by 2 h at 37°C / 5% CO2 before adding effector cells. Effector cells at a 10:1 ratio to target cells were then added at 100,000 cells / well in a volume of 50 μL. Caspase 3 / 7-green was added at a final concentration of 1.25 μM at 50 μL per well. Cells were left at room temperature for 20 min and cell death was analyzed using an Incucyte cell imager. After 30 min of temperature equilibration, target cell killing was assessed by overlap of caspase 3 / 7-green and cyto-ID red every 1.5 h. After overlap masking for target and dead cells, data were plotted and analyzed using GraphPad Prism analysis software.

[0222] As shown in Figures 6A and 6B, treatment of PBMCs with a γδ TCR-targeted IL-2 molecule (1D7xIL-2_X) resulted in an expansion of Vγ9Vδ2 T cells and broad anti-tumor killing activity across a broad range of cell lines, including THP-1 acute monocytic leukemia, HT29 colon adenocarcinoma, Daudi Burkitt's lymphoma, NCI-H460 lung carcinoma, MM1S multiple myeloma, and A375 melanoma cell lines.

[0223] Example 7: Activity of γδTCRxCD20 bispecific molecules The binding activity of a bispecific γδ TCR molecule (cx11498) containing the anti-γδ VHH domain 1D7v9, an anti-CD20 VHH domain and a heterodimeric knob-in-hole Fc region that binds the γδ TCR and a representative target antigen CD20 (γδ TCRxCD20) was assessed by flow cytometry on CD20-expressing Raji target cells and Vγ9Vδ2 T cells. PBMCs and Raji cells were thawed and diluted to 1.0 × 10 in FACS buffer. 6 Cells were incubated with antibody for 30 min at 4°C, spun down and washed 4 times with FACS buffer. Cells were labeled with anti-human Fcγ-A647 (Jackson ImmunoResearch) and PBMCs were also labeled with anti-human γδTCR-FITC (Biolegend) in 50 μL FACS for 30 min at 4°C. Plates were washed twice with FACS buffer, resuspended in FACS buffer and read on an IQue flow cytometer. Mean fluorescence intensity of anti-human Fcγ-A647 was calculated using the on-board software and graphed using Graphpad Prism. γδTCRx antigen molecules bound in a titration-dependent manner to both antigen (CD20)-expressing Raji cells (FIG. 7A) and Vγ9Vδ2 T cells (FIG. 7B).

[0224] The Vγ9Vδ2 T cell mediated killing activity of the γδ TCRxCD20 molecule (cx11498) and a sequence analogue of the anti-CD20 antibody rituximab (Invivogen, hcd20-mab164-43-01) was assessed in a FACS-based killing assay using Raji cells and freshly isolated Vγ9Vδ2 T cells or expanded Vγ9Vδ2 T cells. Freshly isolated Vγ9Vδ2 T cells were isolated from thawed PBMCs washed with CTL thawing buffer in complete medium using the Stemcell EasySep Human Vγ9Vδ2 T cell isolation kit, cells were resuspended in 2% FBS / PBS, stained with Vδ2-PE antibody for 20 min, washed, resuspended in 2% FBS / PBS and sorted for Vδ2 T cells. +Selectively sort T cells and culture the sorted cells at 2 × 10 6 Expanded Vγ9Vδ2 T cells were prepared from thawed PBMCs by briefly washing the PBMCs with CTL thawing buffer in complete medium and resuspending at 1.0 × 10 cells / mL in flasks. 6 The flasks were then resuspended to 20 × 10 cells / mL and dosed with 1 nM of the monovalent γδ TCR-targeted IL-2 molecule 1D7xIL-2_X (cx11026), the flasks were incubated at 37 °C / 5% CO for 7 days, and on day 3 the cells were spun down, resuspended and re-dosed with 1 nM 1D7xIL-2_X, and after 7 days of expansion the cells were diluted to 20 × 10 6 Cells were resuspended at 10000 cells / ml, stained for V52-PE in complete medium for 30 min, washed, resuspended in 2%FBS / PBS+1 mM EDTA and run on a cell sorter to isolate pure Vy9V52 T cells. Raji cells were labeled with Far Red cell tracer (ThermoFisher) before plating 10000 cells per well in 50 μL and sorted Vy9V52 T cells (freshly isolated or expanded) were added in 50 μL at a 4:1 (effector:target) cell ratio and titrated 1:2 across the plate. 25 μL of test article (8x concentration of 5 nM final concentration in RPMI) or medium alone was added to the cells, 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: anti-human CD3-BV785, anti-human V52-PE, Apotracker-green, and PI in FACS buffer for 30 min at 4° C. Afterwards, plates were washed and analyzed by flow cytometry (Quanteon). The percentage of apoptotic cells was determined for labeled Raji target cells with Apotracker green.

[0225] As shown in Figures 7C and 7D, treatment with γδ TCRxCD20 polypeptide (cx11498) resulted in increased γδ T cell-mediated killing of Raji target cells by both freshly isolated (Figure 7C) and anti-Vγ9xIL2-X-expanded Vγ9Vδ2 T cells (Figure 7D). 1D7xIL-2_X-expanded Vγ9Vδ2 T cells exhibited superior killing activity compared to freshly isolated Vγ9Vδ2 T cells in the presence and absence of anti-CD20xVγ9-1D7v9. Furthermore, treatment with rituximab had little effect on Raji cell killing by freshly isolated Vγ9Vδ2 T cells, but increased the killing activity of 1D7xIL-2_X-expanded Vγ9Vδ2 T cells. This indicates that treatment with anti-1D7xIL-2_X enhances tumor cell killing activity by multiple mechanisms, including improved ADCC activity. These data indicate that additional targeting with either bispecific or conventional ADCC antibodies improves the target cell killing activity of Vy9V52 T cells.

[0226] Example 8: Activity of γδTCRxCD33 bispecific molecules The binding activity of a bispecific γδ TCR molecule (cx12083) containing the anti-γδ VHH domain 1D7v9, an anti-CD33 VHH domain and a heterodimeric knob-in-hole Fc region binding to the γδ TCR and representative target antigen CD33 (γδ TCRxCD33) was assessed by flow cytometry on target cells MOLM-13 and MV-411 cell lines expressing CD33, as well as on Vγ9Vδ2 T cells. A monospecific CD33 construct (CD33xUT; cx12056) containing an unrelated second VHH domain in place of the γδ TCR VHH domain was also tested. Briefly, frozen PBMCs (pretreated with zoledronate to expand γδ T cells) were thawed and washed twice with 1× CTL thawing reagent in assay medium. Cells were thawed at 1×10 in FACs buffer. 6Cells were resuspended to 100000 cells / mL and plated in 96-well plates (100000 cells / well). Test substances were added to each well in 50 μL at 4x the final starting concentration of 200 nM, titrated 1:4 overall. 50 μL of FACS buffer was added to a final total volume of 200 μL. Cells were incubated with test substances for 30 min at 4° C., washed three times, and stained with Vd2-PE, CD3-BV785, and anti-human A647 antibodies in FACS buffer for 30 min at 4° C. Cells were then washed twice with FACS buffer, resuspended in 70 μl FACS buffer, and read on a Novocyte. All molecules bound to CD33-expressing MOLM-13 (FIG. 8A) and MV4-11 (FIG. 8B) cells in a titration-dependent manner. However, only the bispecific γδTCRxCD33 molecule bound in a titration-dependent manner to Vγ9Vδ2 T cells (Figure 8C).

[0227] The Vy9V52 T cell-mediated killing activity of the γδTCRxCD33 molecule (cx12083) and the CD33xUT control molecule was assessed in a FACS-based killing assay using MOLM-13 and MV-411 target cells, as well as expanded Vy9V52 T cells. + γδ T cells were expanded from frozen PBMCs isolated from peripheral blood leukopacks of healthy human donors and cultured at 1.0 × 10 6 Cells were resuspended at 10 ... + PI - Purity was assessed by flow cytometry and was >95% Vδ2. +γδ T cells. MOLM-13 and MV-411 cells were removed from culture, washed once with complete RPMI, followed by 0.1% BSA / PBS buffer, and labeled with CellTrace Violet at 1:1000 dilution for 10 min at 37°C. Target cells were resuspended in complete RPMI medium and plated at 100 μL / well (10,000 cells) onto corning 96-well U-bottom plates. Sorted Vγ9Vδ2 T cells were added in a 5:1 effector to target cell ratio in a 50 μL volume. Treatment groups were plated in duplicate. Test article CD33x1D7v9 or non-targeting control CD33xUT was added to each plate at 4x the final concentration of 100 nM in 50 μL of complete RPMI. Cells were incubated overnight at 37°C / 5% CO2. The next day, cells were spun down and stained with the following components: anti-human CD3-BV785, anti-human V52-PE, Apotracker-green, and PI in 50 μL FACS buffer for 30 min at 4° C. Plates were then washed twice with FACS buffer, resuspended in 70 μL FACS buffer, and read on a Quanteon flow cytometer. The percentage of dead cells was determined for labeled MOLM-13 or MV-411 target cells with Apotracker green and PI.

[0228] As shown in Figures 8D and 8E, treatment with CD33x1D7v9 resulted in increased killing of MOLM-13 (Figure 8D) and MV-411 (Figure 8E) target cells by expanded Vy9V52 T cells. Ligation of CD33 to Vy9 was required for killing activity, as CD33xUT molecules lacking the γδ TCR binding domain did not result in increased killing compared to no antibody controls.

[0229] Example 9: Activity of γδTCRx5T4 bispecific molecules of different affinities The Vy9V52 T cell-mediated killing activity of bispecific γδ TCR molecules that contain the anti-5T4 VHH domain, the low affinity humanized anti-γδ VHH domain 1D7v9 (1D7v9x5T4) or the high affinity humanized anti-γδ VHH domain 1D7v158 (1D7v158x5T4), and the heterodimeric knob-in-hole Fc region, binding the γδ TCR and representative target antigen 5T4 (γδ TCRx5T4) was evaluated in a CellTiter-Glo™ 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 in complete RPMI (10000 cells / well) and allowed to adhere for 2 hours at 37°C. Frozen PBMCs (pretreated with zoledronate to enrich for γδ T cells) were thawed in complete RPMI medium and added to target cells at a 5:1 (effector:target) ratio. Treatment groups were plated in duplicate. Test substances were added at an initial concentration of 50 nM and titrated 1:3 across the plate with caspase-3 / 7 green reagent, which fluorescently labels nuclear DNA in cells undergoing apoptosis. Plates were incubated at 37°C for 22 hours. Assay plates were imaged periodically using an IncuCyte™. Target cell death was determined by measuring the total overlapping red / green target area. After 22 hours, supernatants were removed and remaining adherent (viable target) cells were gently washed with PBS before being analyzed using CellTiter-Glo™ 2.0, a luciferase-based reagent that produces bioluminescence in the presence of ATP (viable cells).

[0230] As shown in Figures 9A and 9B, both bispecific γδTCRx5T4 molecules induced γδT cell-mediated caspase-3 / 7 activation in A549 cells, but not in A375Δ5T4 cells. As shown in Figures 9C and 9D, target-dependent caspase activation induced by γδTCRx5T4 molecules correlated with target-dependent γδT cell-mediated cytotoxicity (assessed by cell survival). As can be seen from the EC50 values ​​shown in Figures 9A and 9B, the potency of each molecule was influenced by the affinity of the anti-γδTCR VHH domain, with molecules containing 1D7v158 exhibiting approximately two-fold higher potency than those containing 1D7v9. Thus, anti-γδTCR VHH domains with different affinities can be used to modulate the potency of the molecules.

[0231] Example 10: Anti-γδ TCR binding VHH 1D7 binds to cynomolgus monkey γδ T cells and effectively targets fusion molecules The ability of bivalent molecules comprising 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 derived from cynomolgus monkey PBMCs was tested. Briefly, expanded γδ T cells from cynomolgus donors were thawed, washed twice with CTL thawing reagent and 0.5×10 6Cells were resuspended at 100000 cells / mL. 200 μL of resuspended cells or 100,000 cells / well were added to a non-sterile 96-well U-bottom plate. Cells were spun down and 100 μL of FACS buffer was added to the wells. Antibodies were added to the U-bottom plate at 100 μL at 2x the final starting concentration of 100 nM and the plate was titrated 1:5 in FACs buffer. For wells not receiving test article, 100 μL / well of FACs buffer was added instead. Plates were 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 μL of FACS buffer for 30 minutes at 4° C. After incubation, plates were washed twice with FACS buffer, resuspended in 70 μL of FACS buffer, and binding to γδ T cells was determined by flow cytometry on Novocyte.

[0232] As shown in Figure 10A, both bivalent molecules bound specifically to the Vy9+ γδ T cell subset in a dose-dependent manner. The bivalent molecule containing the 1D7v9 VHH showed lower binding affinity to cynomolgus γδ T cells, and a lower affinity for this humanized variant was also observed for human γδ T cells (see Example 5 above).

[0233] The ability of monovalent γδTCR-targeted IL-2_X molecules (cx11026) containing anti-γδTCR binding VHH 1D7 to enhance IL2-signaling via STAT5 phosphorylation was tested across several cynomolgus PBMC donors. Briefly, PBMCs were isolated from three donors and left overnight in complete medium. Cells were labeled with the following fluorescently conjugated antibodies for 20 min at room temperature: non-competitive anti-Vγ9-FITC, anti-CD3-B421. After washing, 400000 PBMCs were seeded per well in a 96-well plate. Cells were treated with titrations of fusion protein (cx11026) containing IL-2_X fused to the C-terminus of anti-γδTCR-1D7, added at 4x the final starting concentration of 100 nM, titrated 1:5 across the plate in assay medium (50 μL). The final total volume was 200 μL / well. Plates were incubated for 20 min at 37°C / 5% CO2. Cells were fixed in 100 μL / well of BD Fixation / Permeabilization buffer for 45 min, then permeabilized with 100 μL / well of BD Permeabilization buffer III for 1 h and washed three times with FACS buffer. Cells were then stained overnight at 4°C with 50 μL / well of FACS-diluted pSTAT5 antibody. The next day, cells were washed twice with FACS buffer, resuspended in 70 μl of FACS buffer, and stained for Vγ9 + γδ T cells or Vγ9 - γδ - Levels of phosphorylated STAT5 ("pSTAT5") in αβ T cells were measured by flow cytometry on a Quanteon flow cytometer.

[0234] As shown in FIG. 10B, treatment with monovalent anti-γδTCR-1D7-targeted IL-2_X significantly reduced pSTAT5 across all three cynomolgus PBMC donor samples. + Vγ9 + This resulted in a dose-dependent increase in the proportion of γδT cells. There was no detectable pSTAT5 on αβT cells from any donor.

[0235] A monovalent γδ TCR-targeted IL-2_X molecule (cx11026) containing anti-γδ TCR binding VHH 1D7 binds Vγ9 + The ability to enhance γδ T cell proliferation was tested across several cynomolgus PBMC donors. Briefly, PBMCs were freshly isolated from fresh cynomolgus blood samples, labeled with CellTrace Violet, and added at 300,000 per well as described in Example 2. Fusion proteins containing IL-2_X fused to the C-terminus of either anti-γδ TCR-1D7 (cx11026) or a non-targeted control VHH (cx9452) were added in 50 μL at 4x the final starting concentration of 100 nM, all titrated 1:5 in assay medium. 50 μL medium was added to a final total volume of 200 μL / well, and the assay plate was incubated at 37° C. / 5% CO2 for 7 days. Cells were labeled with the following fluorescently conjugated antibodies: CD3-APC, Vγ9-FITC, DNAM1-PE, NKGD-APC / Cy7 and the viability dye propidium iodide for 30 minutes at 4° C. Cells were washed and analyzed by flow cytometry on a Quanteon flow cytometer.

[0236] Data from two representative donors are presented in Figures 10C-10F and show that treatment with anti-γδ TCR-1D7 (cx11026)-targeted IL-2_X resulted in a dose-dependent increase in the proliferation of Vγ9 γδ T cells (Figures 10C and 10E). Furthermore, total CD3 + The percentage of Vγ9γδ T cells in the T cell population was substantially increased by anti-γδ TCR-1D7-targeted IL-2_X treatment (FIGS. 10D and 10F). A non-targeted VHH (cx9452) fused to IL-2_X did not promote proliferation of Vγ9γδ T cells, demonstrating target specificity.

[0237] Taken together these studies show that the human γδTCR-binding VHH 1D7 and humanized versions specifically cross-react with cynomolgus γδ T cells and that these VHH domains effectively target fusion molecules (e.g., γδTCRxIL-2_X) to cynomolgus γδ T cells.

[0238] The present disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics of the present invention. The above-described embodiments are therefore to be considered in all respects as illustrative and not limiting of the present disclosure. The scope of the present disclosure is therefore defined by the appended claims, rather than the above detailed description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

[0239] [Table 3]

[0240] [Table 4] TIFF2025504363000006.tif243170TIFF2025504363000007.tif249170TIFF2025504363000008.tif248170TIFF202 5504363000009.tif248170TIFF2025504363000010.tif248170TIFF2025504363000011.tif249170TIFF20255043630 00012.tif254170TIFF2025504363000013.tif254170TIFF2025504363000014.tif252170TIFF2025504363000015.t if252170TIFF2025504363000016.tif254170TIFF2025504363000017.tif254170TIFF2025504363000018.tif180170

Claims

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

5.

2. At least one VHH domain comprises: (a) 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; (b) a CDR1 comprising the amino acid sequence of SEQ ID NO: 144, 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; (c) a CDR1 comprising the amino acid sequence of SEQ ID NO: 145, 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; (d) a CDR1 comprising the amino acid sequence of SEQ ID NO: 146, 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; (e) a CDR1 comprising the amino acid sequence of SEQ ID NO: 147, 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; (f) a CDR1 comprising the amino acid sequence of SEQ ID NO: 148, 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; (g) a CDR1 comprising the amino acid sequence of SEQ ID NO: 149, 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; (h) a CDR1 comprising the amino acid sequence of SEQ ID NO: 3, a CDR2 comprising the amino acid sequence of SEQ ID NO: 150, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 5; (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 3, a CDR2 comprising the amino acid sequence of SEQ ID NO: 151, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 5; (j) a CDR1 comprising the amino acid sequence of SEQ ID NO: 3, a CDR2 comprising the amino acid sequence of SEQ ID NO: 152, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 5; (k) a CDR1 comprising the amino acid sequence of SEQ ID NO: 3, a CDR2 comprising the amino acid sequence of SEQ ID NO: 153, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 5; (l) a CDR1 comprising the amino acid sequence of SEQ ID NO: 3, a CDR2 comprising the amino acid sequence of SEQ ID NO: 154, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 5; (m) a CDR1 comprising the amino acid sequence of SEQ ID NO: 3, a CDR2 comprising the amino acid sequence of SEQ ID NO: 155, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 5; or (n) CDR1 comprising the amino acid sequence of SEQ ID NO: 3, CDR2 comprising the amino acid sequence of SEQ ID NO: 156, and CDR3 comprising the amino acid sequence of SEQ ID NO: 5 The polypeptide of claim 1, comprising:

3. The polypeptide of claim 1, 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. 2. The polypeptide of claim 1, wherein at least one or each VHH domain is humanized.

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

6. The polypeptide of claim 1, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 158, SEQ ID NO: 99, or SEQ ID NO:

143.

7. A polypeptide described in claim 1, comprising one, two, or three VHH domains.

8. 2. The polypeptide of claim 1, comprising an immune cell-activating cytokine, 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 version thereof.

9. The polypeptide of claim 1 , comprising an Fc region.

10. The polypeptide of claim 9, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NO: 32 to SEQ ID NO:

70.

11. 10. The polypeptide of claim 9, comprising an immune cell-activating cytokine, 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 version thereof.

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

13. The polypeptide of claim 1, comprising at least one antigen-binding domain that binds to an antigen other than γδ TCR.

14. Lag3, TGFBR1, TGFBR2, Fas, TNFR2, 1-92-LFA-3, 5T4, α4 integrin, αV integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis Y, apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, complement C5, 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, CD5 1, CD52, CD56, CD64, CD70, CD71, CD73, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD 137, CD138, 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, ED B, 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 α (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, IL1 1, 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, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine-1-phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM 31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, TROP-2, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, or WISP-3.

15. 14. The polypeptide of claim 13, wherein at least one or each antigen-binding domain that binds to an antigen other than a γδ TCR is a VHH domain.

16. 14. The polypeptide of claim 13, wherein at least one or each antigen-binding domain that binds to an antigen other than a γδ TCR comprises a heavy chain variable region and a light chain variable region.

17. A complex comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is a polypeptide according to any one of claims 9 to 16, the first polypeptide comprising a first Fc region, and the second polypeptide comprising a second Fc region, wherein the first Fc region and the second Fc region are the same or different.

18. The complex of claim 17, 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.

19. 18. The conjugate of claim 17, wherein the first Fc region comprises a knob mutation and the second Fc region comprises a hole mutation.

20. 20. The conjugate of claim 19, wherein the first Fc region comprises a T366W mutation, the second Fc region comprises T366S, L368A, and Y407V mutations, and the second Fc region comprises a H435R or H435K mutation.

21. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

22. A pharmaceutical composition comprising the complex described in claim 17.

23. An isolated nucleic acid encoding a polypeptide according to any one of claims 1 to 16.

24. 24. A vector or host cell comprising the nucleic acid of claim 23.

25. A host cell expressing a polypeptide according to any one of claims 1 to 16.

26. 17. A method for producing a polypeptide according to any one of claims 1 to 16, comprising incubating a host cell expressing said polypeptide under conditions suitable for expression of said polypeptide.

27. 27. The method of claim 26, further comprising isolating the polypeptide.

28. A method for increasing proliferation of γδ T cells, the method comprising contacting T cells with a polypeptide according to any one of claims 1 to 16.

29. A method for increasing proliferation of γδT cells, comprising contacting T cells with the complex of claim 17.

30. A polypeptide described in any one of claims 1 to 16, for use in a method for treating cancer, comprising administering a pharmaceutically effective amount of the polypeptide to a subject with cancer.

31. The complex of claim 17, for use in a method for treating cancer, comprising administering a pharmaceutically effective amount of the polypeptide to a subject with cancer.

32. The cancers include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, gastrointestinal cancer, glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney or renal cancer, laryngeal cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, myeloma, neuroblastoma, oral 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, 31. The polypeptide of claim 30, wherein the polypeptide is selected from 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-dividing cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloblastic leukemia.

33. The cancer is selected from the group consisting of basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, gastrointestinal cancer, glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer or renal carcinoma, laryngeal cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, vulvar cancer, 32. The conjugate of claim 31 , wherein the lymphoma is selected from 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-dividing cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloblastic leukemia.

34. The polypeptide described in claim 30, wherein the polypeptide is administered together with an additional therapeutic agent.

35. The complex of claim 31, wherein the complex is administered together with an additional therapeutic agent.

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