NKp46-binding polypeptides and uses thereof

Abstract

Provided herein are VHH-containing polypeptides that bind to NKp46. 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 / 238,429, filed August 30, 2021, which is incorporated by reference in its entirety for all purposes.

[0002] [Incorporation by reference of sequence listing] This application incorporates by reference the Sequence Listing, entitled 01202-0029-00PCT_ST26, created on August 18, 2022, which was submitted in electronic format herewith and is 99.1 kilobytes in size.

[0003] The present invention relates to NKp46-binding polypeptides and methods of using the NKp46-binding polypeptides to modulate the biological activity of NKp46, including, but not limited to, methods of treating cancer and infectious diseases. In some embodiments, the NKp46-binding polypeptide is a fusion polypeptide comprising an NKp46-binding polypeptide and an immune cell-activating cytokine and / or a polypeptide that binds to an antigen other than NKp46. [Background technology]

[0004] NKp46, also known as CD335, LY94 homolog, or NCR1, is an activating cell surface receptor expressed on natural killer (NK) cells. NKp46 is part of the natural cytotoxicity receptor (NCR) family and functions as a receptor for stress ligands that are commonly presented on virus-infected, fungal, or cancer cells. Ligation and clustering of NKp46 drives activation signals through immunoreceptor tyrosine-based activation motifs (ITAMs), including the co-receptors FcεRI and CD3ζ, inducing interferon-γ expression and NK-mediated cytotoxicity. Expression of NKp46 is restricted to NK cells and not expressed on CD4+ or CD8+ T cells, B cells, monocytes, or granulocytes. The NKp46 gene is conserved from humans to cynomolgus monkeys, rats, and mice, and the expression pattern in these animals is restricted to NK cells, similar to humans. This expression pattern and species conservation make NKp46 an ideal NK-specific marker for NK-targeted therapeutics and also a potent NK activating receptor that drives NK-mediated cytotoxicity.

[0005] NK cells are important immune cells that can kill virus-infected and cancer cells without prior sensitization and enhance the adaptive immune responses of dendritic cells, T cells, and B cells through cytokine and chemokine signals (Non-Patent Document 1). NK cells have several mechanisms to recognize and kill target cells. Stress ligands mediate and activate NK cell degranulation through natural cytotoxicity receptors (NCRs) such as NKG2D, NKp30, NKp44, NKp46, and DNAM-1. Loss of MHC class I is an immune evasion strategy common to many viruses and is also observed in human cancers. NK cells can recognize and kill cells that do not express MHC class I by blocking inhibitory signals from KIR and NKG2A (Non-Patent Document 2). Antibody opsonization of viral or cancer antigens by antibodies agonizes CD16a on NK cells, inducing potent activation of NK cytotoxicity. Degranulation of NK cells releases not only cytotoxic proteins but also immune-stimulating cytokines such as interferon-γ and TNF-α, and immune-enhancing chemokines such as CCL3, CCL4, CCL5, XCL1, and XCL2 (Non-Patent Document 3 and Non-Patent Document 4). These secreted factors activate and recruit DCs, T cells, and B cells to orchestrate efficient adaptive immune responses.

[0006] A multitude of stimulatory and inhibitory signals modulate the overall activation state of NK cells, controlling the threshold and magnitude of the response. Ideal conditions for NK-mediated killing of virus-infected cells require support from cytokines such as interleukin-2 (IL-2) or interleukin-15 (IL-15). IL-2 and IL-15 are potent cytokines that stimulate the proliferation of T cells and NK cells through a common heterodimeric signaling receptor composed of CD122 and CD132. IL-2 can also be linked to a heterotrimeric high-affinity form of the receptor that includes CD25. In addition to enhancing NK cell survival and proliferation, IL-2 and IL-15 can prime NK cells to express effector molecules such as granzyme-B, perforin, and interferon-γ, which are released upon degranulation and act to destroy target cells. Pathological inflammation due to infection or cancer can render NK cells exhausted and ineffective. Stimulation with IL-2 or IL-15 can reactivate exhausted NK cells and overcome inhibitory immune signals. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Vidal et al. Curr Opin Virol 1(6):497-512 (2011) [Non-Patent Document 2] Raulet and Vance Nat Rev Immunol 6(7):520-531 (2006) [Non-Patent Document 3] Fauriat et al. Blood 115(11):2167-2176 (2010) [Non-Patent Document 4] Bottcher et al. Cell 172(5):1022-1037 (2018) Summary of the Invention [Problem to be solved by the invention]

[0008] There is a need for NKp46 binding polypeptides that can specifically target molecules, such as activating molecules, to NK cells, increasing the potency and selectivity of NK cell responses. [Means for solving the problem]

[0009] Provided herein are NKp46-binding polypeptides and methods of using the NKp46-binding polypeptides to treat, for example, cancer or infectious disease. In some embodiments, the NKp46-binding polypeptide comprises at least one VHH domain that binds to NKp46. In some embodiments, the NKp46-binding polypeptide comprises one or more additional binding domains and / or cytokine sequences.

[0010] Some embodiments are presented below.

[0011] Embodiment 1. A polypeptide comprising at least one VHH domain that binds to NKp46 and comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:17, a CDR2 comprising the amino acid sequence of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, and a CDR3 comprising the amino acid sequence of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27.

[0012] Embodiment 2. A polypeptide described in embodiment 1, wherein at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22.

[0013] Embodiment 3. A polypeptide described in embodiment 1, wherein at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22.

[0014] Embodiment 4. A polypeptide described in embodiment 1, wherein at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22.

[0015] Embodiment 5. A polypeptide described in embodiment 1, wherein at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22.

[0016] Embodiment 6. A polypeptide described in embodiment 1, wherein at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 23.

[0017] Embodiment 7. A polypeptide described in embodiment 1, wherein at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 24.

[0018] Embodiment 8. A polypeptide described in embodiment 1, wherein at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25.

[0019] Embodiment 9. A polypeptide described in embodiment 1, wherein at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 26.

[0020] Embodiment 10. A polypeptide described in embodiment 1, wherein at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 27.

[0021] Embodiment 11. A polypeptide according to any one of embodiments 1 to 10, wherein at least one or each VHH domain is humanised.

[0022] Embodiment 12. A polypeptide described in any one of embodiments 1 to 11, wherein at least one VHH domain comprises an amino acid sequence that is at least 85%, 90%, 95%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NO:1 to SEQ ID NO:16.

[0023] Embodiment 13. A polypeptide according to any one of embodiments 1 to 12, wherein at least one VHH domain comprises any one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 16.

[0024] Embodiment 14. A polypeptide according to any one of embodiments 1 to 13, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 15.

[0025] Embodiment 15. A polypeptide according to any one of embodiments 1 to 14, comprising two VHH domains.

[0026] Embodiment 16. A polypeptide according to any one of embodiments 1 to 14, comprising three VHH domains.

[0027] Embodiment 17. A polypeptide according to any one of embodiments 1 to 16, comprising an immune cell-activating cytokine or a functional portion thereof.

[0028] Embodiment 18. The polypeptide of embodiment 17, wherein the immune cell-activating cytokine is fused to the N-terminus or C-terminus of a VHH domain that binds to NKp46.

[0029] Embodiment 19. The polypeptide of embodiment 17 or 18, wherein the immune cell-activating cytokine is IL-2, IL-15, IL-7, IL-6, IL-12, IFNα, IFNβ, or IFNγ, or an attenuated or modified form thereof.

[0030] Embodiment 20. A polypeptide according to any one of embodiments 1 to 19, comprising at least one antigen-binding domain that binds to an antigen other than NKp46.

[0031] Embodiment 21. The polypeptide of embodiment 20, comprising at least one antigen-binding domain that binds to a tumor antigen.

[0032] Embodiment 22. 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, C D24, CD25, CD27, CD28, CD30, CD33, CD38, CD39, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD7 0, CD71, CD73, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166, C D172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), claudin 3, claudin 4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGF Rviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FAS, 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, NKG2A, 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, TGFβ receptor 1 (TGFBR1), TGFβ receptor 2 (TGFBR2), TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, 22. The polypeptide of embodiment 20 or 21, comprising at least one antigen-binding domain that binds to an antigen selected from 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, and WISP-3.

[0033] Embodiment 23. A polypeptide according to any one of embodiments 20 to 22, wherein at least one antigen-binding domain that binds to an antigen other than NKp46 is a VHH domain.

[0034] Embodiment 24 The polypeptide of embodiment 23, wherein each antigen-binding domain that binds to an antigen other than NKp46 is a VHH domain.

[0035] Embodiment 25. A polypeptide according to any one of embodiments 20 to 22, wherein at least one antigen-binding domain that binds to an antigen other than NKp46 comprises a heavy chain variable region and a light chain variable region.

[0036] Embodiment 26 The polypeptide of embodiment 25, wherein each antigen-binding domain that binds to an antigen other than NKp46 comprises a heavy chain variable region and a light chain variable region.

[0037] Embodiment 27. A polypeptide according to any one of embodiments 1 to 22, wherein each VHH domain of the polypeptide binds to NKp46.

[0038] Embodiment 28. The polypeptide of embodiment 27, wherein each VHH domain comprises the same CDR1, CDR2, and CDR3 amino acid sequences.

[0039] Embodiment 29. The polypeptide of embodiment 27, wherein each VHH domain comprises the same VHH sequence.

[0040] Embodiment 30. The polypeptide according to any one of embodiments 1 to 29, wherein the NKp46 is human NKp46.

[0041] Embodiment 31. The polypeptide of embodiment 30, wherein the human NKp46 comprises the sequence of SEQ ID NO:29.

[0042] Embodiment 32. A polypeptide according to any one of embodiments 1 to 31, comprising an Fc region.

[0043] Embodiment 33. The polypeptide according to embodiment 32, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NO:53 to SEQ ID NO:89.

[0044] Embodiment 34. The polypeptide of embodiment 32 or 33, which forms a dimer under physiological conditions.

[0045] Embodiment 35. A polypeptide according to any one of embodiments 32 to 34, comprising an immune cell-activating cytokine fused to the C-terminus of the Fc region.

[0046] Embodiment 36. A complex comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is a polypeptide described in any one of embodiments 1 to 35, the first polypeptide comprises a first Fc region, the second polypeptide comprises a second Fc region, and the first Fc region and the second Fc region are the same or different.

[0047] Embodiment 37. A complex described in embodiment 36, wherein the first or second polypeptide comprises at least one VHH domain that binds to NKp46, at least one immune cell-activating cytokine, and / or at least one antigen-binding domain that binds to an antigen other than NKp46.

[0048] Embodiment 38. The conjugate of embodiment 37, wherein when the antigen-binding domain that binds to an antigen other than NKp46 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region is fused to a heavy chain constant region comprising the first or second Fc region.

[0049] Embodiment 39. A complex described in embodiment 37 or 38, wherein the first or second polypeptide comprises an antigen-binding domain that binds to an antigen other than NKp46 selected from TGFβ receptor 1, TGFβ receptor 2, and NKG2A.

[0050] Embodiment 40. A complex according to any one of embodiments 37 to 39, wherein the first or second polypeptide comprises at least one binding domain that binds to a tumor antigen.

[0051] Embodiment 41. The first or second polypeptide is selected from the group consisting of 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, CD1 9, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD39, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD 56, CD64, CD70, CD71, CD73, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD13 8, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), claudin 3, claudin 4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR , EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FAS, 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, TMEM31, TNFα, T The complex according to any one of embodiments 37 to 40, comprising at least one binding domain that binds to an antigen selected from NFR, 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, and WISP-3.

[0052] Embodiment 42. A complex according to any one of embodiments 36 to 41, wherein the or each VHH domain of the second polypeptide is humanized.

[0053] Embodiment 43. A complex described in any one of embodiments 36 to 42, wherein the first Fc region comprises a knob mutation and the second Fc region comprises a hole mutation, or the first Fc region comprises a hole mutation and the second Fc region comprises a knob mutation.

[0054] Embodiment 44. The conjugate of embodiment 43, wherein the first Fc region comprises a T366W mutation and the second Fc region comprises T366S, L368A, and Y407V mutations, or the first Fc region comprises a hole mutation and the second Fc region comprises a knob mutation.

[0055] Embodiment 45 The conjugate of embodiment 44, wherein the Fc region comprises T366S, L368A, and Y407V mutations and comprises a H435R or H435K mutation.

[0056] Embodiment 46. A complex described in any one of embodiments 36 to 45, wherein the polypeptide is a dimer under physiological conditions or the complex is formed under physiological conditions.

[0057] Embodiment 47. An immune complex comprising a polypeptide according to any one of embodiments 1 to 35 or a complex according to any one of embodiments 36 to 46, and a cytotoxic substance.

[0058] Embodiment 48. The immunoconjugate of embodiment 47, 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.

[0059] Embodiment 49. The immune complex of embodiment 47 or 48, comprising a complex described in any one of embodiments 36 to 46, and the second polypeptide comprising at least one binding domain that binds to CD3, T cell receptor (TCR) α, TCR β, CD28, CD16, CD32A, CD64, CD89, or NKG2D.

[0060] Embodiment 50. A pharmaceutical composition comprising a polypeptide described in any one of embodiments 1 to 35, a complex described in any one of embodiments 36 to 46, or an immune complex described in any one of embodiments 47 to 49, and a pharma- ceutical acceptable carrier.

[0061] Embodiment 51. An isolated nucleic acid encoding a polypeptide according to any one of embodiments 1 to 35 or a complex according to any one of embodiments 36 to 46.

[0062] Embodiment 52. A vector comprising the nucleic acid described in embodiment 51.

[0063] Embodiment 53. A host cell comprising the nucleic acid of embodiment 51 or the vector of embodiment 52.

[0064] Embodiment 54. A host cell expressing a polypeptide according to any one of embodiments 1 to 35 or a complex according to any one of embodiments 36 to 46.

[0065] Embodiment 55. A method for producing a polypeptide described in any one of embodiments 1 to 35 or a complex described in any one of embodiments 36 to 46, comprising incubating a host cell described in embodiment 53 or 54 under conditions suitable for expression of the polypeptide or complex.

[0066] Embodiment 56 The method of embodiment 55, further comprising isolating the polypeptide or complex.

[0067] Embodiment 57. A method for increasing proliferation or activation of NK cells, comprising contacting NK cells with a polypeptide described in any one of embodiments 1 to 35 or a complex described in any one of embodiments 36 to 46.

[0068] Embodiment 58. The method of embodiment 57, wherein the NK cells are present in vitro.

[0069] Embodiment 59. The method of embodiment 57, wherein the NK cells are present in vivo.

[0070] Embodiment 60. A method for treating cancer, comprising administering to a subject with cancer or an infectious disease a pharma- ceutical effective amount of a polypeptide according to any one of embodiments 1 to 35, a complex according to any one of embodiments 36 to 46, an immunoconjugate according to any one of embodiments 47 to 49, or a pharmaceutical composition according to embodiment 50.

[0071] Embodiment 61. 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, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer or renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), melanoma, myeloma, neuroblastoma, oral cancer (lips, tongue, oral cavity, and pharynx), 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, 61. The method of embodiment 60, wherein the patient is selected from cancer of the internal organs, cancer of the vulva, 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), acute myeloid leukemia (AML), hairy cell leukemia, and chronic myeloblastic leukemia.

[0072] Embodiment 62 The method of embodiment 60 or 61, further comprising administering an additional therapeutic agent.

[0073] Embodiment 63 The method of embodiment 62, wherein the additional therapeutic agent is an anti-cancer agent.

[0074] Embodiment 64. The method of embodiment 63, 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.

[0075] Embodiment 65 The method of embodiment 64, wherein the additional therapeutic agent is an anti-cancer biologic.

[0076] Embodiment 66 The method of embodiment 64 or 65, wherein the anti-cancer biologic is an antibody that comprises a binding domain that binds to a tumor antigen.

[0077] Embodiment 67. A method for redirecting an NK cell-mediated cytotoxic response against cancer cells, comprising administering to a subject with cancer a pharma- ceutical effective amount of a polypeptide according to any one of embodiments 22-35, a complex according to any one of embodiments 36-46, an immunoconjugate according to any one of embodiments 47-49, or a pharmaceutical composition according to embodiment 50.

[0078] Embodiment 68. A method for treating an infectious disease, comprising administering to a subject with an infectious disease a pharma- ceutical effective amount of a polypeptide or complex described in any one of embodiments 1 to 46, an immunoconjugate described in any one of embodiments 47 to 49, or a pharmaceutical composition described in embodiment 50.

[0079] Embodiment 69. The method of embodiment 68, wherein the infectious disease is a bacterial infection, a viral infection, or a fungal infection.

[0080] Embodiment 70 The method of embodiment 68 or 69, further comprising administering an additional therapeutic agent.

[0081] Embodiment 71. The method of embodiment 70, wherein the additional therapeutic agent is an antibiotic, an antiviral, or an antifungal agent.

[0082] Embodiment 72. A method for redirecting a natural killer-mediated cytotoxic response against a pathogen, comprising administering to a subject with an infectious disease caused by the pathogen a pharmacologic effective amount of a polypeptide or complex described in any one of embodiments 1 to 46, an immunoconjugate described in any one of embodiments 47 to 49, or a pharmaceutical composition described in embodiment 50.

[0083] Embodiment 73. A method according to any one of embodiments 68 to 72, wherein the polypeptide, complex, or immune complex comprises at least one binding domain that binds to an antigen expressed by the pathogen. [Brief description of the drawings]

[0084] [Figure 1] Figures 1A-1J show binding of polypeptides comprising NKp46-binding VHH domains and Fc domains assessed by flow cytometry. Figures 1A and 1B, and 1I show binding to HEK-293F cells transfected with human NKp46. Figures 1C and 1D show binding to HEK-293F cells transfected with cynomolgus monkey NKp46. Figures 1E and 1F show binding to HEK-293F cells transfected with mouse NKp46. Binding to untransfected HEK-293F cells is shown in Figures 1G and 1H, and 1J. Figures 1I and 1J show binding of NKp46-targeting VHH domains formatted as polypeptides comprising bivalent VHH and homodimeric Fc. [Diagram 2]2A-2I show the effects of polypeptides comprising IL-2 variants fused to the C-terminus of heterodimeric Fc and an NKp46-binding VHH domain (hz5D7v12-Fc xELL-hole and hz5D7v12-Fc xELL-knob-mutant IL-2, or hz5D7v17-Fc xELL-hole and hz5D7v17-Fc xELL-knob-mutant IL-2), polypeptides comprising heterodimeric Fc and an NKp46-targeting VHH domain but without IL-2 (hz5D7v12-Fc xELL-hole and hz5D7v12-Fc xELL-knob-mutant IL-2), on CD56dimCD16+ NK cells (FIGS. 2A and 2B), CD56brightCD16- NK cells (FIGS. 2C and 2D), total NK cells (FIG. 2G), and CD4+ T cells (FIGS. 2E and 2F, and FIG. 2H). Figure 2 shows the activity, as measured by intracellular STAT5 phosphorylation levels, of polypeptides comprising IL-2 mutants and non-targeted VHHs fused to the C-terminus of a heterodimeric Fc (non-targeted VHH-Fc xELL-hole and non-targeted VHH-Fc xELL-knob-mutant IL-2), and wild-type recombinant IL-2. The specificity of the signaling activity is shown in NK cells (Figures 2A-D and 2G), with only wild-type recombinant IL-2 having activity on CD4+ (Figures 2E and 2F, and 2H) or CD8+ T cells (Figure 2I). [Diagram 3] FIG. 1 shows the ADCC activity of polypeptides comprising an IL-2 variant and an NKp46-targeted VHH domain fused to the C-terminus of a heterodimeric Fc (hz5D7v12-Fc xELL-hole and hz5D7v12-Fc xELL-knob-mutant IL-2), polypeptides comprising an IL-2 variant and a non-targeted VHH fused to the C-terminus of a heterodimeric Fc (non-targeted VHH-Fc xELL-hole and non-targeted VHH-Fc xELL-knob-mutant IL-2), and wild-type recombinant IL-2 in combination with a sub-optimal dose of cetuximab (0.2 nM) compared to the activity of an optimal dose of cetuximab (20 nM). [Figure 4]Figures 4A and 4B show the enhancement of NK cell ADCC activity against the Raji B cell lymphoma cell line by a polypeptide comprising an IL-2 mutant fused to the C-terminus of a heterodimeric Fc and an NKp46-targeting VHH domain (hz5D7v17-KiH Fc mutant IL-2) when combined with an anti-CD20 antibody that is a sequence analog of rituximab, or a defucosylated variant thereof. Figure 4A shows the titration of anti-CD20 antibody, and Figure 4B shows the effect of varying the NK cell to target cell (Raji) ratio in the presence of 1 nM anti-CD20 antibody. [Diagram 5] 5A-5D show the effect of a polypeptide (hz5D7v17-KiH) comprising an IL-2 variant fused to the C-terminus of a heterodimeric Fc and an NKp46-targeting VHH domain on multiple myeloma cell line NCI-H929 when combined with 5 nM of an anti-CD38 antibody that is a sequence analog of daratumumab or 5 nM of an anti-BCMA antibody (FIG. 5A); on the B cell lymphoma cell line Raji when combined with 10 nM of an anti-CD19 Fc modified antibody that is a sequence analog of tafasitamab or 1 nM of an anti-CD19 Fc modified antibody that is a sequence analog of obinutuzumab (FIG. 5B); on the lung cancer cell line A549 (FIG. 5C) or on the breast cancer cell line SKBR3 (FIG. 5D) when combined with 5 nM of the anti-EGFR antibody cetuximab or 50 nM of the anti-HER2 antibody trastuzumab. Figure 1 shows enhancement of NK cell ADCC activity by Fc mutants (IL-2), where various NK cell to target cell ratios were compared. cx11314 was used at 1 nM in all combination conditions. [Figure 6] Figures 6A-C show the activity of a single dose of a polypeptide comprising an NKp46-binding VHH domain, a heterodimeric Fc region, and an IL-2 variant fused to the C-terminus of the Fc region (hz5D7v12-KiH Fc mutant IL-2) at 0.3 mg / kg, 1 mg / kg, or 3 mg / kg in cynomolgus monkeys. An increase in subpopulations in the peripheral blood of animals is shown 10 days (Figure 6A) and 14 days (Figure 6B) after administration. Figure 4C shows an increase in granzyme B expression 4 and 10 days after administration. [Figure 7]Figure 1 shows antitumor efficacy as measured by changes in tumor volume induced by a polypeptide comprising an IL-2 mutant fused to the C-terminus of a heterodimeric Fc-containing sdAb and an NKp46-targeting VHH domain (hz5D7v17-KiH Fc mutant IL-2) in a subcutaneous Raji tumor xenograft mouse model. hz5D7v17-KiH Fc mutant IL-2 was administered three times weekly as indicated by the arrows and was administered intravenously as a single agent or in combination with a rituximab analog. Control groups included treatment with vehicle only or with the rituximab analog alone. [Figure 8] Figures 8A and 8B show the restoration of chemotherapy-induced NK cell defects by a polypeptide comprising an NKp46-binding VHH domain, a heterodimeric Fc, and an IL-2 mutant fused to the C-terminus of the Fc region (hz5D7v17-KiH Fc mutant IL-2). Figure 8A shows the effect on NK cell numbers in human peripheral blood as determined by flow cytometry after 3 days of treatment with dexamethasone alone or in combination with lenalidomide and / or hz5D7v17-KiH Fc mutant IL-2. Figure 8B shows the ADCC activity of NK cells pretreated with chemotherapy drugs (dexamethasone and lenalidomide) alone or in combination with hz5D7v17-KiH Fc mutant IL-2 against multiple myeloma target cell lines (MM1S) when combined with a sequence analog of daratumumab (anti-hCD38-hIgG1). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0085] Embodiments provided herein relate to NKp46 binding polypeptides and their uses in various methods of treating, for example, cancer or infectious disease.

[0086] Provided herein is an NKp46 binding polypeptide. In some embodiments, the NKp46 binding polypeptide comprises at least one VHH that binds to NKp46. In some embodiments, the NKp46 binding polypeptide comprises an engineered cytokine for NK targeting cytokine activity. In some embodiments, the NKp46 binding polypeptide also binds to another antigen, e.g., comprises a VHH domain that binds to another antigen. In some such embodiments, the NKp46 binding polypeptide is bispecific. This bispecific NKp46 binding polypeptide can redirect NK-mediated cytotoxicity to cells expressing the other antigen targeted by the polypeptide. In some embodiments, the NKp46 binding polypeptide is a trifunctional polypeptide that binds to NKp46 and another antigen, and comprises an engineered cytokine. This trifunctional polypeptide can focus cytokine activity on NK cells, while a second targeting domain targets the polypeptide to a specific cell type, e.g., cancer cells.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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 an unknown characteristic. 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 a disorder of interest (e.g., a particular cancer). 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.

[0095] 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 some degree of inhibition of disease progression, including slowing and complete halt, reduction in the number of disease episodes and / or symptoms, reduction in lesion size, inhibition (i.e., reduction, slowing, or complete halt) of disease cell invasion into adjacent peripheral organs and / or tissues, inhibition (i.e., reduction, slowing, or complete halt) of disease spread, some degree of relief of one or more symptoms associated with the disorder, disease-free presentation after treatment, such as increased length of progression-free survival, increased overall survival, higher response rate, and / or reduced 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".

[0096] 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.

[0097] 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. This definition includes 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.

[0098] As used herein, "NKp46" refers to any naturally occurring mature NKp46 resulting from processing of an NKp46 precursor in a cell. The term includes NKp46 from any vertebrate origin, including mammals such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. The term also includes naturally occurring variants of NKp46, such as splice variants or allelic variants. A non-limiting exemplary human NKp46 amino acid sequence is shown, for example, in UniProt Accession No. O76036. See SEQ ID NO: 29. A non-limiting exemplary mature human NKp46 sequence can be amino acids 22-304 of SEQ ID NO: 29. In some embodiments, NKp46 is expressed on NK cells, but not on CD4+ or CD8+ T cells, B cells, monocytes, or granulocytes. NKp46 may play a role in activating NK cells and inducing effector function.

[0099] The term "natural killer cell-mediated cytotoxicity" or "NK-mediated cytotoxicity" refers to the killing of target cells by the release of cytotoxic molecules from NK cells. These cytotoxic molecules, such as granzymes and perforins, may be stored in secretory lysosomes (also known as lytic granules) that are released by exocytosis from NK cells upon interaction with target cells. Exemplary endogenous targets of NK cells are virus-infected or tumorigenic cells. Exocytosis of secretory lysosomes by NK cells is regulated to avoid indiscriminate cytotoxicity.

[0100] As used herein, "redirected NK-mediated cytotoxicity" refers to cytotoxicity of a target cell by an NK cell, where the target cell is not endogenously targeted by the NK cell. In other words, redirected NK-mediated cytotoxicity refers to NK cytotoxicity directed to a cell that is not normally a target of an NK cell. Redirected NK-mediated cytotoxicity may also refer to a greater cytotoxicity by an NK cell against a target cell compared to an endogenous NK response against the target cell. Redirected NK-mediated cytotoxicity may be mediated by an agent capable of redirecting an NK cell to a target cell, such as a polypeptide comprising at least one VHH domain that binds to NKp46 and at least one VHH that binds to an antigen on the target cell. Thus, a polypeptide comprising at least one VHH domain that binds to NKp46 and at least one VHH that binds to an antigen on the target cell can be used to direct an NK cell to a target cell and stimulate NK-mediated cytotoxicity against the target cell.

[0101] As used herein, a "cytokine" is a small, non-antibody protein released by a cell that mediates an effect on another cell. As used herein, an "engineered cytokine" refers to a cytokine that has been altered from the native cytokine amino acid sequence to have unique properties. For example, an engineered cytokine can be an attenuated cytokine. As used herein, an "attenuated cytokine" is a cytokine that has a reduced affinity for its receptor and requires targeting to its receptor for activity. Exemplary cytokines include IL-2 and IL-5.

[0102] As used herein, "NKp46 binding polypeptide" and "NKp46 targeting polypeptide" are used interchangeably to refer to a polypeptide that comprises a binding domain that binds, e.g., specifically binds, to NKp46.

[0103] 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 the NKp46 epitope 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 NKp46 epitopes or non-NKp46 epitopes. 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.

[0104] The term "avoid" or "avoiding" 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 "avoid" refers to the decrease, reduction, or cessation of an activity, function, and / or amount compared to a reference. In some embodiments, "reduce" or "avoid" refers to the ability to cause an overall reduction of 10% or more. In some embodiments, "reduce" or "avoid" refers to the ability to cause an overall reduction of 50% or more. In some embodiments, "reduce" or "avoiding" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more. In some embodiments, the amount is abrogated or reduced over a period of time relative to a control over the same period of time.

[0105] 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.

[0106] 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.

[0107] 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).

[0108] The term "antibody" is used in the broadest sense and includes a variety of 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, heavy chain constant domains (C H 1, hinge, C H 2, and C H 3, including C H 1 is typically paired with a light chain constant domain CL, while C H 3 and / or hinge mediates dimerization) and Fc region (hinge, C H 2, and C H 3, including C H3 and / or hinge mediate dimerization).

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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 glycines and optionally serines. In some embodiments, the linker comprises Gly-Gly-Gly-Gly (SEQ ID NO: 45), Gly-Gly-Ser-Gly-Gly-Ser (SEQ ID NO: 46), and / or Gly-Gly-Ser-Ser-Gly-Ser (SEQ ID NO: 47). In some embodiments, when the 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).

[0114] 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.

[0115] The term "CDR" refers to a complementarity determining region defined according to at least one specific manner to one of skill 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. In some embodiments, the CDRs are defined according to the AbM definition.

[0116] 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.

[0117] As used herein, an "Fc region" refers to a H 2 and C H In some embodiments, the Fc region refers to a portion of the heavy chain constant region comprising the hinge, C H 2, and C H In some embodiments, the Fc region does not include a hinge. In various embodiments, when the Fc region includes a hinge, the hinge and / or C H C3 mediates dimerization between two Fc-containing polypeptides. In various embodiments, when the Fc region does not contain a hinge, C H 3 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.

[0118] 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.

[0119] "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.

[0120] 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:

[0121] In some embodiments, the K of the antigen binding molecule dis 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, K d is K d(見かけ) It is.

[0122] 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 NK cell proliferation), induction or increase in cell activation (such as NK cell activation), and induction or increase in cytokine expression.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] "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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] "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.)).

[0131] 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%.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] [Table 1]

[0136] 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.

[0137] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0138] 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.

[0139] "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.

[0140] 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".

[0141] 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.

[0142] As used herein, "disease" or "disorder" refers to a condition for which treatment is needed and / or desired.

[0143] 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. Included in this definition are benign and malignant cancers, blood cancers such as leukemia, lymphoma, and multiple myeloma, polyps, hyperplasias, and occult tumors or micrometastases.

[0144] 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, renal or kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, pulmonary fibrosis ... Cancer of the lung, 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 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, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorder (PTLD), and abnormal blood vessel growth associated with nematoses, edema (such as edema associated with brain tumors), and Meigs syndrome.

[0145] 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.

[0146] 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), NK 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.

[0147] The term "infectious disease", as used herein, refers to a disease caused by a pathogenic virus, bacteria, or fungus.

[0148] 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.

[0149] 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.

[0150] 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, arrest of disease or disease progression, arrest 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.

[0151] "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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] "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.

[0157] 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.

[0158] 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.

[0159] "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.

[0160] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] Exemplary NKp46-Binding Polypeptides NKp46 binding polypeptides are provided herein. In various embodiments, the NKp46 binding polypeptides comprise at least one VHH domain that binds to NKp46. In some embodiments, the NKp46 binding polypeptides inhibit binding of NKp46 to a viral hemagglutinin protein. In some embodiments, the NKp46 binding polypeptides provided herein comprise one, two, three, four, five, six, seven, or eight VHH domains that bind to NKp46. In some embodiments, the NKp46 binding polypeptides provided herein comprise one, two, three, or four VHH domains that bind to NKp46. Such NKp46 binding polypeptides may comprise one or more additional VHH domains that bind to one or more target proteins other than NKp46 and / or may comprise one or more additional polypeptide sequences, such as cytokine sequences.

[0168] In some embodiments, the NKp46-binding polypeptide comprises at least one VHH domain that binds to NKp46 and an Fc region. In some embodiments, the NKp46-binding polypeptide provided herein comprises one, two, three, or four VHH domains that bind to NKp46 and an Fc region. In some embodiments, the Fc region mediates dimerization of the NKp46-binding polypeptide under physiological conditions, such that the dimers are formed, thereby doubling the number of NKp46-binding sites. For example, an NKp46-binding polypeptide comprising three VHH domains that bind to NKp46 and an Fc region is trivalent as a monomer, but under physiological conditions, the Fc region can mediate dimerization, such that the NKp46-binding polypeptide exists as a hexavalent dimer under such conditions.

[0169] In some embodiments, an NKp46-binding polypeptide comprises at least two VHH domains, where a first VHH domain binds to a first epitope of NKp46 and a second VHH domain binds to a second epitope of NKp46. When an NKp46-binding polypeptide comprises a VHH domain that binds to a first epitope of NKp46 and a VHH domain that binds to a second epitope of NKp46, the NKp46-binding polypeptide may be referred to as "dual epitopic" or "dual specific."

[0170] Non-limiting exemplary NKp46 binding polypeptides are shown in Table 2. Sequences of designated single domain antibodies are provided in certain sequence tables herein.

[0171] [Table 2]

[0172] In various embodiments, the VHH domain that binds NKp46 comprises a CDR1 sequence of SEQ ID NO: 17, a CDR2 sequence selected from SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, and a CDR3 sequence selected from SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27. In various embodiments, the VHH domain that binds NKp46 comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence selected from SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 22; SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 22; SEQ ID NO: 17, SEQ ID NO: 20, and SEQ ID NO: 22; SEQ ID NO: 17, SEQ ID NO: 21, and SEQ ID NO: 22; SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 23; SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 24; SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 25; SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 26; and SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 27. In various embodiments, the VHH domain is humanized.

[0173] In some embodiments, a VHH domain that binds NKp46 comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. In some embodiments, a VHH domain that binds NKp46 comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. In some embodiments, the VHH domain that binds to NKp46 comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, wherein the VHH domain comprises a K125D, K125E, or K125R mutation.

[0174] In various embodiments, the NKp46 binding polypeptide comprises one, two, three, or four VHH domains that bind to NKp46.

[0175] In various embodiments, the NKp46 binding polypeptide comprises at least one VHH domain that binds to NKp46 and at least one antigen binding domain that binds to an antigen other than NKp46. In some embodiments, the at least one antigen binding domain that binds to an antigen other than NKp46 is a VHH domain. For example, in some embodiments, the at least one VHH domain that binds to an antigen other than NKp46 binds to a T cell antigen, a natural killer cell antigen that is not NKp46, or a tumor antigen.

[0176] In some such embodiments, the NKp46 binding polypeptide may be referred to as a multispecific antibody. Multispecificity means that the NKp46 binding polypeptide can bind to one or more other antigens in addition to NKp46.

[0177] In some embodiments, an NKp46-binding polypeptide may mediate more than one biological function, where one biological function is binding to NKp46. In some embodiments, an NKp46-binding polypeptide is bifunctional (having two functions) or trifunctional (having three functions). For example, as described below, an NKp46-binding polypeptide may bind to another antigen (such as a tumor antigen) and also have cytokine activity.

[0178] In some embodiments, the NKp46 binding polypeptide comprises at least one binding domain that binds to a cancer cell. In some embodiments, the NKp46 binding polypeptide comprises at least one binding domain that binds to a tumor antigen. In some embodiments, the NKp46 binding polypeptide comprises at least one binding domain that binds to a tumor antigen. In some embodiments, the NKp46 binding polypeptide comprises at least one binding domain that binds to a tumor antigen. In some embodiments, the NKp46 binding polypeptide comprises at least one binding domain that binds to a tumor antigen. In some embodiments, the NKp46 binding polypeptide comprises at least one binding domain that binds to a tumor antigen. 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, CD13 7, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), claudin 3, claudin 4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, 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, FAS, 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, NKG2A, 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, TGFβ receptor 1 (TGFBR1), TGFβ receptor 2 (TGFBR2), TIGIT, TIM-3, TLR2, TLR In some embodiments, the NKp46 binding polypeptide comprises at least one binding domain that binds to a virally infected cell expressing a viral protein on the cell surface.

[0179] In some embodiments, a binding domain contained in an NKp46 binding polypeptide that binds to a cell that is not an NK cell may be referred to as a "secondary targeting domain." In this manner, the secondary targeting domain can target the NKp46 binding polypeptide to a cell of a subject and redirect NK-mediated cytotoxicity to the cell expressing an antigen capable of binding to the secondary targeting domain. For example, the secondary targeting domain of an NKp46 binding polypeptide may be an antibody against a tumor antigen, and binding of the NKp46 binding polypeptide to a cancer cell expressing the tumor antigen redirects NK-mediated cytotoxicity to the cancer cell. In some embodiments, the secondary targeting domain is a VHH, a single domain antibody, a scFv, a Fab, or any other type of antibody. In some embodiments, the secondary targeting domain is not an antibody. In some embodiments, the secondary targeting domain is a natural cognate binding partner, an engineered extracellular binding partner, an Anticalin (engineered lipocalin), a Darpin, a Fynomer, a Centyrin (engineered fibronectin type III domain), a cystine knot domain, an Affilin, an Affibody, or an engineered C H There are three domains.

[0180] In some embodiments, the NKp46 binding polypeptide comprises a binding domain, such as a VHH domain, that binds to TGFβ receptor 1, TGFβ receptor 2, or NKG2A.

[0181] In some embodiments, the NKp46 binding polypeptide comprises a functional domain that is not intended for secondary targeting. This functional domain can serve a variety of purposes. In some embodiments, the NKp46 binding polypeptide comprises a cytokine or a functional portion thereof. In some embodiments, the cytokine is an engineered cytokine. In some embodiments, the engineered cytokine is an attenuated cytokine.

[0182] IL-2 and IL-5 are exemplary cytokines that may be included in the NKp46 binding polypeptide. IL-2 and IL-5 can stimulate NK cell proliferation and prime NK cells to express effector molecules such as granzyme-B, perforin, and interferon-γ. IL-2 and IL-5 can also reactivate NK cells and overcome immunosuppressive signals. In these methods, IL-2 and IL-5 may play a role in enhancing the NK response against cells targeted by redirected NK-mediated cytotoxicity. In some embodiments, the NKp46 binding polypeptide comprises all or a portion of IL-2 or IL-5. In some embodiments, the NKp46 binding polypeptide comprises all or a portion of attenuated IL-2 or IL-5.

[0183] In some embodiments, the NKp46 binding polypeptide comprises (i) a secondary targeting domain that targets a tumor antigen expressed by a cancer cell, and (ii) a cytokine or a functional portion thereof.

[0184] In some embodiments, the NKp46 binding polypeptide comprises at least one VHH domain described herein fused to an Fc region. In some embodiments, the Fc region has a sequence selected from SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89. In some such embodiments, the Fc region further comprises a C-terminal lysine. In some embodiments, the C-terminal amino acid of the Fc region is an amino acid other than lysine.

[0185] In some embodiments, the VHH domain that binds NKp46 is 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 immune responses 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.

[0186] 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).

[0187] 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".

[0188] In various embodiments, the Fc region comprised in the NKp46 binding polypeptide is a human Fc region or is derived from a human Fc region.

[0189] In some embodiments, the Fc region included in the NKp46 binding polypeptide is derived from a human Fc region and contains 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.

[0190] In some embodiments, the Fc region included in the NKp46 binding polypeptide is derived from a human Fc region and contains 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.

[0191] In some embodiments, the Fc region included in the NKp46 binding polypeptide is derived from a human Fc region and includes mutations designed for heterodimerization, referred to herein as "knob" and "hole." As used herein, "knob-in-hole" and "KiH" refer to such heterodimeric Fc regions. In some embodiments, the "knob" Fc region includes the mutation T366W. In some embodiments, the "hole" Fc region includes the mutations T366S, L368A, and Y407V. In some embodiments, the Fc region used for heterodimerization includes 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 includes 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.

[0192] Non-limiting exemplary Fc regions that can be used in NKp46-binding polypeptides include Fc regions comprising the amino acid sequences of SEQ ID NO:53 to SEQ ID NO:89.

[0193] Exemplary Activities of NKp46 Binding Polypeptides In various embodiments, the NKp46 binding polypeptides provided herein stimulate NK cells in vitro and / or in vivo. In vitro and / or in vivo NK cell stimulation or activity may, in some embodiments, be determined using the methods provided in the Examples herein.

[0194] In some embodiments, the NKp46 binding polypeptides provided herein comprise an antigen binding domain that binds to an immune cell activating cytokine and / or an antigen other than NKp46 and stimulates NK cells. In some embodiments, the NK cell stimulating activity of the antigen binding domain that binds to an immune cell activating cytokine and / or an antigen other than NKp46 is increased over that when used alone and / or is more specifically targeted to cytotoxic NK cells when fused to an NKp46 binding VHH. In some embodiments, the toxicity of the antigen binding domain that binds to an immune cell activating cytokine and / or an antigen other than NKp46 is reduced by specifically targeting NK cells.

[0195] In some embodiments, immune cell activating cytokines provided herein and / or NKp46 binding polypeptides comprising an antigen binding domain that binds to an antigen other than NKp46 increase NK cell proliferation in vitro and / or in vivo.

[0196] In some embodiments, the NKp46 binding polypeptides provided herein comprise an NKp46 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, the NKp46 binding polypeptides comprising the immune cell activating cytokine stimulate the activation and proliferation of NK cells in vivo. In some embodiments, the NKp46 binding polypeptides comprising the immune cell activating cytokine are used in methods of treating cancer or infectious disease.

[0197] The increase in proliferation of activated NK cells can be determined by any method in the art. A non-limiting exemplary assay is as follows: NK cells can be isolated from one or more healthy human donors and / or one or more human donors with a particular disease or disorder. The NK cells are stained and then contacted with a polypeptide comprising a cytokine, e.g., an NKp46-binding polypeptide comprising a cytokine, and then analyzed by FACS. Loss of staining indicates proliferation. In some embodiments, the increase in proliferation of NK cells is determined as an average from a series of experiments or pooled NK cells, e.g., by measuring the proliferation of NK cells isolated from different human donors. In some embodiments, the increase in proliferation of NK cells is determined as an average from experiments performed using NK cells from at least 5 or at least 10 different healthy donors, or from a pool of NK cells from at least 5 or at least 10 different healthy donors. In some embodiments, the increase in NK cell proliferation is determined as an average from experiments performed using NK cells from at least 5 or at least 10 different donors with a particular disease or disorder, or from a pool of NK cells from at least 5 or at least 10 different donors with a particular disease or disorder.

[0198] Polypeptide Expression and Production Nucleic acid molecules are provided that include a polynucleotide encoding a NKp46 binding polypeptide. In some embodiments, the nucleic acid molecule can also encode a leader sequence that directs secretion of the NKp46 binding polypeptide, and the leader sequence is typically cleaved so that it is not present in the secreted polypeptide. The leader sequence can be the native heavy chain (or VHH) leader sequence, or can be another heterologous leader sequence.

[0199] 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.

[0200] Vectors are provided that contain nucleic acids encoding the NKp46 binding polypeptides described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, vectors are selected that are optimized for expression of the polypeptide in a desired cell type, such as CHO cells or CHO-derived cells, or NSO cells. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0201] In some embodiments, NKp46 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 to express 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, NKp46 binding polypeptides may be expressed in yeast. See, for example, U.S. 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.

[0202] 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.

[0203] Also provided are host cells comprising any of the nucleic acids or vectors described herein. In some embodiments, host cells are provided that express the NKp46-binding polypeptides described herein. The NKp46-binding polypeptides expressed in the host cells can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices 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 NKp46-binding polypeptides that include 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.

[0204] In some embodiments, the NKp46 binding polypeptide is 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).

[0205] In some embodiments, an NKp46 binding polypeptide produced by the above method is provided. In some embodiments, the NKp46 binding polypeptide is produced in a host cell. In some embodiments, the NKp46 binding polypeptide is produced in a cell-free system. In some embodiments, the NKp46 binding polypeptide is purified. In some embodiments, a cell culture medium comprising the NKp46 binding polypeptide is provided.

[0206] In some embodiments, a composition is provided that comprises an antibody produced by the above method. In some embodiments, the composition comprises an NKp46 binding polypeptide produced in a host cell. In some embodiments, the composition comprises an NKp46 binding polypeptide produced in a cell-free system. In some embodiments, the composition comprises a purified NKp46 binding polypeptide.

[0207] Exemplary Methods of Treating Disease Using NKp46 Binding Polypeptides In some embodiments, a method of treating a disease in an individual is provided, comprising administering an NKp46 binding polypeptide. Such diseases include any disease that would benefit from increased proliferation and activation of NK cells. In some embodiments, a method of treating cancer or infectious disease in an individual is provided. In some embodiments, a method of increasing proliferation of NK cells in an individual is provided, comprising administering an NKp46 binding polypeptide. In some embodiments, a method of enhancing ADCC activity of a therapeutic antibody in an individual is provided, comprising administering an NKp46 binding polypeptide in combination with the therapeutic antibody. In some embodiments, a method of enhancing cytotoxicity of NK cells in an individual is provided, comprising administering an NKp46 binding polypeptide alone or in combination with a therapeutic antibody. In some embodiments, a method of overcoming chemotherapy suppression of NK cell activity in an individual is provided, comprising administering an NKp46 binding polypeptide before, during, or after treatment with a chemotherapeutic agent. In some embodiments, methods are provided for enhancing ADCC activity of a therapeutic antibody in an individual undergoing chemotherapy comprising administering an NKp46 binding polypeptide in combination with the therapeutic antibody before, during, or after treatment with a chemotherapeutic agent.

[0208] The method includes administering to the individual an effective amount of an NKp46 binding polypeptide provided herein.

[0209] In some embodiments, the NKp46 binding polypeptide is used to redirect NK-mediated cytotoxicity. In some such embodiments, the NKp46 binding polypeptide also comprises a binding domain that binds to a cytotoxic T cell or another NK cell antigen. In some such embodiments, the binding domain binds to CD3, T cell receptor (TCR) alpha, TCR beta, CD28, CD16, CD32A, CD64, CD89, or NKG2D. The binding domain may in some embodiments be a VHH domain, or an antibody binding domain comprising a heavy chain variable region and a light chain variable region, such as a VH / VL, scFv, Fab fragment, etc.

[0210] In some such embodiments, the NKp46 binding polypeptide also comprises a binding domain that binds to a cancer cell. The binding domain that binds to a cancer cell may be referred to as a "targeting domain" or a "secondary targeting domain." In some embodiments, the binding domain that binds to a cancer cell redirects NK-mediated cytotoxicity to the cancer cell.

[0211] In some embodiments, the NKp46 binding polypeptide is linked to a cytokine. In some embodiments, the cytokine is IL-2 or IL-5.

[0212] In some embodiments, the NKp46 binding polypeptide is linked to a cytotoxic agent to form an immunoconjugate. A variety of cytotoxic agents for use in immunoconjugates are known in the art and include, but are not limited to, calicheamicins, auristatins, dolastatins, tubulysins, maytansinoids, cryptophycins, duocarmycins, esperamicins, pyrrolobenzodiazepines, and enediyne antibiotics.

[0213] Non-limiting exemplary cancers that can be treated using the NKp46 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, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, renal 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, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, 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, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloblastic leukemia.

[0214] The NKp46 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, and the like. In some embodiments, an effective dose of the NKp46 binding polypeptide is administered to the subject one or more times. In some embodiments, an effective dose of the NKp46 binding polypeptide is administered to the subject daily, twice weekly, weekly, biweekly, monthly, and the like. An effective dose of the NKp46 binding polypeptide is administered to the subject at least once. In some embodiments, an effective dose of the NKp46 binding polypeptide may be administered multiple times, including multiple times over at least one month, at least six months, or at least one year.

[0215] In some embodiments, the pharmaceutical composition is administered in an amount effective to treat (including prevent) cancer or infectious disease, enhance the cytotoxic potential of NK cells, increase NK cell proliferation or activation, and / or overcome chemotherapeutic suppression of NK cell activity. 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.

[0216] In some embodiments, the NKp46 binding polypeptide may be administered in vivo by various routes, including but not limited to, intravenous, intraarterial, parenteral, intraperitoneal, or subcutaneous. Depending on the intended use, an appropriate formulation and administration route may be selected.

[0217] In some embodiments, therapeutic treatment using NKp46 binding polypeptides is achieved by increasing the proliferation and / or activation of NK cells and / or by contacting NK cells with cancer cells. In some embodiments, increasing the proliferation and / or activation of NK cells inhibits the growth of cancer. In some embodiments, therapeutic treatment using NKp46 binding polypeptides is achieved by increasing the proliferation and / or activation of NK cells. In some embodiments, therapeutic treatment using NKp46 binding polypeptides is achieved by increasing the cytotoxic capacity of NK cells.

[0218] Pharmaceutical Compositions In some embodiments, compositions comprising NKp46 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, 7th ed. (2004)). 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.

[0219] In some embodiments, the pharmaceutical composition comprises the NKp46 binding polypeptide at a concentration of at least 10 mg / mL.

[0220] Combination therapy The NKp46 binding polypeptide may be administered alone or in combination with other therapeutic modalities, such as other anti-cancer agents. The NKp46 binding polypeptide may be provided before, substantially simultaneously with, or after (i.e., simultaneously or sequentially with) other therapeutic modalities. In some embodiments, the therapeutic methods described herein may further include 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.

[0221] In some embodiments, the NKp46 binding polypeptide is administered before, during, or after treatment with a chemotherapeutic agent. In some embodiments, the NKp46 binding polypeptide is administered in combination with an antibody that comprises a binding domain that binds to a tumor antigen, non-limiting examples of tumor antigens to which such domains may bind are provided herein. In some embodiments, the NKp46 binding polypeptide is administered in combination with an antibody that comprises a binding domain that binds to tumor antigens BCMA, CD19, CD20, CD38, CD70, EGFR, or HER2. In some embodiments, the NKp46 binding polypeptide is administered in combination with an antibody that comprises a binding domain that binds to tumor antigens BCMA, CD19, CD20, CD38, CD70, EGFR, or HER2, such administration being before, during, or after treatment with a chemotherapeutic agent.

[0222] In some embodiments, the NKp46 binding polypeptides provided herein are given simultaneously 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 NKp46 binding polypeptides provided herein are given simultaneously with an agonist of OX40, GITR, 41BB, CD27, HVEM, CD28, and / or ICOS, such as an agonist antibody.

[0223] In some embodiments, the NKp46 binding polypeptides provided herein are given concomitantly with one or more chemotherapeutic agents, CAR-T therapies, oncolytic virus therapies, cytokine therapies, and / or agents targeting other checkpoint molecules, such as VISTA, gpNMB, B7H4, HHLA2, CD73, CTLA4, TIGIT, and the like.

[0224] In some embodiments, the NKp46 binding polypeptides or engineered cells provided herein are given simultaneously with a PD-1 / PD-L1 therapy. 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).

[0225] In some embodiments, the NKp46 binding polypeptide and the additional agent are combined in a single therapeutic composition, and the NKp46 binding polypeptide and the additional agent are administered simultaneously. Alternatively, the NKp46 binding polypeptide and the additional agent are separate from each other, e.g., each is combined in a separate therapeutic composition, and the NKp46 binding polypeptide and the additional agent are administered simultaneously, or the NKp46 binding polypeptide and the additional agent are administered at different times during the treatment regimen. For example, the NKp46 binding polypeptide is administered before the administration of the additional agent, the NKp46 binding polypeptide is administered after the administration of the additional agent, or the NKp46 binding polypeptide and the additional agent are administered in alternation. The NKp46 binding polypeptide and the additional agent may be administered in a single dose or multiple doses.

[0226] In some embodiments, the NKp46 binding polypeptide and the additional agent(s) are administered simultaneously. For example, the NKp46 binding polypeptide and the additional agent(s) can be combined in a single composition or administered as two or more separate compositions. In some embodiments, the NKp46 binding polypeptide and the additional agent(s) are administered sequentially, or the NKp46 binding polypeptide and the additional agent(s) are administered at different times during a treatment regimen.

[0227] 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.

[0228] 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.

[0229] 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 polypeptide is used as an adjuvant therapy in the treatment of cancer. In some embodiments, the polypeptide is used as the sole adjuvant therapy in the treatment of cancer. In some embodiments, the polypeptide 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.

[0230] In some embodiments, the polypeptide 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 polypeptide is used as a neoadjuvant therapy in the treatment of cancer. In some embodiments, the use is before resection.

[0231] In some embodiments, the tumor microenvironment contemplated by the methods described herein is or comprises 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, NK cells, macrophages, other lymphoid cells, neutrophils, and other immune cells located in proximity to the tumor.

[0232] kit Also provided are articles of manufacture and kits comprising any of the NKp46-binding polypeptides described herein and suitable packaging. In some embodiments, the invention includes a kit comprising (i) an NKp46-binding polypeptide and (ii) instructions for using the kit to administer the NKp46-binding polypeptide to an individual.

[0233] 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.

[0234] 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

[0235] 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.

[0236] Example 1: NKp46 single domain antibody Single domain antibodies targeting human NKp46 were generated by immunization of llamas and alpacas with recombinant human NKp46 extracellular domain. The amino acid sequences of the NKp46 VHH domains are shown in the tables of certain sequences presented below. It is shown that the lysine at residue 125 (K125) of any of the disclosed VHH domains may be substituted with aspartic acid (K117D), glutamic acid (K125E), or arginine (K125R). The VHH named hz5D7v17 (SEQ ID NO: 15) contains an arginine (R) at residue 125 (shown in bold and underlined in the tables of certain sequences).

[0237] Binding of NKp46-binding polypeptides formatted as VHH-Fc fusion proteins was assessed by flow cytometry. Hinge-less IgG1-Fc or homodimeric Fc (Figures 1I and 1J) were used (hinge-less Fc is Fc *(Annotated as NKp46-NKp46-GFP). HEK293F cells were transiently transfected with plasmids encoding full-length human NKp46, cynomolgus monkey NKp46, or mouse NKp46 followed by an IRES and GFP. Polypeptide binding was measured using transfected cells expressing NKp46 and GFP. Transfected cells were plated in 96-well plates at 30,000 cells per well in FACS buffer (PBS, 1% BSA, 0.1% NaN3, pH 7.4). Untransfected HEK293F cells were used as an NKp46 negative control and plated at 30,000 cells per well in a separate plate. Test polypeptides were then diluted 2-fold to a final concentration of 1000 nM, followed by 3-, 4-, and 5-fold serial dilutions. The last column was left with only FACS buffer as a secondary-only control. Dilutions of test substances were added to equal volumes 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 α-hFc-647 secondary diluted 1000-2000 times. The assay plate was then incubated at 4°C for 20-30 minutes. After one further wash with 150 μL of FACS buffer, bound antibodies were detected by flow cytometry. Flow cytometric detection was performed on an Intellicyt iQue Plus or Accuri iQue. NKp46 transfected cells were gated as GFP positive and polypeptide binding was measured as median fluorescence at 647 nm. Data were plotted and analyzed using GraphPad Prism analysis software and the results are shown in the following table and in Figure 1.

[0238] [Table 3]

[0239] [Table 4]

[0240] [Table 5]

[0241] [Table 6]

[0242] [Table 7]

[0243] [Table 8]

[0244] [Table 9]

[0245] As shown in Figures 1A and 1B, 1I and Tables 3, 4 and 9, the NKp46-binding polypeptides bound to human NKp46 with an affinity of less than 1 nM. Figures 1C and 1D and Tables 5 and 6 show that the NKp46-binding polypeptides bound to cynomolgus monkey NKp46 with an affinity of less than 2 nM. Figures 1E and 1F and Tables 7 and 8 show that nearly all of the NKp46-targeting polypeptides bound to mouse NKp46 with an affinity of less than 2 nM. Figures 1G and 1H and 1J show that the polypeptides did not bind to non-transfected HEK-293F cells.

[0246] Example 2: Specific IL-2 signaling induced by a polypeptide comprising an NKp46-binding VHH and an IL-2 variant The NKp46-targeting IL-2 activity of polypeptides comprising an NKp46-binding VHH domain (hz5D7v12 or hz5Dv12), a heterodimeric knob-in-hole Fc region ("KiH Fc"), and an attenuated IL-2 mutant fused to the C-terminus of the Fc region was evaluated in a phospho-STAT5 assay. As shown in the table below, control proteins included a polypeptide comprising hz5D7v12 and a heterodimeric knob-in-hole Fc region without IL-2, a polypeptide comprising a non-targeted VHH and an attenuated IL-2 mutant fused to the C-terminus of the heterodimeric knob-in-hole Fc region, and wild-type recombinant IL-2. Increased levels of phosphorylated STAT5 (pSTAT5) were measured by intracellular flow cytometry as a proximity readout of IL-2 receptor ligation and signaling. Human PBMCs were plated in 96-well plates at 1 million cells per well in complete growth medium (RPMI, 10% FBS, 1% anti-anti). Test polypeptides were then diluted 2-fold to a final concentration of 100 nM, and serial 5-fold dilutions were performed. Serial dilutions were added to the cells and incubated at 37° C. for 15 minutes. Cells were then fixed in 100 μL of Cytofix fixation buffer (BD) for 30 minutes at 4° C. Cells were then washed once in 200 μL of FACS buffer and permeabilized in Perm buffer III (BD Phosflow) for 30 minutes at 4° C. Permeabilized cells were washed a total of three times in 1x Permeabilization Buffer (eBioscience) and then incubated overnight at 4°C in 1x Permeabilization Buffer containing fluorescently labeled antibodies against CD4 (OKT4, 1:100), CD3 (SP34-2, 1:50), CD16 (3G8, 1:1000), pSTAT5 (SRBCZX, 1:70), CD56 (NCAM16.2, 1:500), and CD8 (RPA-T8, 1:4000). The next day, cells were washed with 150 μL of FACS buffer and analyzed using an ACEA Biosciences Novocyte-Quanteon flow cytometer. IL-2 signaling was measured in NK cells (CD3-CD56 dimCD16+ or CD3-CD56 bright pSTAT5 was quantified by the increase in median frequency and fluorescence intensity levels of fluorescently labeled antibodies detecting pSTAT5 on CD4 T cells (CD16-, or total NK cells), CD3+CD4+, or CD8 (CD3+CD8+). Data were plotted and analyzed using GraphPad Prism analysis software.

[0247] As shown in FIG. 2, a polypeptide comprising hz5D7v12 or hz5D7v17 and an attenuated IL-2 mutant fused to the C-terminus of a heterodimeric knob-in-hole Fc region inhibits CD56 dim EC<0.4 nM on CD16+ NK cells (Figures 2A and 2B) and total NK cells (Figure 2G) 50 induced a concentration-dependent increase in pSTAT5 levels, which was less than the activity of wild-type recombinant IL-2. bright In CD16-NK cells, EC 50 was less than 0.08 nM (Figures 2C and 2D), which is comparable to the EC of wild-type recombinant IL-2 of approximately 0.06 nM. 50 No detectable increase in pSTAT5 on CD4 or CD8 T cells was induced by NKp46-targeted mutant IL-2 (Figures 2E and 2F, 2H and 2I). None of the control polypeptides induced a detectable increase in pSTAT5 levels in any of the cell types tested, indicating that attenuated IL-2 requires targeting to cells via the binding domain to achieve IL-2 receptor signaling activity.

[0248] [Table 10]

[0249] Example 3: Enhancement of antibody-dependent cellular cytotoxicity induced by a polypeptide comprising an NKp46-binding VHH and an IL-2 mutant The NKp46-targeting IL-2 activity of polypeptides comprising the NKp46-binding VHH domains hz5D7v12 or hz5D7v17, a heterodimeric knob-in-hole Fc region, and an attenuated IL-2 mutant fused to the C-terminus of the Fc region (hz5D7v12-Fc xELL-hole and hz5D7v12-Fc xELL-knob-mutant IL-2, also referred to herein as "hz5D7v12-KiH Fc mutant IL-2") was further evaluated in an antibody-dependent cellular cytotoxicity (ADCC) assay in combination with the anti-EGFR antibody cetuximab, which exhibits ADCC activity. Polypeptides containing a non-targeted VHH and an attenuated IL-2 mutant fused to the C-terminus of a heterodimeric knob-in-hole Fc region (non-targeted VHH-Fc xELL-hole and non-targeted VHH-Fc xELL-knob-mutant IL-2) and wild-type recombinant IL-2 were used as controls. A431 cells were labeled with CYTO-ID red long-term cell tracer (Enzo) and then plated in 96-well flat-bottom plates at 10000 cells per well in 100 μL and allowed to adhere for 4 hours. PBMCs obtained from human donors were thawed and tested for NK cell frequency by flow cytometry. Each well received 25 μL of Incucyte™ Caspase-3 / 7 Green Dye for Apoptosis (Sartorius) at a final dilution of 2000 times per 25 μL of medium, or 20 nM or 0.2 nM final concentrations of cetuximab, 1 nM final concentration of wild-type recombinant IL-2, or 1 nM final concentration of IL-2 mutant fusion polypeptide, and 25 μL of human PBMC adjusted to a concentration of 10 NK cells per A431 cell. Cells were allowed to settle for 10 minutes at room temperature, after which the plates were placed in an Incucyte imager at 37° C. for 24 hours, with images acquired every 30 minutes. A431 killing was determined by overlap of Caspase-3 / 7 and CYTO-ID red, with maximum killing defined by the level observed when 20 nM cetuximab was used. Data was plotted and analyzed using GraphPad Prism analysis software.

[0250] As shown in Figure 3, when 0.2 nM cetuximab was used, the ADCC activity of cetuximab was reduced to approximately 65% ​​of the maximum activity. The killing activity of this suboptimal cetuximab dose was not enhanced by polypeptides comprising an attenuated IL-2 mutant, a heterodimeric Fc, and a non-targeted VHH (non-targeted VHH-Fc xELL-hole and non-targeted VHH-Fc xELL-knob-mutant IL-2), whereas wild-type recombinant IL-2 and polypeptides comprising an attenuated IL-2 mutant fused to the C-terminus of a heterodimeric Fc and an NKp46-targeted VHH domain (hz5D7v12-Fc xELL-hole and hz5D7v12-Fc xELL-knob-mutant IL-2) were both able to enhance the activity of 0.2 nM cetuximab such that maximum killing was achieved.

[0251] In additional studies, target cell killing was assessed using the PBMC ADCC bioassay (Promega). The kit includes target cells expressing a HiBit fusion protein that is released upon cell lysis and generates a luminescent signal upon binding to the complementary polypeptide LgBiT. In this assay, target cells were mixed with the indicated test substances (anti-BCMA antibodies alone or in combination with cx11314, cetuximab (anti-EGFR), trastuzumab (anti-HER2), the following sequence analogs: rituximab (anti-CD20), defucosylated variants of rituximab (anti-CD20), daratumumab (anti-CD38), tafasitamab (anti-CD19), obinutuzumab (anti-CD19)) and human PBMCs in a 96-well white U-bottom plate at the ratio of 0.01 to 0.01. The assay plate was then incubated in a 37°C incubator for 5 hours. Detection reagent containing the polypeptide LgBiT was then added to each well and luminescence was read on a plate reader. A maximum lysis control in which 100 μg / mL digitonin was added to wells containing PBMCs and target cells, as well as an untreated control, were included in each experiment. The % specific lysis was calculated based on the relative light units of each sample compared to the untreated and digitonin controls.

[0252] As shown in Figures 4 and 5, an NKp46-binding polypeptide (hz5D7v12-KiH Fc mutant IL-2) comprising an IL-2 mutant fused to the C-terminus of a heterodimeric Fc and an NKp46-targeting VHH domain significantly enhanced the ADCC activity of various antibodies targeting cell surface antigens including CD20 (Figures 4A and 4B, and Figure 5B), CD19 (Figure 5B), CD38 (Figure 5A), BCMA (Figure 5A), HER2 (Figures 5C and 5D), and EGFR (Figures 5C and 5D).

[0253] Example 4: Cell expansion of cynomolgus monkey PBMC subpopulations induced by polypeptides comprising NKp46-binding VHH and IL-2 variants The effect of polypeptides comprising the NKp46-binding VHH domain hz5D7v12, a heterodimeric Fc region, and an attenuated IL-2 mutant fused to the C-terminus of the Fc region (hz5D7v12-Fc xELL-hole and hz5D7v12-Fc xELL-knob-mutant IL-2) on in vivo cell expansion was tested in non-human primates. Cynomolgus monkeys were intravenously bolus-injected with 0.3 mg / kg, 1 mg / kg, or 3 mg / kg of the polypeptide. Whole blood samples were taken from the study animals before dosing and 4, 10, and 14 days after dosing. PBMCs at each time point were isolated using density centrifugation on Lymphoprep (STEMCELL Technologies), and cells were stained with a combination of fluorescently labeled cell type-specific antibodies. T cells were stained with CD3 T cells, which do not express the B cell marker CD20, and CD20 T cells, which do not express the B cell marker CD20. + Regulatory T cells (T reg ") also expressed CD25 and had reduced levels of CD127 and CD4 + NK cells were defined as CD3 T cells expressing NKG2A. -Cells were defined as non-T and non-B cells and were either positive or negative for CD16. CD20 positive staining populations were classified as B cells. Fold changes were calculated by dividing the number of cells per mL of whole blood 10 days after treatment by the baseline number per mL of whole blood before treatment. Granzyme B expression was measured in the above PBMC subpopulations using additional fixation, permeabilization, and staining steps. Briefly, cells were stained for cell surface markers with a combination of fluorescently labeled cell type specific antibodies, followed by fixation and permeabilization using FoxP3 Transcription Factor Staining Buffer Set (eBioscience). Granzyme B was then detected with a specific fluorescently labeled antibody. Flow cytometry detection was performed on an ACEA Biosciences Novocyte-Quanteon flow cytometer. Data were plotted and analyzed using GraphPad Prism analysis software. Fold changes were calculated by dividing the median granzyme B fluorescence on days 4 and 10 by the median fluorescence of baseline (pre-treatment) NK cells. The results are shown in Table 11 and Figures 6A to 6C.

[0254] [Table 11]

[0255] As shown in Figures 6A and 6B and Table 11, a single administration of a polypeptide comprising the NKp46-binding VHH domain hz5D7v12, a heterodimeric Fc region, and an attenuated IL-2 variant fused to the C-terminus of the Fc region resulted in a dose-dependent increase in NK cells, with a higher increase occurring on day 10 at doses of 0.3 mg / kg and 1 mg / kg, and on day 14 at a dose of 3 mg / kg. CD16 + and CD16 - A greater than 2-fold increase was seen in both NK cell compartments at all dose levels tested, with the greatest fold increase seen at the 3 mg / kg dose. regNo increase was observed in non-NK cell populations, including the NKp46-binding VHH domain hz5D7v12, a heterodimeric Fc region, and an attenuated IL-2 mutant fused to the C-terminus of the Fc region. Figure 6C shows that treatment with a single dose of a polypeptide comprising the NKp46-binding VHH domain hz5D7v12, a heterodimeric Fc region, and an attenuated IL-2 mutant fused to the C-terminus of the Fc region also resulted in an increase in NK cell killing capacity, as demonstrated by upregulation of granzyme B, a surrogate marker of cytotoxicity. Granzyme B expression peaked 4 days after treatment but remained elevated above pretreatment levels 10 days after treatment. The maximum increase in granzyme B expression was 3.9-, 4.1-, and 5.1-fold in the 0.3 mg / kg, 1 mg / kg, and 3 mg / kg groups, respectively. These data indicate that a polypeptide comprising an NKp46-binding VHH domain (e.g., hz5D7v12), a heterodimeric Fc region, and an attenuated IL-2 mutant fused to the C-terminus of the Fc region specifically induced cell proliferation and activity of NK cell populations in vivo.

[0256] Example 5: Antitumor efficacy induced by polypeptides comprising NKp46-binding VHH and IL-2 mutants in human xenograft tumor mouse models The in vivo antitumor activity of polypeptides comprising an NKp46-binding VHH domain (hz5D7v17), a heterodimeric Fc region, and an attenuated IL-2 mutant fused to the C-terminus of the Fc region (hz5D7v17-Fc xELL-hole and hz5D7v17-Fc xELL-knob-mutant IL-2, also referred to herein as "hz5D7v17-KiH Fc mutant IL-2") was tested in a xenograft mouse model. Seven-week-old female BALB-Scid mice (8 per group) were injected with 3.5 x 10 6 Raji cells were inoculated subcutaneously. The tumor volume was an average of 100 mm 3Upon reaching a tumor volume of 1000 mg / kg, animals were administered either 1 mg / kg hz5D7v17-KiH Fc mutant IL-2, 5 mg / kg sequence analog of the therapeutic antibody rituximab, a combination of both regimens, or vehicle control. All treatments were administered once a week for three consecutive weeks. hz5D7v17-KiH Fc mutant IL-2 and vehicle were administered intravenously, and rituximab analog was administered intraperitoneally. Tumor volume was determined by measuring the length and width of the subcutaneous tumor mass three times a week. Tumor volume was calculated using the formula V=L×W×W / 2. Data were plotted and analyzed using GraphPad Prism analysis software.

[0257] As shown in Figure 5, monotherapy with hz5D7v17-KiH Fc mutant IL-2 delays the growth of Raji xenografts in BALB-Scid mice compared to vehicle controls. An analog of the therapeutic antibody rituximab, which can recognize CD20 on Raji tumor cells and redirect effector cells, including NK cells, to recognize and kill tumor cells, induces a remarkable antitumor response, resulting in tumor stasis that lasts approximately 3 weeks before tumor volume begins to increase again. In combination therapy, hz5D7v17-KiH Fc mutant IL-2 further enhances the activity of the rituximab analog, resulting in complete and sustained tumor regression in 7 / 8 animals. These data indicate that a polypeptide comprising an NKp46-binding VHH domain (e.g., hz5D7v17), a heterodimeric Fc region, and an attenuated IL-2 mutant fused to the C-terminus of the Fc region induces in vivo functional responses that lead to antitumor activity as a single agent and potently enhances the antitumor response of therapeutic antibodies such as rituximab.

[0258] Example 6: Rescue of chemotherapy-induced defects in NK cell health and antitumor activity by a polypeptide comprising an NKp46-binding VHH and an IL-2 mutant The activity of a polypeptide comprising the NKp46-binding VHH domain hz5D7v17, a heterodimeric Fc region, and an attenuated IL-2 mutant fused to the C-terminus of the Fc region (hz5D7v17-KiH Fc mutant) was further evaluated in combination with the standard of care chemotherapy reagents dexamethasone and lenalidomide. PBMCs obtained from human donors were thawed and treated for 3 days with a combination of 500 nM dexamethasone, 2 μM lenalidomide, and 5 nM hz5D7v17-KiH Fc mutant. All pretreatment conditions also included 2 ng / mL IL-2 to support NK cell survival. NK cell frequencies in each treated PBMC sample were quantified by flow cytometry and normalized to media-only controls. To assess antibody-dependent cellular cytotoxicity (ADCC) capacity of NK cells from pretreated PBMCs, NK cells were enriched and co-cultured with CellTrace™ Violet-labeled MM1S (multiple myeloma) target cells at a ratio of 10 NK cells to 1 MM1S cells and 1 nM of daratumumab sequence analog (anti-hCD38-hIgG1) for 18 hours. Pretreatment conditions were continued throughout the co-culture. MM1S killing was determined by flow cytometry by quantifying the percentage of MM1S cells staining positive with the live / dead stain Zombie Aqua and / or the apoptosis marker Apotracker green.

[0259] As shown in Figure 8A, treatment of human PBMCs with standard of care dexamethasone and lenalidomide results in approximately 50% fewer NK cells after 3 days compared to treatment with media alone. Co-treatment of hz5D7v17-KiH Fc mutants with chemotherapy regimens restores NK cell viability and / or proliferation, resulting in NK cell numbers similar to or greater than media controls. Figure 8B shows that ADCC activity of NK cells in the presence of a daratumumab analog (anti-hCD38-hIgG1) is reduced by approximately 20% when cells are pretreated with dexamethasone and lenalidomide. However, adding hz5D7v17-KiH Fc mutants to the pretreatment regimen rescues or even increases ADCC activity. These data indicate that a polypeptide comprising the NKp46-binding VHH domain hz5D7v17, a heterodimeric Fc region and an attenuated IL-2 mutant fused to the C-terminus of the Fc region can overcome the suppression of NK cells by standard of care chemotherapeutic drugs such as dexamethasone and lenalidomide.

[0260] 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.

[0261] [Table 12] TIFF2024534838000014.tif255169TIFF2024534838000015.tif250170TIFF20245348380 00016.tif252170TIFF2024534838000017.tif247170TIFF2024534838000018.tif255169

Claims

1. binds to NKp46, and (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 24; (b) a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (c) a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (d) a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (e) a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (f) a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 23; (g) a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25; (h) a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 26; or (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 27; A polypeptide comprising at least one VHH domain comprising:

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

3. The polypeptide of claim 1, wherein at least one VHH domain comprises an amino acid sequence that is at least 85%, 90%, 95%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1 to 16.

4. The polypeptide of claim 1, wherein at least one VHH domain comprises an amino acid sequence of any one of SEQ ID NOs: 1 to 16.

5. The polypeptide of claim 1, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO:

15.

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

7. 2. The polypeptide of claim 1, comprising an immune cell-activating cytokine or a functional portion thereof, optionally fused to the N-terminus or C-terminus of a VHH domain that binds to NKp46, and further optionally, the immune cell-activating cytokine is IL-2, IL-15, IL-7, IL-6, IL-12, IFNα, IFNβ, or IFNγ, or an attenuated or modified form thereof.

8. The polypeptide of claim 1 , comprising at least one antigen-binding domain that binds to an antigen other than NKp46.

9. The tumor antigen comprises at least one antigen-binding domain that binds to a tumor antigen, and the tumor antigen may be 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, CD 6, 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, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB , EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FAS, 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, NKG2A, 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, TGFβ receptor 1 (TGFBR1), TGFβ receptor 2 (TGFBR2), TIGIT, TIM-3, TLR2, TLR 10. The polypeptide of claim 8, wherein the polypeptide is selected from TLR4, TLR6, TLR7, 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, and WISP-3.

10. at least one antigen-binding domain that binds to an antigen other than NKp46 is a VHH domain; and / or The polypeptide according to claim 8 , wherein each antigen-binding domain that binds to an antigen other than NKp46 is a VHH domain.

11. The polypeptide of claim 8, wherein at least one antigen-binding domain that binds to an antigen other than NKp46 comprises a heavy chain variable region and a light chain variable region, or each antigen-binding domain that binds to an antigen other than NKp46 comprises a heavy chain variable region and a light chain variable region.

12. The polypeptide of claim 1, wherein each VHH domain of the polypeptide binds to NKp46.

13. 13. The polypeptide of claim 12, wherein each VHH domain comprises the same CDR1, CDR2, and CDR3 amino acid sequence, and optionally each VHH domain comprises the same VHH sequence.

14. The polypeptide according to claim 1, wherein the NKp46 is human NKp46.

15. The polypeptide of claim 14, wherein the human NKp46 comprises the sequence of SEQ ID NO:

29.

16. The polypeptide of claim 1 , comprising an Fc region. (a) the Fc region comprises an amino acid sequence selected from SEQ ID NO: 53 to SEQ ID NO: 89; (b) the polypeptide forms dimers under physiological conditions, and / or (c) the polypeptide comprises an immune cell-activating cytokine fused to the C-terminus of the Fc region. The polypeptide of claim 16.

18. A complex comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is a polypeptide according to any one of claims 1 to 17, 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.

19. The complex of claim 18, wherein the second polypeptide comprises at least one VHH domain that binds to NKp46, at least one immune cell-activating cytokine, and / or at least one antigen-binding domain that binds to an antigen other than NKp46.

20. The complex of claim 19, wherein when the antigen-binding domain that binds to an antigen other than NKp46 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region is fused to a heavy chain constant region comprising the second Fc region.

21. The second polypeptide may be selected from the group consisting of 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, C D20, 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, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FAS, 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, NKG2A, 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, TGFβ receptor 1 (TGFBR1), TGFβ receptor 2 (TGFBR2), TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7 , 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, and WISP-3.

22. 19. The conjugate of claim 18, wherein the first Fc region comprises a knob mutation and the second Fc region comprises a hole mutation, or the first Fc region comprises a hole mutation and the second Fc region comprises a knob mutation; optionally, the first Fc region comprises a T366W mutation and the second Fc region comprises T366S, L368A, and Y407V mutations, or the first Fc region comprises a hole mutation and the second Fc region comprises a knob mutation.

23. 18. An immunoconjugate comprising the polypeptide of any one of claims 1 to 17 and a cytotoxic agent, optionally 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.

24. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

25. An isolated nucleic acid encoding the polypeptide of any one of claims 1 to 17.

26. An isolated nucleic acid encoding the complex of claim 18.

27. A vector comprising the nucleic acid of claim 25.

28. A vector comprising the nucleic acid described in claim 26.

29. A host cell expressing a polypeptide according to any one of claims 1 to 17.

30. 18. A method for producing a polypeptide according to any one of claims 1 to 17, comprising incubating a host cell expressing said polypeptide under conditions suitable for expression of said polypeptide, and optionally isolating said polypeptide.

31. 20. A method of treating cancer, comprising administering a pharmaceutically effective amount of a polypeptide according to any one of claims 1 to 17 to a subject with cancer or an infectious disease, optionally wherein 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, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer or renal carcinoma, 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 (lip, tongue, oral cavity, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, and the like. Liquid gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, vulvar cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, 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 The method is selected from aggressive 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), acute myeloid leukemia (AML), hairy cell leukemia, and chronic myeloblastic leukemia.

32. A method of treating cancer using the conjugate of claim 18, optionally wherein 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, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer or renal carcinoma, 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 (lip, tongue, oral cavity, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, The method is selected from testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, 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), acute myeloid leukemia (AML), hairy cell leukemia, and chronic myeloblastic leukemia.

33. A pharmaceutical composition containing the complex described in claim 18.

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