CD8-binding polypeptides and uses thereof

Abstract

Provided herein are VHH-containing polypeptides that bind to CD8. 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 / 223,786, filed July 20, 2021, and U.S. Provisional Application No. 63 / 288,111, filed December 10, 2021, each of which is incorporated by reference in its entirety for all purposes.

[0002] The present invention relates to CD8 binding polypeptides and methods of using CD8 binding polypeptides to modulate the biological activity of CD8, including, but not limited to, methods of treating cancer. In some embodiments, the CD8 binding polypeptide is a fusion polypeptide comprising a CD8 binding polypeptide and a polypeptide that binds to an antigen other than CD8. [Background technology]

[0003] CD8 is a transmembrane glycoprotein expressed on the surface of cytotoxic T cells (CD8+ T cells) as well as other cells of the lymphoid system, including a subset of natural killer cells, γδ T cells, cortical thymocytes, and dendritic cells. CD8 is typically a heterodimer composed of the CD8α and CD8β chains, but may exist as a CD8α homodimer in some circumstances. In cytotoxic T cells, CD8 acts as a coreceptor for the T cell receptor (TCR), enhancing antigen recognition and T cell activation. Activation of cytotoxic T cells is governed by the interaction of the TCR with peptide antigens bound to class I major histocompatibility complex (MHC) proteins. CD8 helps stabilize the TCR / peptide-MHC interaction by binding to the constant region of class I MHC proteins. CD8 also enhances TCR signaling by recruiting Lck to the cytoplasmic domain of CD8α, resulting in a cascade that amplifies T cell activation signals.

[0004] T cell activation is also controlled by other molecules such as IL-2, IL-15, IL-7, IL-6, IL-12, IFNα, IFNβ, and IFNγ. The cytokine interleukin-2 (IL-2), synthesized and secreted by activated T cells themselves, is a pleiotropic cytokine that regulates the differentiation of helper T cells, enhances the cytolytic activity of natural killer cells, and controls the generation of CD8+ T cells. IL-2 binds to a high-affinity receptor composed of three subunits (IL-2α, IL-2β, and γc) on the surface of T cells. Signaling through the IL-2 receptor complex drives T cell division and clonal expansion of activated T cells. Summary of the Invention [Problem to be solved by the invention]

[0005] There is a need for CD8 binding polypeptides that can specifically target activating molecules to CD8+ T cells, increasing the potency and selectivity of cytotoxic T cell responses. [Means for solving the problem]

[0006] Provided herein are CD8 binding polypeptides and methods of using the CD8 binding polypeptides to treat, for example, cancer. In some embodiments, the CD8 binding polypeptide comprises one or more additional binding domains and / or cytokine sequences. Certain numbered embodiments are provided below.

[0007] Embodiment 1 A polypeptide comprising at least one VHH domain which binds to CD8 and comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:73, or SEQ ID NO:74, a CDR2 comprising the amino acid sequence of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:22, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, or SEQ ID NO:80, and a CDR3 comprising the amino acid sequence of SEQ ID NO:5, SEQ ID NO:16, or SEQ ID NO:18.

[0008] Embodiment 2 A polypeptide according to embodiment 1, wherein at least one VHH domain comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:12 and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:14 and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:16; SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:18; SEQ ID NO:3, SEQ ID NO:22 and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:14 and SEQ ID NO:18; SEQ ID NO:3, SEQ ID NO:27 and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:29 and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:31 and SEQ ID NO:5; SEQ ID NO:73, SEQ ID NO:14 and SEQ ID NO:18; SEQ ID NO:74, SEQ ID NO:14 and SEQ ID NO:18; SEQ ID NO:3, SEQ ID NO:75 and SEQ ID NO:18; SEQ ID NO:3, SEQ ID NO:76 and SEQ ID NO:18; SEQ ID NO:3, SEQ ID NO:77 and SEQ ID NO:18; SEQ ID NO:3, SEQ ID NO:78 and SEQ ID NO:18; SEQ ID NO:3, SEQ ID NO:79 and SEQ ID NO:18; or SEQ ID NO:3, SEQ ID NO:80 and SEQ ID NO:18.

[0009] Embodiment 3: A polypeptide described in embodiment 1 or 2, wherein at least one VHH domain comprises a CDR1 having the amino acid sequence of SEQ ID NO: 3, a CDR2 having the amino acid sequence of SEQ ID NO: 78, and a CDR3 having the amino acid sequence of SEQ ID NO: 18.

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

[0011] Embodiment 5 A polypeptide according to any one of embodiments 1 to 4, wherein at least one VHH domain comprises an amino acid sequence that is at least 85%, 90%, 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO:2, 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:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, or SEQ ID NO:100.

[0012] Embodiment 6 A polypeptide described in any one of embodiments 1 to 5, wherein at least one VHH domain comprises the amino acid sequence of 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:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, or SEQ ID NO:99, or SEQ ID NO:100.

[0013] Embodiment 7 The polypeptide according to any one of embodiments 1 to 6, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 100.

[0014] Embodiment 8. A polypeptide according to any one of embodiments 1 to 7, comprising two VHH domains.

[0015] Embodiment 9. A polypeptide according to any one of embodiments 1 to 7, comprising three VHH domains.

[0016] Embodiment 10. The polypeptide according to any one of embodiments 1 to 9, comprising an immune cell-activating cytokine.

[0017] Embodiment 11. The polypeptide of embodiment 10, wherein the immune cell-activating cytokine is fused to the N-terminus or C-terminus of the VHH domain that binds to CD8.

[0018] Embodiment 12. The polypeptide of embodiment 10 or 11, wherein the immune cell-activating cytokine is IL-2, IL-15, IL-7, IL-6, IL-12, IFNα, IFNβ, or IFNγ, or an attenuated or modified form thereof.

[0019] Embodiment 13. The polypeptide according to any one of embodiments 1 to 12, comprising an Fc region.

[0020] Embodiment 14 The polypeptide according to embodiment 13, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NO: 32 to SEQ ID NO: 70, or SEQ ID NO: 101 to SEQ ID NO: 111.

[0021] Embodiment 15. A polypeptide described in embodiment 13 or 14, comprising an immune cell activating cytokine.

[0022] Embodiment 16. The polypeptide of embodiment 15, wherein the immune cell-activating cytokine is IL-2, IL-15, IL-7, IL-6, IL-12, IFNα, IFNβ, or IFNγ, or an attenuated or modified form thereof.

[0023] Embodiment 17. The polypeptide of embodiment 16, wherein the immune cell-activating cytokine is fused to the C-terminus of the Fc region.

[0024] Embodiment 18. The polypeptide according to any one of embodiments 1 to 17, comprising at least one antigen-binding domain that binds to an antigen other than CD8.

[0025] Embodiment 19. The polypeptide of embodiment 18, comprising at least one antigen-binding domain that binds to Lag3, CTLA4, TGFBR1, TGFBR2, Fas, TNFR2, PD1, PDL1, or TIM3.

[0026] TGFBR1, TGFBR2, Fas, TNFR2, 1-92-LFA-3, 5T4, α4 integrin, αV integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis Y, apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, complement C5, C-242, CA9, CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD1 1a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD39, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, C D52, 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, CTLA4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB , EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FcRH5, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor 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, 20. The polypeptide of embodiment 18 or 19, comprising at least one antigen-binding domain that binds to TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, TROP-2, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, or WISP-3.

[0027] Embodiment 21 The polypeptide according to any one of embodiments 18 to 20, wherein at least one antigen-binding domain that binds to an antigen other than CD8 is a VHH domain.

[0028] Embodiment 22. The polypeptide of embodiment 21, wherein each antigen-binding domain that binds to an antigen other than CD8 is a VHH domain.

[0029] Embodiment 23 The polypeptide of any one of embodiments 18 to 21, wherein at least one antigen-binding domain that binds to an antigen other than CD8 comprises a heavy chain variable region and a light chain variable region.

[0030] Embodiment 24 The polypeptide of embodiment 23, wherein each antigen-binding domain that binds to an antigen other than CD8 comprises a heavy chain variable region and a light chain variable region.

[0031] Embodiment 25: A complex comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is a polypeptide described in any one of embodiments 13 to 24, the first polypeptide comprises a first Fc region, and the second polypeptide comprises a second Fc region, and the first Fc region and the second Fc region are the same or different.

[0032] Embodiment 26: The complex described in embodiment 25, wherein the second polypeptide comprises at least one VHH domain that binds to CD8, at least one immune cell activating cytokine, and / or at least one antigen binding domain that binds to an antigen other than CD8.

[0033] Embodiment 27. The conjugate of embodiment 26, wherein when the antigen-binding domain that binds to an antigen other than CD8 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region is fused to a heavy chain constant region that comprises the second Fc region.

[0034] Embodiment 28. The conjugate according to any one of embodiments 25 to 27, wherein the first Fc region comprises a knob mutation and the second Fc region comprises a hole mutation.

[0035] Embodiment 29. The conjugate of embodiment 28, wherein the first Fc region comprises a T366W mutation and the second Fc region comprises T366S, L368A, and Y407V mutations.

[0036] Embodiment 30. The conjugate of embodiment 29, wherein the second Fc region comprises a H435R or H435K mutation.

[0037] Embodiment 31 A polypeptide or complex described in any one of embodiments 13 to 30, wherein the polypeptide is a dimer under physiological conditions or the complex is formed under physiological conditions.

[0038] Embodiment 32. The polypeptide or complex according to any one of embodiments 1 to 31, wherein said CD8 is human CD8.

[0039] Embodiment 33. The polypeptide or complex described in embodiment 32, wherein the human CD8 comprises the sequence of SEQ ID NO:1.

[0040] Embodiment 34 An immune complex comprising the polypeptide or complex according to any one of embodiments 1 to 33 and a cytotoxic substance.

[0041] Embodiment 35. The immunoconjugate of embodiment 34, 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.

[0042] Embodiment 36 A pharmaceutical composition comprising a polypeptide or complex according to any one of embodiments 1 to 33, or an immunoconjugate according to embodiment 34 or 35, and a pharma- ceutically acceptable carrier.

[0043] Embodiment 37. An isolated nucleic acid encoding a polypeptide or complex according to any one of embodiments 1 to 33.

[0044] Embodiment 38. A vector comprising the nucleic acid described in embodiment 37.

[0045] Embodiment 39. A host cell comprising the nucleic acid described in embodiment 37 or the vector described in embodiment 38.

[0046] Embodiment 40. A host cell expressing a polypeptide or complex according to any one of embodiments 1 to 33.

[0047] Embodiment 41: A method for producing a polypeptide or complex described in any one of embodiments 1 to 33, comprising incubating a host cell described in embodiment 38 or 39 under conditions suitable for expression of the polypeptide or complex.

[0048] Embodiment 42. The method of embodiment 41, further comprising isolating the polypeptide or complex.

[0049] Example 43 CD8 + A method for increasing proliferation of T cells, comprising contacting T cells with a polypeptide or complex described in any one of embodiments 1 to 33.

[0050] Embodiment 44 The CD8 + 44. The method of embodiment 43, wherein the T cell is present in vitro.

[0051] Embodiment 45 The CD8 + 44. The method of embodiment 43, wherein the T cell is present in vivo.

[0052] Embodiment 46 A method for treating cancer, comprising administering to a subject with cancer a pharma- ceutical effective amount of a polypeptide or complex described in any one of embodiments 1 to 33, or a pharmaceutical composition described in embodiment 36.

[0053] The cancer may be basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, gastrointestinal cancer, glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer or renal cancer, laryngeal cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, vulvar cancer, 47. The method of embodiment 46, wherein the patient is selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-dividing cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloblastic leukemia.

[0054] Embodiment 48. The method of embodiment 46 or 47, further comprising administering an additional therapeutic agent.

[0055] Embodiment 49. The method of embodiment 48, wherein the additional therapeutic agent is an anti-cancer agent.

[0056] Embodiment 50. The method of embodiment 49, wherein the anti-cancer agent is selected from a chemotherapeutic agent, an anti-cancer biologic, a radiotherapy agent, a CAR-T therapy agent, and an oncolytic virus.

[0057] Embodiment 51 The method of embodiment 48, wherein the additional therapeutic agent is an anti-cancer biologic.

[0058] Embodiment 52. The method of embodiment 51, wherein the anticancer biologic is an agent that inhibits PD-1 and / or PD-L1.

[0059] Embodiment 53. The method of embodiment 51, wherein the anticancer biologic is an agent that inhibits VISTA, gpNMB, B7H3, B7H4, HHLA2, CTLA4, or TIGIT.

[0060] Embodiment 54. The method of any one of embodiments 49, wherein the anticancer agent is an antibody.

[0061] Embodiment 55. The method of embodiment 51, wherein the anticancer biologic is a cytokine.

[0062] Embodiment 56: The method described in embodiment 49, wherein the anticancer agent is a CAR-T therapeutic agent.

[0063] Embodiment 57 The method described in embodiment 49, wherein the anticancer agent is an oncolytic virus.

[0064] Embodiment 58. The method described in any one of embodiments 46 to 57, further comprising tumor resection and / or radiation therapy. [Brief description of the drawings]

[0065] [Figure 1] Figures 1A and 1B show binding of CD8a-targeting sdAbs formatted as VHH-hIgG1-Fc fusion proteins assessed by flow cytometry. Figure 1A shows binding to isolated human T cells. Figure 1B shows binding to HEK293FS cells as a CD8a negative control. [Diagram 2]Figures 2A and 2B show binding of CD8a-targeting sdAbs formatted as VHH-hIgG1-Fc fusion proteins assessed by flow cytometry. Figure 2A shows binding to isolated human T cells. Figure 2B shows binding to HEK293FS cells as a CD8a negative control. [Diagram 3] Figures 3A and 3B show binding of CD8a-targeting sdAbs formatted as VHH-hIgG1-Fc fusion proteins assessed by flow cytometry. Figure 3A shows binding to human CD8a-FL cells (expressing full-length CD8a). Figure 3B shows binding to cynomolgus monkey CD8a-FL cells. [Figure 4] Figures 4A and 4B show binding of CD8a-targeting sdAbs formatted as VHH-hIgG1-Fc fusion proteins assessed by flow cytometry. Figure 4A shows binding to isolated human CD3+CD4- T cells. Figure 4B shows binding to isolated cynomolgus monkey CD3+CD4- peripheral blood mononuclear cells (PBMCs). [Diagram 5] Figures 5A and 5B show the binding of the fusion protein CD8a-hzB7v15 xELL-Fc assessed by flow cytometry: Figure 5A shows binding to human CD3+CD4-Leuko 29 T cells; Figure 5B shows binding to cynomolgus monkey CD3+CD4-CD16- T cells. [Figure 6] Figures 6A-C show the IL-2 activity of wild type IL-2 and CD8a targeted VHH-hIgG1 fusion proteins comprising CD8a-hzB7v15 and attenuated IL-2 on IL-2 reporter cells. Figure 6A shows IL-2 activity on reporter cells not expressing CD8. Figure 6B shows IL-2 activity on IL-2 reporter cells expressing CD8. Figure 6C shows the activity of wild type IL-2, fusion proteins comprising CD8a-hzB7v31 and attenuated IL-2 mutants, and non-targeted attenuated IL-2 mutants containing the same mutations on IL-2 reporter cells expressing CD8. [Figure 7]FIG. 1 shows cell expansion in peripheral blood of cynomolgus monkeys after a single administration of a fusion protein comprising CD8a-hzB7v15 and attenuated IL-2. [Figure 8] Figures 8A and 8B show binding of CD8a-targeting sdAbs formatted as VHH-homodimeric Fc fusion proteins or VHH-knob-in-hole Fc fusion proteins containing attenuated IL-2 mutants assessed by flow cytometry. Figure 8A shows binding to HEK293F cells transfected with full-length human CD8a (CD8a-FL). Figure 8B shows binding to HEK293F cells transfected with full-length human CD8b (CD8b-FL). [Figure 9] Figures 9A and 9B show binding of CD8a-targeting sdAbs formatted as VHH-homodimeric Fc fusion proteins or VHH-knob-in-hole Fc fusion proteins containing attenuated IL-2 mutants assessed by flow cytometry. Figure 9A shows binding to CD8 T cells within pan T cells enriched from human whole blood. Figure 9B shows lack of binding to CD4 T cells within pan T cells enriched from human whole blood. [Figure 10] Figures 10A-H show binding of CD8a-targeting sdAbs formatted as VHH-hIgG1-Fc fusion proteins assessed by flow cytometry. Figures 10A and 10B and 10E and 10F show binding to pan T cells enriched from human whole blood (Figures 10A and 10B) or CD8 T cells (Figures 10A and 10D) or CD4 T cells (Figures 10B and 10F) within peripheral blood mononuclear cells (PBMCs) (Figures 10E and 10F). Figures 10C and 10D and 10F-H show binding to CD8 T cells (Figures 10C and 10G) or CD4 T cells (Figures 10D and 10H) within peripheral blood mononuclear cells (PBMCs) isolated from cynomolgus monkey whole blood. [Figure 11]Figures 11A and 11B show STAT5 signaling cell populations in peripheral blood of human donors. The levels of phosphorylated STAT5 (pSTAT5) (Figures 11A and 11B) or the percentage of cells expressing pSTAT5 (Figures 11C and 11D) in CD8 T cells (Figures 11A, 11C) or regulatory T cells (Treg, Figure 11B) or CD4 T cells (Figure 11D) in pan-T cells enriched from human whole blood are shown. Cells were treated with fusion proteins comprising CD8a-hzB7v31 or CD8aB7v41, an Fc region, and mutant attenuated IL-2; fusion proteins comprising CD8a-hzB7v31 and an Fc region (no IL-2); fusion proteins comprising non-targeted VHH, an Fc region, and attenuated IL-2; or wild-type IL-2. [Figure 12] 12A-12C show the expansion of CD8 T cells (FIGS. 12A and 12C) or CD4 T cells (FIG. 12B) in dissociated tumor cell preparations from human tumor samples (two head and neck or renal cancer cases and one colon cancer case, FIGS. 12A and 12B) or PBMCs from healthy blood donors (FIG. 12C) treated ex vivo with fusion proteins comprising CD8a-hzB7v31, an Fc region, and mutant attenuated IL-2; fusion proteins comprising CD8a-hzB7v31 and an Fc region (without IL-2); fusion proteins comprising untargeted VHH, an Fc region, and attenuated IL-2; or wild-type IL-2. [Figure 13] Figures 13A and 13B show the activity of a single dose (1 mg / kg) of a fusion protein comprising CD8a-hzB7v15, an Fc region, and mutant attenuated IL-2 in cynomolgus monkeys. Figure 13A shows the increase in certain PBMC subpopulations as fold change over baseline in cell number 7 days after dosing. Figure 13B shows the percentage of Ki67+ cells in these subpopulations before dosing (baseline) and 7 days after dosing. [Figure 14]Figures 14A and 14B show the cytotoxic activity of enriched pre-stimulated CD8 T cells (Figure 14A) or antibody-dependent cellular cytotoxicity (ADCC) of PBMCs (Figure 14B) against A431 epidermoid carcinoma cells. Cells were treated with a fusion protein comprising CD8a-hzB7v31, an Fc region, and an attenuated IL-2 mutant; a fusion protein comprising a non-targeted VHH, an Fc region, and an attenuated IL-2 mutant; or wild-type IL-2 as indicated. CD8 T cells or PBMCs were added at different effector to target cell ratios (20:1, 10:1, or 5:1) as indicated. The EGFR-specific therapeutic antibody cetuximab was added to the cell cultures in Figure 14B. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] The embodiments presented herein relate to CD8 binding polypeptides and their uses in various methods of treating, for example, cancer.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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 or CD8-associated disorder). 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.

[0075] 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".

[0076] 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.

[0077] 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 include natural or non-natural amino acid residues and 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 a protein that includes 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 mutation of the host that produces the protein or by errors due to PCR amplification. In some embodiments, the polypeptide is a "complex" of a first polypeptide and a second polypeptide.

[0078] The terms "CD8a" or "CD8" are used interchangeably herein and refer to any naturally occurring mature CD8 resulting from processing of a CD8 precursor in a cell. The term includes CD8 from any vertebrate source, 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 CD8, such as splice variants or allelic variants. A non-limiting exemplary mature human CD8 amino acid sequence is shown, for example, in NCBI Accession No. NP_001369627.1. See SEQ ID NO:1.

[0079] 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 a CD8 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 CD8 epitopes or non-CD8 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.

[0080] 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.

[0081] As used herein, the term "direct inhibition" and similar terms refer to an inhibition profile in which increasing antibody concentration results in increasing inhibition. In some embodiments, after a certain concentration, maximum inhibition is reached and the inhibition profile plateaus. Maximum inhibition does not have to be 100% inhibition, but may be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

[0082] 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.

[0083] 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.

[0084] 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).

[0085] The term "antibody" is used in the broadest sense and includes various polypeptides, including but not limited to conventional antibodies (typically comprising at least one heavy chain and at least one light chain), single domain antibodies (sdAbs, comprising at least one VHH domain and an Fc region), VHH-containing polypeptides (polypeptides comprising at least one VHH domain), and antibody-like antigen-binding domains, including fragments of any of the above, so long as they exhibit the desired antigen-binding activity. In some embodiments, the antibody comprises a dimerization domain. Such dimerization domains include but are not limited to a heavy chain constant domain (comprising CH1, hinge, CH2, and CH3, where CH1 is typically paired with a light chain constant domain CL, while the hinge mediates dimerization) and an Fc region (comprising hinge, CH2, and CH3, where the hinge mediates dimerization).

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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: 112), Gly-Gly-Ser-Gly-Gly-Ser (SEQ ID NO: 113), and / or Gly-Gly-Ser-Ser-Gly-Ser (SEQ ID NO: 114). 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).

[0091] 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.

[0092] 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.

[0093] 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.

[0094] As used herein, "Fc region" refers to a portion of a heavy chain constant region comprising CH2 and CH3. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. In various embodiments, when the Fc region comprises a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4. In some embodiments, when the Fc region comprises a hinge, the hinge is of the same isotype as the Fc region. In some embodiments, the IgG4 hinge comprises a S228P stabilizing mutation.

[0095] 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.

[0096] "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.

[0097] 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:

[0098] 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.

[0099] The term "biological activity" refers to any one or more biological properties of a molecule (whether naturally occurring as found in vivo or provided or made possible by recombinant means). Biological properties include, but are not limited to, binding of a ligand, induction or increase in cell proliferation (such as T cell proliferation), and induction or increase in expression of a cytokine.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] "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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] "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.)).

[0108] 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%.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] [Table 1]

[0113] 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.

[0114] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0115] 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.

[0116] "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.

[0117] 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".

[0118] 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.

[0119] As used herein, "disease" or "disorder" refers to a condition for which treatment is necessary and / or desirable.

[0120] The terms "tumor cell," "cancer cell," "cancer," "tumor," and / or "neoplasm" are used interchangeably herein, unless otherwise specified, to refer to a cell (or cells) that exhibit uncontrolled proliferation and / or abnormally increased cell survival and / or inhibited apoptosis that interferes with the normal functioning of bodily organs and systems. This definition includes benign and malignant cancers, polyps, hyperplasias, and occult tumors or micrometastases.

[0121] The terms "cancer" and "tumor" include solid cancers and hematological / lymphatic cancers, as well as malignant tumors such as dysplasia, premalignant tumors, and benign tumors. Exemplary cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney or renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), melanoma, myeloma, neuroblastoma, oral cancer (lips, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, vulvar cancer, Hodgkin's lymphoma, and non-Hodgkin's lymphoma lymphomas, including non-Hodgkin's lymphoma, as well as B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-dividing cell NHL, bulky disease NHL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, as well as post-transplant lymphoproliferative disorders (PTLD), and abnormal blood vessel growth associated with nematoses, edema (such as edema associated with brain tumors), and Meigs syndrome.

[0122] 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.

[0123] The term "cells or tissues in the tumor microenvironment" as used herein refers to cells, molecules, extracellular matrix and / or blood vessels that surround and / or nourish tumor cells. Exemplary cells or tissues in the tumor microenvironment include, but are not limited to, tumor vasculature, tumor-infiltrating lymphocytes, fibroblastic reticular cells, endothelial progenitor cells (EPCs), cancer-associated fibroblasts, pericytes, other stromal cells, components of the extracellular matrix (ECM), dendritic cells, antigen-presenting cells, T cells, regulatory T cells (Treg cells), macrophages, neutrophils, myeloid-derived suppressor cells (MDSCs) and other immune cells located in the vicinity of the tumor. Methods for identifying tumor cells and / or cells / tissues located in the tumor microenvironment are well known in the art, as described herein below.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] "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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] "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.

[0133] 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.

[0134] 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.

[0135] "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.

[0136] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] Exemplary CD8 Binding Polypeptides Antagonist CD8 binding polypeptides are provided herein. In various embodiments, the CD8 binding polypeptide comprises at least one VHH domain that binds to CD8. In some embodiments, the CD8 binding polypeptides provided herein comprise one, two, three, four, five, six, seven, or eight VHH domains that bind to CD8. In some embodiments, the CD8 binding polypeptides provided herein comprise one, two, three, or four VHH domains that bind to CD8. Such CD8 binding polypeptides may comprise one or more additional antigen binding domains (e.g., VHH domains) that bind to one or more target proteins other than CD8, and / or may comprise one or more additional polypeptide sequences, such as cytokine sequences.

[0144] In some embodiments, the CD8 binding polypeptide comprises at least one VHH domain that binds to CD8 and an Fc region. In some embodiments, the CD8 binding polypeptide provided herein comprises one, two, three, or four VHH domains that bind to CD8 and an Fc region. In some embodiments, the Fc region mediates dimerization of the CD8 binding polypeptide under physiological conditions, such that the formation of dimers doubles the number of CD8 binding sites. For example, a CD8 binding polypeptide comprising three VHH domains that bind to CD8 and an Fc region is trivalent as a monomer, but under physiological conditions, the Fc region can mediate dimerization such that the CD8 binding polypeptide exists as a hexavalent dimer under such conditions.

[0145] In some embodiments, a CD8 binding polypeptide comprises at least two VHH domains, where a first VHH domain binds to a first epitope of CD8 and a second VHH domain binds to a second epitope of CD8. When a CD8 binding polypeptide comprises a VHH domain that binds to a first epitope of CD8 and a VHH domain that binds to a second epitope of CD8, the CD8 binding polypeptide may be referred to as "dual epitopic" or "dual specific."

[0146] CD8-binding polypeptide In various embodiments, the VHH domain that binds to CD8 comprises a CDR1 sequence selected from SEQ ID NO:3, SEQ ID NO:73, and SEQ ID NO:74, a CDR2 sequence selected from SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:22, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, and a CDR3 sequence selected from SEQ ID NO:5, SEQ ID NO:16, and SEQ ID NO:18. In various embodiments, the VHH domain that binds to CD8 comprises CDR1, CDR2, and CDR3 sequences selected from SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:12, and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:14, and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:16; SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:18; SEQ ID NO:3, SEQ ID NO:22, and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:14, and SEQ ID NO:18; SEQ ID NO:3, SEQ ID NO:27, and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:29, and SEQ ID NO:5; SEQ ID NO:3, SEQ ID NO:31, and SEQ ID NO:5; SEQ ID NO:73, SEQ ID NO:14, and SEQ ID NO:18; SEQ ID NO:74, SEQ ID NO:14, and SEQ ID NO:18; SEQ ID NO:3, SEQ ID NO:75, and SEQ ID NO:18; SEQ ID NO:3, SEQ ID NO:76, and SEQ ID NO:18; SEQ ID NO:3, SEQ ID NO:77, and SEQ ID NO:18; SEQ ID NO:3, SEQ ID NO:78, and SEQ ID NO:18; SEQ ID NO:3, SEQ ID NO:79, and SEQ ID NO:18; and SEQ ID NO:3, SEQ ID NO:80, and SEQ ID NO:18. In various embodiments, the VHH domain is humanized.

[0147] In some embodiments, the VHH domain that binds CD8 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to an amino acid sequence selected from SEQ ID NO:2, 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:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, and SEQ ID NO:100. In some embodiments, the VHH domain that binds CD8 comprises an amino acid sequence selected from SEQ ID NO:2, 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:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, and SEQ ID NO:100. In some embodiments, the VHH domain that binds CD8 comprises an amino acid sequence selected from SEQ ID NO:2, 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:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:30, where residue XX is absent.In some embodiments, the VHH domain that binds CD8 comprises an amino acid sequence selected from SEQ ID NO:2, 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:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, where residues XX are Gly-Gly. In some embodiments, the VHH domain that binds CD8 comprises an amino acid sequence selected from SEQ ID NO:2, 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:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99, wherein the VHH domain comprises the mutation K117D, K117E, or K117R.

[0148] In some embodiments, the VHH domain that binds CD8 comprises a CDR1 sequence of SEQ ID NO: 3, a CDR2 sequence of SEQ ID NO: 14, and a CDR3 of SEQ ID NO: 18. In some embodiments, the VHH domain that binds CD8 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 the amino acid sequence of SEQ ID NO: 25. In some embodiments, the VHH domain that binds CD8 comprises a CDR1 sequence of SEQ ID NO: 3, a CDR2 sequence of SEQ ID NO: 78, and a CDR3 of SEQ ID NO: 18. In some embodiments, the VHH domain that binds CD8 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 the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 100. In some embodiments, the CD8-binding VHH domain comprises the amino acid sequence of SEQ ID NO: 92. In some embodiments, the CD8-binding VHH domain comprises the amino acid sequence of SEQ ID NO: 100.

[0149] In various embodiments, the CD8 binding polypeptide comprises one, two, three, or four VHH domains that bind to CD8.

[0150] In some embodiments, the VHH domain that binds CD8 may be humanized. Humanized antibodies (such as sdAbs or VHH-containing polypeptides) are useful as therapeutic molecules because they reduce or eliminate human immune responses to non-human antibodies that may result in an immune response to antibody therapeutics and reduce the efficacy of the therapeutics. Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0151] 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).

[0152] 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".

[0153] In various embodiments, the Fc region comprised in the CD8 binding polypeptide is a human Fc region or is derived from a human Fc region.

[0154] In some embodiments, the Fc region comprised in the CD8 binding polypeptide is derived from a human Fc region and comprises a three amino acid deletion in the lower hinge corresponding to IgG1 E233, L234, and L235, and is referred to herein as "Fc xELL". Fc xELL polypeptides do not bind FcγR and are therefore referred to as "effector silent" or "effector null", however, in some embodiments, the xELL Fc region binds to FcRn, with associated extended half-life and transcytosis associated with FcRn mediated recycling.

[0155] In some embodiments, the Fc region included in the CD8 binding polypeptide is derived from a human Fc region and includes 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.

[0156] In some embodiments, the Fc region included in the CD8 binding polypeptide is derived from a human Fc region and includes mutations designed for heterodimerization, referred to herein as "knob" and "hole." 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.

[0157] In some embodiments, the Fc region comprised in the CD8 binding polypeptide is derived from a human Fc region and lacks the C-terminal lysine residue (ΔK447).

[0158] Non-limiting exemplary Fc regions that may be used in CD8 binding polypeptides include Fc regions comprising the amino acid sequences of SEQ ID NO: 32-70, and SEQ ID NO: 101-111. In some embodiments, the CD8 binding polypeptide comprises an Fc region comprising an amino acid sequence selected from SEQ ID NO: 33, SEQ ID NO: 36-52, SEQ ID NO: 68-70, and SEQ ID NO: 101-111.

[0159] Exemplary Activities of CD8 Binding Polypeptides In various embodiments, the CD8 binding polypeptides provided herein stimulate CD8+ cells in vitro and / or in vivo. In vitro and / or in vivo stimulation or activity of CD8+ cells can, in some embodiments, be determined using the methods provided in the Examples herein.

[0160] In some embodiments, the CD8 binding polypeptides provided herein comprise an antigen binding domain that binds to an immune cell activating cytokine or an antigen other than CD8 and stimulates CD8+ T cells. In some embodiments, the CD8+ stimulatory activity of the immune cell activating cytokine or antigen binding domain that binds to an antigen other than CD8 is increased compared to when used alone and / or is more specifically targeted to cytotoxic T cells when fused to a CD8 binding VHH. In some embodiments, the toxicity of the immune cell activating cytokine or antigen binding domain that binds to an antigen other than CD8 is reduced by specifically targeting CD8+ T cells.

[0161] In some embodiments, the immune cell activating cytokines provided herein, or CD8 binding polypeptides that contain an antigen binding domain that binds to an antigen other than CD8, increase T cell proliferation in vitro and / or in vivo.

[0162] In some embodiments, a CD8 binding polypeptide provided herein comprises a CD8 binding VHH provided herein and an immune cell activating cytokine. In some such embodiments, the immune cell activating cytokine is IL-2, IL-15, IL-7, IL-6, IL-12, IFNα, IFNβ, or IFNγ. In some such embodiments, the immune cell activating cytokine is a wild-type immune cell activating cytokine. In some embodiments, the immune cell activating cytokine comprises a mutation that attenuates the activity of the immune cell activating cytokine compared to the activity of the wild-type cytokine. In some embodiments, a CD8 binding polypeptide comprising an immune cell activating cytokine stimulates the activation and proliferation of CD8+ T cells in vivo. In some embodiments, a CD8 binding polypeptide comprising an immune cell activating cytokine is used in a method of treating cancer.

[0163] activated CD8 + Increased proliferation of T cells can be determined by any method in the art, such as, for example, the methods shown in the Examples herein. Non-limiting exemplary assays are as follows: CD8 + T cells can be isolated from one or more healthy human donors. T cells are stained with CellTrace Violet (CTV), activated with anti-CD3 antibodies, contacted with a polypeptide provided herein, and then analyzed by FACS. Loss of CTV staining indicates proliferation. In some embodiments, CD8 + Increased proliferation of T cells was observed, for example, in CD8 + By measuring the proliferation of T cells, the proliferation is determined as an average from a series of experiments or from pooled T cells. In some embodiments, CD8 + The increase in T cell proliferation is determined as an average from experiments performed using T cells from at least 5 or at least 10 different healthy donors, or from a pool of T cells from at least 5 or at least 10 different healthy donors.

[0164] In some embodiments, the CD8 binding polypeptides provided herein comprise a CD8 binding VHH and an antigen binding domain that binds to an antigen other than CD8. In some such embodiments, the antigen is Lag3, CTLA4, TGFBR1, TGFBR2, Fas, TNFR2, PD1, PDL1, or TIM3. In some embodiments, the antigen is 1-92-LFA-3, 5T4, alpha4 integrin, alphaV integrin, alpha4beta1 integrin, alpha4beta7 integrin, AGR2, anti-Lewis Y, apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, complement C5, C-242, CA9, CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD 19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD39, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD73, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD 138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), claudin 3, claudin 4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, E GFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FcRH5, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GPRC5D, GRP78, HAVCAR1, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MICA, MICB, MRP4, MUC1, mucin 16 (MUC16, CA-125), Na / K ATPase, NGF, nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, 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, T LR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, TROP-2, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, or WISP-3. In some embodiments, the CD8 binding polypeptide comprises a CD8 binding VHH and an antigen binding domain that binds to a tumor cell antigen.

[0165] Polypeptide Expression and Production Nucleic acid molecules are provided that comprise a polynucleotide encoding a CD8 binding polypeptide. In some embodiments, the nucleic acid molecule can also encode a leader sequence that directs secretion of the CD8 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.

[0166] 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.

[0167] Vectors are provided that contain a nucleic acid encoding a CD8 binding polypeptide as described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, a vector is selected that is optimized for expression of the polypeptide in a desired cell type, such as a CHO cell or CHO-derived cell, or an NSO cell. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0168] In some embodiments, CD8 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, CD8 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 a polypeptide. For example, in some embodiments, CHO cells produce polypeptides with higher levels of sialylation than the same polypeptides produced in 293 cells.

[0169] 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.

[0170] 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 CD8 binding polypeptides described herein. The CD8 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 matrix or hydrophobic interaction chromatography. Suitable affinity ligands include agents that bind to the ROR1 ECD and Fc region. For example, Protein A, Protein G, Protein A / G, or antibody affinity columns can be used to purify CD8 binding polypeptides comprising an Fc region by binding to the Fc region. Hydrophobic interaction chromatography, e.g., butyl or phenyl columns, can also be suitable for purifying some polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange and / or cation exchange chromatography) can 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.) can also be suitable for purifying some polypeptides, such as antibodies. Many methods for purifying polypeptides are known in the art.

[0171] In some embodiments, the CD8 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).

[0172] In some embodiments, a CD8 binding polypeptide produced by the above method is provided. In some embodiments, the CD8 binding polypeptide is produced in a host cell. In some embodiments, the CD8 binding polypeptide is produced in a cell-free system. In some embodiments, the CD8 binding polypeptide is purified. In some embodiments, a cell culture medium comprising the CD8 binding polypeptide is provided.

[0173] In some embodiments, compositions are provided that include an antibody produced by the above method. In some embodiments, the composition includes a CD8 binding polypeptide produced in a host cell. In some embodiments, the composition includes a CD8 binding polypeptide produced in a cell-free system. In some embodiments, the composition includes a purified CD8 binding polypeptide.

[0174] Exemplary Methods of Treating Disease Using CD8 Binding Polypeptides In some embodiments, methods are provided for treating a disease in an individual comprising administering a CD8 binding polypeptide. Such diseases include diseases that involve T cell, e.g., CD8 + Any disease that benefits from increased T cell proliferation and activation is included. In some embodiments, a method for treating cancer in an individual is provided. In some embodiments, the method for treating cancer comprises increasing proliferation and / or activation of CD8+ T cells by administering a CD8 binding polypeptide comprising a CD8 binding VHH and an antigen binding domain that binds to an immune cell activating cytokine or a tumor cell antigen other than CD8.

[0175] The method comprises administering to the individual an effective amount of a CD8 binding polypeptide provided herein. Such a method of treatment can be in a human or an animal. In some embodiments, a method of treatment in a human is provided. Non-limiting exemplary cancers that can be treated using the CD8 binding polypeptides provided herein include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, glioblastoma, hepatic cancer, hepatocellular carcinoma, intraepithelial neoplasia, renal or kidney cancer, laryngeal cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, These include testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, and vulvar cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-dividing cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloblastic leukemia.

[0176] The CD8 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 CD8 binding polypeptide is administered to the subject one or more times. In some embodiments, an effective dose of the CD8 binding polypeptide is administered to the subject daily, twice weekly, weekly, biweekly, monthly, and the like. An effective dose of the CD8 binding polypeptide is administered to the subject at least once. In some embodiments, an effective dose of the CD8 binding polypeptide may be administered multiple times, including multiple times over at least one month, at least six months, or at least one year.

[0177] In some embodiments, the pharmaceutical composition is administered in an amount effective to treat cancer (including cancer prevention) and / or increase T cell proliferation. The therapeutically effective amount typically depends on the weight of the subject being treated, the physical or health condition of the subject, the extent of the condition being treated, or the age of the subject being treated. In general, the antibody may be administered in an amount ranging from about 0.05 mg / kg (body weight) to about 100 mg / kg (body weight) per dose.

[0178] In some embodiments, the CD8 binding polypeptide may be administered in vivo by a variety of routes, including, but not limited to, intravenous, intraarterial, parenteral, intraperitoneal, or subcutaneous. Depending on the intended use, the appropriate formulation and route of administration may be selected.

[0179] In some embodiments, therapeutic treatment with CD8 binding polypeptides is achieved by increasing T cell proliferation and / or activation and / or by contacting CD8+ T cells with cancer cells, in some embodiments, increasing T cell proliferation and / or activation inhibits cancer growth.

[0180] Pharmaceutical Compositions In some embodiments, compositions comprising CD8 binding polypeptides are provided in formulations that include a wide variety of pharma- ceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 th ed., Lippencott Williams and Wilkins (2004), Kibbe et al., Handbook of Pharmaceutical Excipients, 3 rd ed., Pharmaceutical Press (2000). A variety of pharma- ceutically acceptable carriers, including excipients, adjuvants, and diluents, are available. In addition, a variety of pharma- ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like, are also available. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0181] In some embodiments, the pharmaceutical composition comprises the CD8 binding polypeptide at a concentration of at least 10 mg / mL.

[0182] Combination therapy The CD8 binding polypeptide may be administered alone or in combination with other therapeutic modalities, such as other anti-cancer agents. The CD8 binding polypeptide may be provided prior to, substantially simultaneously with, or subsequent to (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.

[0183] In some embodiments, a CD8 binding polypeptide provided herein is given simultaneously with a second therapeutic agent, eg, a PD-1 or PD-L1 therapeutic agent. Examples of PD-1 / PD-L1 therapies include nivolumab (BMS), pidilizumab (CureTech, CT-011), pembrolizumab (Merck), durvalumab (Medimmune / AstraZeneca), atezolizumab (Genentech / Roche), avelumab (Pfizer), AMP-224 (Amplimmune), BMS-936559, AMP-514 (Amplimmune), MDX-1105 (Merck), TSR-042 (Tesaro / AnaptysBio, ANB-011), STI-A1010 (Sorrento Therapeutics), STI-A1110 (Sorrento Therapeutics), and other agents directed against programmed death-1 (PD-1) or programmed death-ligand 1 (PD-L1).

[0184] In some embodiments, the CD8 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 CD8 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.

[0185] In some embodiments, the CD8 binding polypeptides provided herein are given concomitantly with CAR-T (chimeric antigen receptor T cell) therapy, oncolytic virus therapy, cytokine therapy, and / or agents targeting other checkpoint molecules such as VISTA, gpNMB, B7H3, B7H4, HHLA2, CTLA4, TIGIT, etc.

[0186] 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.

[0187] 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.

[0188] In some embodiments, the evaluation may prescribe treatment, including treatment with the antibodies described herein. In some embodiments, the evaluation may prescribe the use or withholding of adjuvant therapy after resection. Adjuvant therapy, also called adjuvant therapy, is a treatment given in addition to the primary, main, or initial treatment. As a non-limiting example, adjuvant therapy may be an additional treatment, usually given after surgery, when all detectable disease has been removed but there remains a statistical risk of relapse due to latent disease. In some embodiments, the antibody is used as an adjuvant therapy in the treatment of cancer. In some embodiments, the antibody is used as the sole adjuvant therapy in the treatment of cancer. In some embodiments, the antibody described herein is withheld as an adjuvant therapy in the treatment of cancer. For example, when a patient is unlikely to respond or has a minimal response to the antibodies described herein, treatment may not be administered for quality of life and to avoid unnecessary toxicity from ineffective chemotherapy. In such cases, palliative care may be used.

[0189] In some embodiments, the molecule is administered as a neoadjuvant therapy before resection. In some embodiments, neoadjuvant therapy refers to a therapy that shrinks and / or downgrades the tumor before any surgery. In some embodiments, neoadjuvant therapy refers to a chemotherapy drug administered to a cancer patient before surgery. In some embodiments, neoadjuvant therapy refers to an antibody administered to a cancer patient before surgery. Cancer types for which neoadjuvant chemotherapy is usually considered include, for example, breast cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, and lung cancer. In some embodiments, the antibody is used as a neoadjuvant therapy in the treatment of cancer. In some embodiments, the use is before resection.

[0190] In some embodiments, the tumor microenvironment contemplated by the methods described herein is one or more of tumor vasculature, tumor-infiltrating lymphocytes, fibroblastic reticular cells, endothelial progenitor cells (EPCs), cancer-associated fibroblasts, pericytes, other stromal cells, components of the extracellular matrix (ECM), dendritic cells, antigen-presenting cells, T cells, regulatory T cells, macrophages, neutrophils, and other immune cells located in proximity to the tumor.

[0191] kit Also provided are articles of manufacture and kits comprising any of the CD8 binding polypeptides described herein and suitable packaging. In some embodiments, the invention includes a kit comprising (i) a CD8 binding polypeptide and (ii) instructions for using the kit to administer the CD8 binding polypeptide to an individual.

[0192] 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.

[0193] 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

[0194] 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.

[0195] Example 1: Development of CD8a-binding VHH domains Single domain antibodies targeting human CD8a were generated by immunizing llamas with the extracellular domain of human CD8a fused to llama Fc (SEQ ID NO: 72). After the development of specific anti-CD8a antibody titers, llama peripheral blood mononuclear cells (PBMCs) were isolated from 500 mL of blood of the immunized animals, and total mRNA was isolated using Qiagen RNeasy Maxi Kit and subsequently converted to first-strand cDNA using Thermo Superscript IV Reverse Transcriptase and oligo dT priming. VHH sequences were specifically amplified by PCR using cDNA as template and cloned into yeast surface display vectors as VHH-Fc-AGA2 fusion proteins. Fc was human IgG1 Fc (SEQ ID NO: 32) or, in some cases, a mutant IgG1 Fc with reduced effector function (e.g., Fc xELL; SEQ ID NO: 33).

[0196] A yeast library displaying VHH-Fc-AGA2 fusion proteins was enriched with a recombinant form of the CD8a ECD by magnetic bead isolation followed by fluorescence-activated cell sorting (FACS). Sorted yeast were plated out and isolated colonies were picked into 96-well blocks and grown in medium that switches expression from surface-displayed VHH-Fc to secretion into the medium. Supernatants from 96-well yeast secretion cultures were applied to 293F cells transiently transfected with CD8a (CD8a positive) or non-transfected 293F cells (CD8a negative), washed, treated with a fluorophore-labeled anti-human IgG1 Fc secondary antibody, and analyzed by 96-well flow cytometry.

[0197] Nucleic acid sequences encoding VHHs that bind to CD8a-positive cells but not to CD8a-negative cells were cloned in frame with the human Fc xELL coding region into a mammalian expression vector and expressed by transient transfection in HEK293 Freestyle cells (293F cells) or CHO cells using polyethyleneimine. After 3 to 7 days, the supernatant was collected, and the secreted recombinant protein was purified by protein A chromatography, and the concentration was calculated from the absorbance at 280 nm and the extinction coefficient.

[0198] One VHH domain (clone B7) that binds to CD8a was humanized. Briefly, various humanized versions of B7 were generated based on the human heavy chain framework. Certain amino acids were backmutated to donor amino acids and certain mutations were tested, for example, in the CDRs for their binding properties. The amino acid sequence of B7 and the various humanized forms are shown in certain sequence tables presented below. It is noted that the sequences of B7 VHH (SEQ ID NO:2) and humanized versions hzB7v1 to hzB7v18 (SEQ ID NO:6 to SEQ ID NO:30) may include an optional Gly-Gly (GG) linker (represented by XX in certain sequence tables) at the C-terminus. In addition, it is indicated that the lysine at residue 117 (K117) of any of the disclosed VHH domains may be substituted with aspartic acid (K117D), glutamic acid (K117E), or arginine (K117R). The humanized VHH named hzB7v41 (SEQ ID NO: 100) contains a K117R substitution (shown in bold and underlined in the table of certain sequences).

[0199] Binding of CD8a binding polypeptides formatted as CD8a VHH-hIgG1-Fc was assessed by flow cytometry on isolated human CD8+ T cells. Isolated T cells were plated in 96-well plates at 30000 cells per well in FACS buffer (PBS, 1% BSA, 0.1% NaN3, pH 7.4). Non-transfected HEK293F cells were used as a CD8a negative control and plated at 30000 cells per well in a separate plate. Test polypeptides were then diluted 2-fold to a final concentration of 1000 nM, followed by 3-, 4-, and 5-fold serial dilutions. FACS buffer without polypeptide was used as a secondary antibody only control. Polypeptide dilutions were added to an equal volume of cells and the assay plate was incubated at 4°C for 30 minutes. After two washes with 150 μL FACS buffer per well, cells were resuspended in FACS buffer containing fluorescently labeled anti-human Fc antibody diluted 1:2000 to detect binding and fluorescently labeled anti-CD4 antibody (clone OKT4) diluted 1:100 as counterstain. Assay plates were incubated for 20 min at 4°C. After one further wash with 150 μL FACS buffer per well, bound polypeptides were detected by flow cytometry in CD4- cells. CD8a binding was measured in this population as median fluorescence at 647 nm. Data were plotted and analyzed using GraphPad Prism analysis software. Flow cytometric detection was performed on Intellicyt's iQue Plus. The obtained maximum binding (Bmax) values ​​and binding affinity (K d ) are shown in Tables 2 and 3, and the binding curves are shown in Figures 1A and 1B and 2A and 2B. Higher Bmax values ​​generally indicate slower off-rates, and similarly, lower Bmax values ​​indicate faster off-rates.

[0200] [Table 2]

[0201] [Table 3]

[0202] As shown in Figures 1A and 2A and in the tables above, the CD8 binding polypeptides tested bound to human CD8+ T cells with affinities less than 0.2 nM, and in most cases less than 0.1 nM. As shown in Figures 1B and 2B, all of the tested polypeptides, except for the parental B7-xELL-Fc, showed no significant binding to 293 control cells, and B7-xELL-Fc bound to control cells with over 2000-fold lower affinity compared to binding to CD8+ T cells. These results demonstrate that the CD8a binding polypeptides specifically bind to CD8.

[0203] Example 2: Binding of CD8-binding polypeptides to human and cynomolgus monkey CD8 The binding of the parental and two humanized CD8a-binding polypeptides described above was assessed by flow cytometry in transfected HEK293F cells. HEK293F cells were transiently transfected with plasmids encoding full-length human or cynomolgus CD8a followed by IRES and GFP. Transfected cells were plated in 96-well plates at 30000 cells per well in FACS buffer (PBS, 1% BSA, 0.1% NaN3, pH 7.4). Test polypeptides were then diluted 2-fold to a final concentration of 500 nM, followed by 3-, 4-, and 5-fold serial dilutions. FACS buffer without polypeptide was used as a secondary antibody-only control. Test polypeptides were added to an equal volume of cells, and the assay plate was incubated for 30 min at 4°C. After washing twice with 150 μL FACS buffer per well, cells were resuspended in FACS buffer containing a 2000-fold diluted fluorescently labeled anti-human Fc antibody. The assay plate was incubated for 20 minutes at 4°C. After one further wash with 150 μL FACS buffer, bound polypeptides were detected by flow cytometry. Flow cytometric detection was performed on Intellicyt's iQue Plus. CD8a expressing 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. The results are shown in Tables 4 and 5 and in Figures 3A and 3B.

[0204] [Table 4]

[0205] [Table 5]

[0206] As shown in Figure 3A and Table 4, the CD8 binding polypeptides tested bound to transfected HEK293F cells expressing human CD8a with an affinity of less than 0.6 nM. As shown in Figure 3B and Table 5, the CD8 binding polypeptides tested bound to HEK293F cells expressing cynomolgus monkey CD8a with an affinity of less than 0.1 nM.

[0207] Example 3: CD8-binding polypeptides bind to human and cynomolgus immune cells The binding of the above parent polypeptides and two humanized CD8a binding polypeptides was assessed by flow cytometry on isolated human T cells and isolated cynomolgus monkey PBMCs. Isolated cells were plated in 96-well plates in FACS buffer (PBS, 1% BSA, 0.1% NaN3, pH 7.4) at 200,000 cells per well for cynomolgus monkey PBMCs and 50,000 cells per well for human T cells. Test polypeptides were then diluted 2-fold to a final concentration of 250 nM and serial 4-fold dilutions were performed. FACS buffer alone was used as a secondary antibody only control. Polypeptide dilutions were added to an equal volume of cells and the assay plate was incubated at 4° C. for 30 minutes. After washing twice with 150 μL of FACS buffer per well, the cells were resuspended in FACS buffer containing fluorescently labeled anti-human antibody diluted 1:1000, fluorescently labeled anti-CD3 antibody (clone SP34.2) diluted 1:40, and anti-CD4 antibody (clone OKT4) diluted 1:100 to detect CD8a binding. The assay plate was incubated at 4° C. for 20 minutes. After one further wash with 150 μL of FACS buffer, CD8a binding was detected by flow cytometry in CD3+CD4− cells. Binding was measured as the mean fluorescence at 647 nm in these cell populations. Data were plotted and analyzed using GraphPad Prism analysis software. Flow cytometric detection was performed on a Novocyte-Quanteon flow cytometer from ACEA Biosciences. The results are shown in Tables 6 and 7 and in Figures 4A and 4B.

[0208] [Table 6]

[0209] [Table 7]

[0210] As shown in Figure 4A and Table 6, the CD8 binding polypeptides tested bound to human CD3+CD4- T cells with an affinity of less than 0.1 nM. As shown in Figure 4B and Table 7, the CD8 binding polypeptides tested bound to cynomolgus monkey CD3+CD4- cells with an affinity of less than 0.08 nM.

[0211] Binding of the CD8a binding polypeptide hzB7v15-xELL-Fc was assessed by flow cytometry in human leukopak T cells and cynomolgus monkey PBMCs. Leukopak T cells were thawed in CTL anti-aggregate wash thawing solution and plated into 96-well U-bottom assay plates. Cells were centrifuged at 400 x g for 5 min and the supernatant was discarded. hzB7v15-xELL-Fc was serially diluted 3-fold across 10 wells from an initial concentration of 200 nM. FACS buffer was used as a non-binding control and plates were incubated at 4°C for 30 min. Assay plates were centrifuged at 400 x g for 5 min and the supernatant was discarded. Cells were washed once and resuspended in staining panel (anti-CD3 antibody clone OKT3-BV605 (1:200), anti-CD4 antibody clone OKT4-BV785 (1:200), and fluorescently labeled anti-human Fc antibody (1:500)) for 30 minutes at 4°C. Assay plates were centrifuged at 400 x g for 5 minutes and the supernatant was discarded. Cells were washed with 150 μL FACS buffer, resuspended in 70 μL FACS buffer, and read on a Novocyte flow cytometer. Results are shown in Table 8 below and Figure 5A.

[0212] Cynomolgus monkey PBMCs were thawed in CTL anti-aggregate wash thaw solution and plated at 500,000 cells per well in 96-well U-bottom assay plates. Cells were centrifuged at 400×g for 5 min and the supernatant was discarded. Alexa Fluor 647 chemically labeled hzB7v15-xELL-Fc (AF647-hzB7v15-xELL-Fc) was serially diluted 3-fold across 10 wells from an initial assay concentration of 30 nM. FACS buffer was used as a non-binding control and plates were incubated at 4°C for 20 min. Assay plates were centrifuged at 400×g for 5 min and the supernatant was discarded. The cells were washed with 150 μL of PBS buffer, resuspended in 40 μL of FACS buffer, and 10 μL of BV staining buffer (Brilliant Stain Buffer Plus; BD Biosciences) in FACS buffer and 50 μL of antibody mixture (anti-CD3 antibody clone SP34-BV421 (1:25), anti-CD4 antibody clone OKT4-BV785 (1:100), and anti-CD16 antibody clone 3G8-PE (1:100)) were added to the cells. The cells were stained for 20 minutes at 4° C. The assay plate was centrifuged at 400×g for 5 minutes and the supernatant was discarded. The cells were washed with 150 μL of FACS buffer, resuspended in 70 μL of FACS buffer, and read on a Novocyte flow cytometer. The results are shown in Table 9 below and in FIG. 5B.

[0213] [Table 8]

[0214] [Table 9]

[0215] As shown in Figure 5A and Table 8, hzB7v15-xELL-Fc bound to human CD3+CD4- T cells with an affinity of less than 0.1 nM. As shown in Figure 5B and Table 9, hzB7v15-xELL-Fc bound to cynomolgus monkey CD3+CD4-CD16- cells with an affinity of less than 0.06 nM.

[0216] Example 4: Targeting CD8a of attenuated IL-2 restores activity The CD8a-targeting IL-2 activity of polypeptides comprising a CD8a-binding VHH hzB7v15 or VHH hzB7v31 domain, an Fc region, and an attenuated IL-2 fused to the C-terminus of the Fc region was evaluated in IL-2 reporter cells. The fusion proteins were dimeric and contained a VHH hzB7v15 domain or a VHH hzB7v31 domain fused to a knob Fc region, an attenuated IL-2, and a VHH hzB7v15 domain or a VHH hzB7v31 domain fused to a hole Fc region. Thus, the dimeric fusion protein contained two CD8a-binding VHH domains, two Fc regions, and one attenuated IL-2. HEK-Blue IL2 reporter cells or CD8a-expressing HEK-Blue IL2 reporter cells were detached, transferred to a 50 mL conical tube, pelleted at 400 × g for 5 min, and diluted to 0.5 × 10 in fresh pre-warmed assay medium (DMEM + 4.5 g / L glucose, 2 mM L-glutamine + 10% heat-inactivated FBS + 100 U / mL penicillin + 100 μg / mL streptomycin + 100 μg / mL normocin). 6 Cells were resuspended at a density of 10000 cells / ml. Polypeptide dilution series were prepared in assay medium at 2x the final concentration and 100 μL was added per well. 50,000 cells in 100 μL were added to each well of a flat-bottom 96-well tissue culture treated plate. Plates were incubated for 20 hours at 37°C in a CO2 incubator. Quanti-Blue solution was prepared according to the manufacturer's instructions (resuspended in water and warmed to 37°C in a water bath for 30 minutes). Assay plates were spun down at 400×g for 5 minutes. 100 μL of supernatant was transferred to a new flat-bottom 96-well tissue culture plate and 100 μL / well of Quanti-Blue solution was added and incubated for 1-2 hours at 37°C in a 5% CO2 incubator. Absorbance was read at 650 nm on an EMax plate reader.

[0217] As shown in FIG. 6A, the CD8a-targeting polypeptide containing attenuated IL-2 showed significantly lower activity in cells not expressing CD8a than the polypeptide containing the untargeted VHH domain and wild-type IL-2. As shown in FIG. 6B and FIG. 6C, in cells expressing CD8a, the polypeptide containing the CD8a-binding VHH and attenuated IL-2 showed strong IL-2 activity similar to the polypeptide containing the untargeted VHH domain and wild-type IL-2. The polypeptide containing the untargeted VHH domain and attenuated IL-2 showed significantly lower activity in CD8a-expressing reporter cells. These results indicate that IL-2 activity can be specifically targeted to CD8a-expressing cells in this reporter assay over a wide concentration range of approximately 0.01 nM to 1 nM.

[0218] Example 5: T cell proliferation induced by a polypeptide comprising a CD8a-binding VHH and attenuated IL-2 The effect of a fusion protein comprising attenuated IL-2 fused to the C-terminus of the CD8a-binding VHH hzB7v15 on CD8+ T cell expansion was tested in non-human primates. Cynomolgus monkeys were given an intravenous bolus injection of 0.3 mg / kg of the fusion protein. Whole blood samples were taken from the study animals before and 7 days after fusion protein administration. Peripheral blood mononuclear cells (PBMCs) at each time point were isolated using density centrifugation on Lymphoprep™, and cells were stained with combinations of fluorescently labeled cell type specific antibodies. T cells were classified as CD3+ cells that expressed CD4 or CD8a and did not express the B cell marker CD20. Regulatory T cells ("Treg") were defined as CD4+ T cells that also expressed CD25 and had reduced levels of CD127. CD4+ conventional T cells ("CD4+Tcon") were defined as CD4+ T cells that did not express CD25 and had normal levels of CD127. NK cells were defined as non-T and non-B cells expressing NKG2A. CD20 positive staining populations were classified as B cells. Absolute cell numbers of each PBMC subpopulation were determined using flow cytometry, and fold increases were calculated by dividing the absolute cell numbers 7 days after treatment by the baseline numbers before treatment.

[0219] As shown in Figure 7, a single dose of 0.3 mg / kg CD8-targeted attenuated IL-2 resulted in a 5.6-fold increase in CD8+ T cells and a 2.8-fold increase in NK cells, but had no significant effect on CD8- cell populations, including Tregs, CD4+ conventional T cells, and B cells. Additionally, the increase in CD8+ T cell numbers resulted in a 3.2-fold increase in total T cells, and the total PBMC numbers were increased 2.7-fold over the cell numbers before administration. These data indicate that CD8a-targeted attenuated IL-2 specifically induces cell proliferation of CD8+ cell populations in vivo.

[0220] Example 6: Binding of CD8a-binding polypeptides to human CD8 chains expressed on 293F cells Binding of polypeptides comprising a humanized CD8a-binding VHH domain, an Fc region, and, in certain polypeptides, a mutant attenuated IL-2 fused to the C-terminus of the Fc region was assessed by flow cytometry in HEK293F cells transiently transfected with plasmids encoding the human CD8a or CD8b chain. Complexes or polypeptides designated "KiH" contain knob-in-hole heterodimeric Fc regions in which the indicated CD8a-binding VHH domain is fused to the N-terminus of each Fc region and mutant IL-2 is fused to the C-terminus of only the "knob" Fc region. Complexes or polypeptides not designated "KiH" form homodimers under physiological conditions. Transfected cells were plated in 96-well plates at 50,000 cells per well in FACS buffer (PBS, 1% BSA, 0.1% NaN3, pH 7.4). Test polypeptides were then diluted 2-fold to a final concentration of 500 nM, with 6-fold serial dilutions. FACS buffer without polypeptide was used as a secondary antibody-only control. Test polypeptides were added to an equal volume of cells, and the assay plate was incubated at 4°C for 30 min. After two washes with 150 μL FACS buffer per well, cells were resuspended in FACS buffer containing fluorescently labeled anti-human Fc antibody diluted 1000-fold to detect CD8 binding. The assay plate was incubated at 4°C for 30 min. After one further wash with 150 μL FACS buffer, polypeptides bound to CD8 were detected by flow cytometry in cells positive for the transfection marker citrine. Binding was measured in these cell populations as mean fluorescence intensity (MFI) at 647 nm. Flow cytometric detection was performed with IntelliCyt's iQue Screener Plus. Data were plotted and analyzed using GraphPad Prism analysis software. The results are shown in Table 10 and in Figures 8A and 8B.

[0221] [Table 10]

[0222] As shown in Figure 8A and Table 10, the CD8a binding polypeptides tested bound to human CD8a with affinities in the low nanomolar range. Figure 8B shows that the polypeptides bound to human CD8b with low to negligible affinity.

[0223] Example 7: Binding of CD8a-binding polypeptides to T cells Binding of polypeptides comprising a humanized CD8a-binding VHH domain, an Fc region, and, in certain polypeptides, a mutant attenuated IL-2 fused to the C-terminus of the Fc region was assessed by flow cytometry in isolated human T cells. Complexes or polypeptides designated "KiH" contain knob-in-hole heterodimeric Fc regions in which the designated CD8a-binding VHH domain is fused to the N-terminus of each Fc region and mutant IL-2 is fused to the C-terminus of only the "knob" Fc region. Complexes or polypeptides not designated "KiH" form homodimers under physiological conditions. Isolated cells were plated in 96-well plates at 50,000 cells per well in FACS buffer (PBS, 1% BSA, 0.1% NaN3, pH 7.4). Test polypeptides were then diluted 2-fold to a final concentration of 200 nM, with 5-fold serial dilutions. FACS buffer without polypeptide was used as a secondary antibody only control. Test polypeptides were added to equal volumes of cells and assay plates were incubated for 30 min at 4°C. After washing twice with 150 μL FACS buffer per well, cells were resuspended in FACS buffer containing fluorescently labeled anti-human IgG antibody diluted 1:1000 to detect CD8a binding and fluorescently labeled anti-CD4 antibody (clone OKT4, 1:200). Propidium iodide (PI) was added at 1:2000 to distinguish live from dead cells. Assay plates were incubated for 30 min at 4°C. After one further wash with 150 μL FACS buffer, polypeptides bound to CD8a were detected by flow cytometry in PI-CD4- and PI-CD4+ cells. Binding was measured in these cell populations as mean fluorescence intensity (MFI) at 647 nm. Flow cytometric detection was performed on an ACEA Biosciences Novocyte-Quanteon flow cytometer. Data was plotted and analyzed using GraphPad Prism analysis software, and the results are shown in Table 11 and Figures 9A and 9B.

[0224] [Table 11]

[0225] As shown in Figure 9A and Table 11, the CD8a binding polypeptides tested bound to human CD8 T cells with affinities in the low nanomolar to subnanomolar range, and Figure 9B shows that the polypeptides did not bind to human CD4 T cells.

[0226] Example 8: Binding of CD8a-binding polypeptides to human and cynomolgus CD8a Binding of four polypeptides containing humanized CD8a-binding VHH domains fused to the xELL fc region was assessed by flow cytometry in isolated human T cells and isolated human or cynomolgus peripheral blood mononuclear cells (PBMCs). Isolated cells were plated in 96-well plates at 200,000 cells per well for cynomolgus PBMCs and 100,000 cells per well for human T cells in FACS buffer (PBS, 1% BSA, 0.1% NaN3, pH 7.4). Test polypeptides were then diluted 2-fold to a final concentration of 25 nM or 50 nM, and 3- or 5-fold serial dilutions were prepared. FACS buffer alone was used as a secondary antibody-only control. Polypeptide dilutions were added to an equal volume of cells and the assay plate was incubated at 4°C for 30 minutes. After washing twice with 150 μL FACS buffer per well, cells were resuspended in FACS buffer containing fluorescently labeled anti-human IgG antibody diluted 1:1000 to detect CD8a binding, fluorescently labeled anti-CD3 antibody (clone SP34.2, 1:50, for PBMC preparation only), and fluorescently labeled anti-CD4 antibody (clone OKT4, 1:100). Propidium iodide (PI) was added at 1:2000 to distinguish live from dead cells. Assay plates were incubated for 30 min at 4°C. After one further wash with 150 μL FACS buffer, CD8a binding was detected by flow cytometry in PI− (CD3+)CD4− cells. Binding was measured in these cell populations as mean fluorescence intensity (MFI) at 647 nm. Flow cytometric detection was performed on an ACEA Biosciences Novocyte-Quanteon flow cytometer. Data was plotted and analyzed using GraphPad Prism analysis software, and the results are shown in Tables 12 and 13 and Figures 10A-10D.

[0227] [Table 12]

[0228] [Table 13]

[0229] As shown in Figures 10A and 10E and Table 12, the CD8a binding polypeptides tested bound to human CD8 T cells with an affinity of 0.1 nM or less. Figures 10B and 10F show that these polypeptides did not bind to human CD4 T cells. As shown in Figure 10C and Table 13, the CD8a binding polypeptides tested bound to cynomolgus CD8 T cells with an affinity of less than 0.04 nM. Figures 10D and 10H show that the polypeptides did not bind to cynomolgus CD4 T cells.

[0230] Example 9: Specific IL-2 signaling induced by a polypeptide comprising a CD8a-binding VHH and attenuated IL-2 The CD8a-targeted IL-2 activity of polypeptides comprising a CD8-binding VHH domain (hzB7v31, SEQ ID NO: 92, or hzB7v41, SEQ ID NO: 100), an Fc region, and a mutant attenuated IL-2 fused to the C-terminus of the Fc region was evaluated in a pSTAT5 assay. Control proteins included a polypeptide comprising a CD8a-binding VHH domain and an Fc region but without IL-2, a fusion protein comprising a non-targeting VHH, an Fc region, and a mutant attenuated IL-2, and wild-type IL-2. Increases in the levels of phosphorylated STAT5 (pSTAT5) or the percentage of cells expressing pSTAT5 were measured by intracellular flow cytometry as a proximity readout of IL-2 receptor ligation and signaling. Enriched human T cells were plated in 96-well plates at 500,000 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 200 nM or 50 nM, and serial 4-fold dilutions were performed. Serial dilutions were added to 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), FoxP3 (236A / E7, 1:40), pSTAT5 (SRBCZX, 1:70), CD25 (M-A251, 1:500) and CD8 (RPA-T8, 1:4000). The next day, cells were washed with 150 μL FACS buffer and analyzed using an ACEA Biosciences Novocyte-Quanteon flow cytometer.IL-2 signaling was quantified by the increase in median fluorescence intensity or the percentage of positive cells stained with a fluorescently labeled antibody detecting pSTAT5 on CD8 T cells (CD3+CD8+) or regulatory T cells (Treg, CD3+CD4+FoxP3+). Data were plotted and analyzed using GraphPad Prism analysis software.

[0231] As shown in Figures 11A and 11C, the tested polypeptides comprising the CD8a-binding VHH hzB7v31 or VHH hzB7v41 domain, an Fc region, and a mutant attenuated IL-2 fused to the C-terminus of the Fc region had an EC 50 IL-2 induced a concentration-dependent increase in the levels of pSTAT5 or the percentage of pSTAT5-positive CD8 T cells at 1.6 nM. Wild-type IL-2 (non-targeted) had an EC 50 Wild-type IL2 showed approximately 50-fold lower activity at EC 50 Although CD8a-targeted attenuated IL-2 induced IL-2 receptor signaling in Tregs, no detectable increase in Treg pSTAT5 or the percentage of pSTAT5-positive CD4 T cells was induced by CD8a-targeted attenuated IL-2 (Figures 11A-11D). Neither the polypeptide containing CD8a-hzB7v31, without IL-2, nor the polypeptide containing the untargeted VHH, Fc region, and mutant attenuated IL-2 induced a detectable increase in pSTAT5 levels in any of the cell types tested, indicating that attenuated IL-2 requires targeting to cells to induce IL-2 receptor signaling activity (Figures 11A-11D).

[0232] Example 10: T cell proliferation of human tumor-infiltrating T cells and healthy donor T cells induced by a polypeptide comprising a CD8a-binding VHH and attenuated IL-2 The activity of the polypeptides comprising the CD8a-binding VHH hzB7v31 domain, an Fc region, and a mutant attenuated IL-2 fused to the C-terminus of the Fc region was further evaluated in a proliferation assay using dissociated tumor cell (DTC) samples from human cancer patients or PBMCs from healthy human donor blood. DTC single cell suspensions were generated from biopsies of head and neck, kidney, or colon tumors using a human tumor dissociation kit (Miltenyi Biotec). DTCs or PBMCs were then labeled with the proliferation dye CellTrace Violet (Thermo) according to the manufacturer's recommended protocol. Cells were incubated in complete growth medium (RPMI, 10% FBS, 1% anti-anti) supplemented with 10 nM of test polypeptide or 5-fold dilutions of test polypeptide starting at a concentration of 200 nM. Control proteins included a polypeptide containing CD8a-hzB7v31 formatted as VHH-hIgG1-xELL Fc, a fusion protein containing non-targeting VHH-hIgG1-xELL Fc and mutant attenuated IL-2, and wild-type IL-2. After 6 or 7 days of culture, cell subpopulations were labeled with fluorescently tagged antibodies against CD3 (Hit3, 1:100), CD4 (OKT4, 1:200), CD8 (RPA-T8, 1:200), and CD45 (HI30, 1:100), as well as propidium iodide (PI, 1:2000) to distinguish live from dead cells. T cells were classified as CD45+CD3+PI- cells expressing either CD4 or CD8a. The cell numbers of these T cell subpopulations were quantified using flow cytometry on days 6 or 7. Flow cytometric detection was performed on an ACEA Biosciences Novocyte-Quanteon flow cytometer. Data were plotted and analyzed using GraphPad Prism analysis software. Cell numbers were normalized to samples treated with CD8a-hzB7v31-Fc-xELL, and the fold increase in cell numbers was determined relative to a control polypeptide that does not contain IL-2 and does not induce cell proliferation. Percent proliferation was determined by quantifying the percent of cells with CellTrace Violet fluorescence intensity lower than the parental non-dividing cell peak.

[0233] As shown in Figures 12A and 12C, the tested polypeptides comprising the CD8a-binding VHH hzB7v31 domain, an Fc region, and mutant attenuated IL-2 fused to the C-terminus of the Fc region induced proliferation of CD8 T cells in dissociated tumor samples and healthy PBMCs. Also, wild-type IL2 induced proliferation of both CD8 (Figures 12A and 12C) and CD4 (Figure 12B) T cells, whereas CD8a-targeted mutant attenuated IL-2 did not induce proliferation of CD4 T cells. Neither the polypeptide comprising CD8a-hzB7v31 without IL-2 nor the polypeptide comprising the non-targeted VHH, an Fc region, and mutant attenuated IL-2 induced a detectable increase in proliferation of CD8 or CD4 T cells, indicating that attenuated IL-2 requires targeting to cells to induce IL-2 receptor signaling activities such as proliferation.

[0234] Example 11: Cell expansion of a subpopulation of cynomolgus monkey PBMCs induced by a polypeptide comprising a CD8a-binding VHH and attenuated IL-2 The effect of a fusion protein comprising the CD8a-binding VHH hzB7v15, xELL P329G, a knob-in-hole heterodimeric Fc region, and attenuated IL-2 fused to the C-terminus of the "knob" Fc on in vivo cell expansion was tested in non-human primates. Cynomolgus monkeys were given an intravenous bolus injection of 1.0 mg / kg of the fusion protein. Whole blood samples were taken from the study animals before and 7 days after fusion protein administration. PBMCs at each time point were isolated using density centrifugation on Lymphoprep™, and cells were stained with a combination of fluorescently labeled cell type-specific antibodies. T cells were classified as CD3+ cells that expressed CD4 or CD8a and did not express the B cell marker CD20. Regulatory T cells ("Treg") were defined as CD4+ T cells that also expressed CD25 and had reduced levels of CD127. CD4+ conventional T cells ("CD4+Tcon") were defined as CD4+ T cells that did not express CD25 and had normal levels of CD127. NK cells were defined as non-T and non-B cells that expressed NKG2A and were either positive or negative for CD16. CD20 positive staining populations were classified as B cells. Absolute cell numbers of each PBMC subpopulation were determined using flow cytometry, and fold increases were calculated by dividing the absolute cell numbers 7 days after treatment by the baseline numbers before treatment. Ki67 expression was measured in the above PBMC subpopulations using additional fixation, permeabilization, and staining steps. Briefly, cells were stained with a combination of fluorescently labeled cell type specific antibodies for cell surface markers, followed by fixation and permeabilization using FoxP3 Transcription Factor Staining Buffer Set (eBioscience). FoxP3 and Ki67 were then detected with specific fluorescently labeled antibodies. T cells were classified as CD3+ cells that expressed CD4 or CD8a and did not express the NK cell marker NKG2A. Regulatory T cells ("Treg") were defined as CD4+ T cells that also expressed CD25 and FoxP3. CD4+ conventional T cells ("CD4+Tcon") were defined as CD4+ T cells that did not express CD25 or FoxP3. NK cells were defined as non-T cells that expressed NKG2A and were either positive or negative for CD16.Flow cytometry detection was performed on an ACEA Biosciences Novocyte-Quanteon flow cytometer. Data were plotted and analyzed using GraphPad Prism analysis software. Fold change was calculated by dividing the number of cells per mL of whole blood on day 7 by the number of cells per mL of whole blood at baseline (pre-dose).

[0235] As shown in Figure 13A, a single dose of 1 mg / kg CD8a-targeted attenuated IL-2 resulted in a 5-fold increase in CD8 T cells, as well as a 3.9-fold and 4.7-fold increase in CD8a-expressing CD16+ or CD16- NK cells, respectively. The numbers of CD8a-negative cell populations, including Tregs, CD4+ conventional T cells, and B cells, did not increase significantly between the pre-dose blood draw and day 7. Figure 13B shows that the specific increase in CD8a-expressing cell populations in vivo was accompanied by a specific increase in the proliferation marker Ki67. The percentage of Ki67+ proliferating CD8 T cells increased from 6% at baseline to 58% at day 7, while the CD16+ and CD16- NK cell populations showed a mean increase of Ki67+ cells of 40% to 53% over the same time frame. The percentage of Ki67+ populations within the CD8a-negative cell population, including Tregs and CD4+ conventional T cells, did not change. These data indicate that CD8a-targeted attenuated IL-2 specifically induced cell proliferation of CD8a-positive cell populations in vivo.

[0236] Example 12: Enhancement of CD8 T cell cytotoxic activity and antibody-dependent cellular cytotoxicity against human cancer cells induced by a polypeptide comprising a CD8a-binding VHH and attenuated IL-2 The activity of fusions comprising the CD8a-binding VHH hzB7v31 domain, the xELL knob-in-hole heterodimeric Fc region, and attenuated IL-2 fused to the C-terminus of the "knob" Fc region was further evaluated in tumor cell killing assays using enriched CD8 T cells and in antibody-dependent cellular cytotoxicity (ADCC) assays in combination with cetuximab. Control proteins included a fusion protein comprising the non-targeting VHH-hIgG1-xELL Fc and mutant attenuated IL-2, and wild-type IL-2. For the CD8 T cell killing assay, PBMCs from healthy human donor blood were used to isolate CD8 T cells, and enriched cells were stimulated for 3 days with antibodies against CD3 (clone: ​​OKT3) coated on culture plates at 1 μg / mL in the presence or absence of additional cytokine support from wild-type IL-2 or a fusion protein containing CD8a-binding VHH hzB7v31-hIgG1-xELL Fc and mutant attenuated IL-2 (1 nM each). On the day of the target cell killing assay, A431 cells were labeled with CYTO-ID red long-term cell tracer (Enzo) and then plated in 96-well flat-bottom plates at 4000 cells per well in 100 μL and allowed to adhere for 4 hours. Pre-stimulated CD8 T cells were washed once in PBS and added to the labeled A431 target cells at different effector to target cell ratios (20:1, 10:1 and 5:1) as indicated. Caspase-3 / 7 Green Dye (Sartorius) was added to each well to detect cell death, and A431 death was determined after 20 h by quantifying the overlap of Caspase-3 / 7 and CYTO-ID red using an Incucyte imager.

[0237] For the ADCC assay, A431 cells were labeled with CYTO-ID red long-term cell tracer (Enzo) and then plated in 96-well flat-bottom plates at 10,000 cells per well in 100 μL and allowed to adhere for 4 hours. Human PBMCs 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 1:2000 per 25 μL of medium, or 25 μL of ADCC antibody cetuximab at a final concentration of 20 nM, wild-type recombinant IL-2 at a final concentration of 1 nM, or IL-2 mutant fusion polypeptide at a final concentration of 1 nM per 25 μL of medium, and 25 μL of human PBMCs adjusted to a concentration of 10 or 5 NK cells per A431 cell. Cells were allowed to settle for 10 minutes at room temperature, and then the plates were placed in an Incucyte imager at 37° C. for imaging. A431 killing was determined after 15 hours by quantifying the overlap of Caspase-3 / 7 and CYTO-ID red, with maximum killing defined by 20 nM cetuximab. All data were plotted and analyzed using GraphPad Prism analysis software.

[0238] As shown in Figures 14A and 14B, the tested polypeptides containing the CD8a-binding VHH hzB7v31 domain, an Fc region, and mutant attenuated IL-2 fused to the C-terminus of the Fc region enhanced the relative cytotoxicity of CD8 T cells at different effector-to-target cell ratios (Figure 14A) and helped improve cetuximab-driven ADCC activity of PBMCs against EGFR-positive A431 target cells at suboptimal effector-to-target cell ratios (Figure 14B). The extent of activity by CD8 T cells was 3-4 times higher than that observed with wild-type IL-2, but was comparable in the ADCC assay. A fusion protein containing non-targeted VHH-hIgG1-xELL Fc and mutant attenuated IL-2 failed to improve ADCC activity at lower effector-to-target cell ratios, indicating that attenuated IL-2 requires targeting to effector cells to induce IL-2 receptor signaling activity and enhanced cytotoxicity.

[0239] 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.

[0240] [Table 14] TIFF2024526835000016.tif253170TIFF2024526835000017.tif255169TIFF2024526835000018.tif253170TIFF2024526835000019.tif253170TIFF2024526835000020.tif255169TIFF2024526835000021.tif255169TIFF2024526835000022.tif255170TIFF2024526835000023.tif255170TIFF2024526835000024.tif255169TIFF2024526835000025.tif161170

Claims

**Claim 1** A polypeptide comprising at least one VHH domain, wherein the VHH domain comprises CDR1, CDR2, and CDR3, and CDR1 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 78, and SEQ ID NO: 18; or CDR1 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 14, and SEQ ID NO: 18; or CDR1 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; or CDR1 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 12, and SEQ ID NO: 5; or CDR1 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 14, and SEQ ID NO: 5; or CDR1 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 16; or CDR1 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 18; or CDR1 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 22, and SEQ ID NO: 5; or CDR1 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 27, and SEQ ID NO: 5; or CDR1 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 29, and SEQ ID NO: 5; or CDR1 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 31, and SEQ ID NO: 5; or CDR1 comprises the amino acid sequence of SEQ ID NO: 73, SEQ ID NO: 14, and SEQ ID NO: 18; or CDR1 comprises the amino acid sequence of SEQ ID NO: 74, SEQ ID NO: 14, and SEQ ID NO: 18; or CDR1 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 75, and SEQ ID NO: 18; or CDR1 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 76, and SEQ ID NO: 18; or CDR1 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 77, and SEQ ID NO: 18; or CDR1 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 79, and SEQ ID NO: 18; or CDR1 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 80, and SEQ ID NO: 18; CDR2 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 14, and SEQ ID NO: 18; or CDR2 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; or CDR2 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 12, and SEQ ID NO: 5; or CDR2 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 14, and SEQ ID NO: 5; or CDR2 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 16; or CDR2 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 18; or CDR2 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 22, and SEQ ID NO: 5; or CDR2 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 27, and SEQ ID NO: 5; or CDR2 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 29, and SEQ ID NO: 5; or CDR2 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 31, and SEQ ID NO: 5; or CDR2 comprises the amino acid sequence of SEQ ID NO: 73, SEQ ID NO: 14, and SEQ ID NO: 18; or CDR2 comprises the amino acid sequence of SEQ ID NO: 74, SEQ ID NO: 14, and SEQ ID NO: 18; or CDR2 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 75, and SEQ ID NO: 18; or CDR2 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 76, and SEQ ID NO: 18; or CDR2 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 77, and SEQ ID NO: 18; or CDR2 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 79, and SEQ ID NO: 18; or CDR2 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 80, and SEQ ID NO: 18; CDR3 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 16; or CDR3 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 18; or CDR3 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 22, and SEQ ID NO: 5; or CDR3 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 27, and SEQ ID NO: 5; or CDR3 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 29, and SEQ ID NO: 5; or CDR3 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 31, and SEQ ID NO: 5; or CDR3 comprises the amino acid sequence of SEQ ID NO: 73, SEQ ID NO: 14, and SEQ ID NO: 18; or CDR3 comprises the amino acid sequence of SEQ ID NO: 74, SEQ ID NO: 14, and SEQ ID NO: 18; or CDR3 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 75, and SEQ ID NO: 18; or CDR3 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 76, and SEQ ID NO: 18; or CDR3 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 77, and SEQ ID NO: 18; or CDR3 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 79, and SEQ ID NO: 18; or CDR3 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 80, and SEQ ID NO:

18. **Claim 2** The polypeptide according to claim 1, wherein the at least one VHH domain comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 3, CDR2 comprising the amino acid sequence of SEQ ID NO: 78, and CDR3 comprising the amino acid sequence of SEQ ID NO:

18. **Claim 3** The polypeptide according to claim 1, wherein the at least one VHH domain or each VHH domain is humanized. **Claim 4** The polypeptide according to claim 1, wherein the at least one VHH domain comprises an amino acid sequence that is at least 85%, 90%, 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2, 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: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, or SEQ ID NO:

100. **Claim 5** The polypeptide according to claim 1, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO:

100. **Claim 6** The polypeptide according to claim 1, comprising one, two, or three VHH domains. **Claim 7** The polypeptide according to claim 1, comprising an Fc region. **Claim 8** The polypeptide according to claim 7, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NOs: 32 to 70 and SEQ ID NOs: 101 to 111. **Claim 9** The polypeptide according to claim 1, comprising at least one antigen-binding domain that binds to an antigen other than CD8. **Claim 10** Comprising at least one antigen-binding domain that binds to Lag3, CTLA4, TGFBR1, TGFBR2, Fas, TNFR2, PD1, PDL1, or TIM3, and / or TGFBR1, TGFBR2, Fas, TNFR2, 1-92-LFA-3, 5T4, α4 integrin, αV integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis Y, apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, complement C5, C-242, CA9, CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD39, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD73, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), claudin 3, claudin 4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, Cyr61, DLL4, DLK1, DLL3, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FcRH5, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor α (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPThe polypeptide according to claim 9, comprising at least one antigen-binding domain that binds to IIb / 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, LRRRC26, 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, TNFR, TNFR12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, TROP-2, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, or WISP-3. **Claim 11** The polypeptide according to claim 9, wherein at least one antigen-binding domain or each antigen-binding domain that binds to an antigen other than CD8 is a VHH domain. **Claim 12** The polypeptide according to claim 9, wherein at least one antigen-binding domain or each antigen-binding domain that binds to an antigen other than CD8 comprises a heavy chain variable region and a light chain variable region. **Claim 13** A complex comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is the polypeptide according to any one of claims 7 to 12, 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, complex. **Claim 14** The complex according to claim 13, wherein the second polypeptide comprises at least one VHH domain that binds to CD8 and / or at least one antigen-binding domain that binds to an antigen other than CD8. **Claim 15** The complex according to claim 14, wherein when the antigen-binding domain that binds to an antigen other than CD8 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. **Claim 16** The complex according to claim 13, wherein the first Fc region comprises a knob mutation and the second Fc region comprises a hole mutation. **Claim 17** The first Fc region contains a T366W mutation, and the second Fc region contains T366S, L368A, and Y407V mutations, or the first Fc region contains a T366W mutation, and the second Fc region contains T366S, L368A, Y407V, and H435R or H435K mutations, the complex according to claim 16.

18. An immune complex comprising the polypeptide according to any one of claims 1 to 12 and a cytotoxic substance.

19. The cytotoxic substance is selected from calicheamicin, auristatin, dolastatin, tubulysin, maytansinoid, cryptophycin, zuocarmycin, esperamicin, pyrrolobenzodiazepine, and enediyne antibiotics, the immune complex according to claim 18.

20. An immune complex comprising the complex according to any one of claims 13 to 17 and a cytotoxic substance.

21. The cytotoxic substance is selected from calicheamicin, auristatin, dolastatin, tubulysin, maytansinoid, cryptophycin, zuocarmycin, esperamicin, pyrrolobenzodiazepine, and enediyne antibiotics, the immune complex according to claim 20.

22. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

23. A pharmaceutical composition comprising the complex according to any one of claims 13 to 17.

24. A pharmaceutical composition comprising the immune complex of claim 18.

25. A pharmaceutical composition comprising the immune complex of claim 20.

26. An isolated nucleic acid encoding the polypeptide according to any one of claims 1 to 12.

27. A vector or host cell comprising the nucleic acid according to claim 26.

28. A host cell expressing the polypeptide according to any one of claims 1 to 12.

29. A method for producing the polypeptide according to any one of claims 1 to 12, comprising incubating a host cell that expresses the polypeptide under conditions suitable for the expression of the polypeptide.

30. The method according to claim 29, further comprising isolating the polypeptide.

31. The polypeptide according to any one of claims 1 to 12 for use in a method for treating cancer, comprising administering the polypeptide to a subject with cancer in a pharmaceutically effective amount. The conjugate according to any one of claims 13 to 17 for use in a method of treating cancer comprising administering the polypeptide to a subject with cancer in a pharmaceutically effective amount. The immunoconjugate according to claim 18 for use in a method of treating cancer comprising administering the polypeptide to a subject with cancer in a pharmaceutically effective amount. The immunoconjugate according to claim 20 for use in a method of treating cancer comprising administering the polypeptide to a subject with cancer in a pharmaceutically effective amount. The polypeptide according to claim 31, wherein the method of treating the cancer comprises administering the polypeptide together with an additional therapeutic agent. The conjugate according to claim 32, wherein the method of treating the cancer comprises administering the conjugate together with an additional therapeutic agent. The immunoconjugate according to claim 33, wherein the method of treating the cancer comprises administering the immunoconjugate together with an additional therapeutic agent. The immunoconjugate according to claim 34, wherein the method of treating the cancer comprises administering the immunoconjugate together with an additional therapeutic agent.

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