Multifunctional molecules binding to tcr and uses thereof

Abstract

Provided herein are multifunctional molecules comprising T cell receptor variable beta-binding moieties and cytokines and methods of treating conditions or diseases in a subject using the same. Also provided herein is a combination therapy using the multifunctional molecules comprising T cell receptor variable beta-binding moieties and cytokines and an HDAC inhibitor.

Description

MULTIFUNCTIONAL MOLECULES BINDING TO TCR AND USES THEREOFCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 633,193, filed April 12, 2024, the content of which is incorporated by reference herein in its entirety.

[0002] This invention was made with government support under NIH Z01 Project number ZIA BC 010666 awarded by the National Institute of Health (NIH). The government has certain rights in the invention.BACKGROUND

[0003] Currently available molecules designed to redirect T cells to promote tumor cell lysis for cancer immunotherapy typically target the CD3 epsilon (CD3e) subunit of the T cell receptor (TCR). However, there are limitations to this approach. Previous studies have shown that, e.g., low doses of anti-CD3e monoclonal antibody (mAb) can cause T cell dysfunction and exert immunosuppressive effects.Epigenetic silencing and poor T cell infiltration and function in the tumor microenvironment (TME) are prevalent in solid malignancies and associate with lack of response to immune checkpoint inhibitors. Thus, there is a need for improved T cell receptor-binding molecules that redirect T cells and epigenetic modulator for cancer immunotherapy.SUMMARY

[0004] Provided herein is a composition for treating a disease or condition in a subject in need thereof comprising: a first agent comprising a first domain that binds to a TCR P variable (TCR V) region; and a second agent comprising a histone deacetylase (HDAC) inhibitor.

[0005] Also provided herein is a kit for treating a disease or condition in a subject in need thereof comprising: a first agent comprising a first domain that binds to a TCR variable (TCRpV) region; and a second agent comprising a histone deacetylase (HDAC) inhibitor.

[0006] In some embodiments, the composition or the kit comprises a therapeutically effective amount of the first agent and the second agent.

[0007] In some embodiments, the subject exhibits an improved therapeutic effect after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

[0008] In some embodiments, the subject comprises more activated and / or expanded T cells after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

[0009] In some embodiments, the subject comprises less exhausted T cells after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

[0010] In some embodiments, the subject exhibits more anti -tumor efficacy after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

[0011] In some embodiments, the subject comprises T cells that are more cytotoxic against tumor cells or cancer cells after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

[0012] In some embodiments, the subject comprises more T cells that are cytotoxic against tumor cells or cancer cells after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

[0013] In some embodiments, the subject exhibits more cytotoxic activity against tumor cells or cancer cells after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

[0014] In some embodiments, the subject comprises tumors that are more immune infiltrated after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

[0015] In some embodiments, the subject comprises more immune permissive tumor environment after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

[0016] In some embodiments, the T cells are tumor-infiltrated T-cells. In some embodiments, the T cells express one or more of TCRPV6-5, TCRp VI 0-1, and TCRp VI 0-2. In some embodiments, the T cells are CD8+ or CD4+. In some embodiments, the T cells are granzyme B+. In some embodiments, T cells are IFNy+ or TNFa+.

[0017] In some embodiments, the anti-tumor efficacy, the cytotoxic activity, the immune infiltration, or the immune permissive tumor environment is measured by number of CD8+ T cells or CD4+ T cells.

[0018] In some embodiments, the CD8+ T cells are granzyme B+.

[0019] In some embodiments, the anti-tumor efficacy, the cytotoxic activity, the immune infiltration, or the immune permissive tumor environment is measured by number of Ml macrophages.

[0020] In some embodiments, the Ml macrophages are MHCII+.

[0021] In some embodiments, the anti-tumor efficacy, the cytotoxic activity, the immune infiltration, or the immune permissive tumor environment is measured by IFNy or TNFa level.

[0022] In some embodiments, the subject exhibits an improved therapeutic effect after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

[0023] In some embodiments, the subject comprises more activated and / or expanded T cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

[0024] In some embodiments, the subject comprises less exhausted T cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

[0025] In some embodiments, the subject exhibits more anti -tumor efficacy after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

[0026] In some embodiments, the subject comprises T cells that are more cytotoxic against tumor cells or cancer cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

[0027] In some embodiments, the subject comprises more T cells that are cytotoxic against tumor cells or cancer cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

[0028] In some embodiments, the subject exhibits more cytotoxic activity against tumor cells or cancer cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

[0029] In some embodiments, the subject comprises tumors that are more immune infiltrated after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

[0030] In some embodiments, the subject comprises more immune permissive tumor environment after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

[0031] In some embodiments, the T cells are tumor-infiltrated T-cells. In some embodiments, the T cells express one or more of TCRPV6-5, TCRp VI 0-1, and TCRp VI 0-2. In some embodiments, the T cells are CD8+ or CD4+. In some embodiments, the T cells are granzyme B+. In some embodiments, the T cells are IFNy+ or TNFa+.

[0032] In some embodiments, the anti-tumor efficacy, the cytotoxic activity, the immune infiltration, or the immune permissive tumor environment is measured by number of CD8+ T cells or CD4+ T cells. In some embodiments, the CD8+ T cells are granzyme B+.

[0033] In some embodiments, the anti-tumor efficacy, the cytotoxic activity, the immune infiltration, or the immune permissive tumor environment is measured by number of Ml macrophages. In some embodiments, the Ml macrophages are MHCII+.

[0034] In some embodiments, the anti-tumor efficacy, the cytotoxic activity, the immune infiltration, or the immune permissive tumor environment is measured by IFNy or TNFa level.

[0035] In some embodiments, the TCRpV region is a human TCRpV region. In some embodiments, the first domain binds to one or more of a TCRpV subfamily selected from the group consisting of TCRP V6 subfamily or TCRP V10 subfamily. In some embodiments, the first domain binds to one or more of a TCRpV subfamily selected from the group consisting of: TCRP V6 subfamily comprising one or more selected from TCRp V6-l*01, TCRp V6-2*01, TCRp V6-3*01, TCRp V6-4*01, TCRp V6-4*02, TCRp V6-5*01, TCRP V6-6*01, TCRp V6-6*02, TCRp V6-6*03, TCRp V6-6*04, TCRp V6-6*05, TCRp V6- 7*01, TCRp V6-8*01, and TCRp V6-9*01; or TCRp V10 subfamily comprising one or more selectedfrom TCRp V10-l*01, TCR V10-l*02, TCRp V10-l*03, TCRp V10-2*01, TCRp V10-2*02, TCRp V10-3*01, TCRP V10-3*02, TCRp V10-3*03, and TCRp V10-3*04.

[0036] In some embodiments, the first domain is a full antibody, an antigen binding domain thereof, or a functional fragment or variant thereof, a bivalent antibody, a bispecific antibody, or a biparatopic antibody that binds to a TCRpV region.

[0037] In some embodiments, the full antibody, an antigen binding domain thereof, or a functional fragment or variant thereof is a Fab, a Fab', a F(ab’)2, a F(ab)2, a variable fragment (Fv), a single domain antibody, a diabody, a nanobody, a camelid single domain antibody, or a single chain variable fragment (scFv).

[0038] In some embodiments, the first agent comprises one or more heavy chain constant regions selected from the group consisting of IgGl heavy chain constant region or fragment thereof, IgG2 heavy chain constant region or fragment thereof, IgG3 heavy chain constant region or fragment thereof, IgGAl heavy chain constant region or fragment thereof, IgGA2 heavy chain constant region or fragment thereof, IgG4 heavy chain constant region or fragment thereof, IgJ heavy chain constant region or fragment thereof, IgM heavy chain constant region or fragment thereof, IgD heavy chain constant region or fragment thereof, and IgE heavy chain constant region or fragment thereof.

[0039] In some embodiments, the first agent comprises a kappa light chain constant region or fragment thereof, a lambda light chain constant region or fragment thereof, or a combination thereof.

[0040] In some embodiments, the first agent is a multifunctional molecule.

[0041] In some embodiments, the first agent comprises at least two non-contiguous polypeptide chains; wherein a first polypeptide chain of the at least two non-contiguous polypeptide chains comprises a first Fc region, and a second polypeptide chain of the at least two non-contiguous polypeptide chains comprises a second Fc region.

[0042] In some embodiments, the first Fc region and the second Fc region comprise an Fc interface with a knob-in-a hole.

[0043] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises an Asn297Ala mutation, a Leu234Ala / Leu235Ala mutation, or a combination thereof.

[0044] In some embodiments, the first agent is a multispecific molecule that further comprises one or more of a tumor-targeting moiety, a cytokine molecule, a stromal modifying moiety, or an immune cell engager.

[0045] In some embodiments, the first agent is a multispecific molecule that further comprises a cytokine molecule.

[0046] In some embodiments, the first agent comprises a dimerization module comprising a first immunoglobulin chain constant region and a second immunoglobulin chain constant region.

[0047] In some embodiments, the first immunoglobulin chain constant region comprises the first fragment crystallizable region (Fc region) and the second first immunoglobulin chain constant region comprises the second fragment crystallizable region (Fc region).

[0048] In some embodiments, dimerization of the first Fc region and the second Fc region is enhanced by providing an Fc interface of the first Fc region and the second Fc region with one or more of a paired cavity-protuberance, an electrostatic interaction, or a strand-exchange, such that a greater ratio of heteromultimer to homomultimer forms relative to a non-engineered interface.

[0049] In some embodiments, the immune cell engager is selected from the group consisting of a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, a macrophage cell engager, and any combination thereof.

[0050] In some embodiments, the cytokine molecule is selected from the group consisting of inter- leukin-2 (IL-2) or functional variant thereof, interleukin-7 (IL-7) or functional variant thereof, in- terleukin-12 (IL-12) or functional variant thereof, interleukin- 15 (IL-15) or functional variant thereof, interleukin- 18 (IL- 18) or functional variant thereof, interleukin-21 (IL-21) or functional variant thereof, interferon gamma or functional variant thereof, and any combination thereof.

[0051] In some embodiments, the cytokine molecule comprises interleukin-2 (IL-2) or functional variant thereof. In some embodiments, the interleukin-2 (IL-2) or functional variant thereof comprises the sequence of SEQ ID NO : 2270 or SEQ ID NO : 2191.

[0052] In some embodiments, the second agent is a small molecule inhibitor. In some embodiments, the second agent is a Class I HDAC inhibitor. In some embodiments, the second agent is a HDAC1 inhibitor. In some embodiments, the second agent is a HDAC3 inhibitor. In some embodiments, the second agent is a HDAC1 and HDAC3 inhibitor.

[0053] In some embodiments, the second agent is a benzamide HDAC inhibitor.

[0054] In some embodiments, the second agent is selected from the group consisting of: entinostat, tacedinaline, mocetinostat, vorinostat, belinostat, LAQ824, and romidepsin panobinostat.

[0055] In some embodiments, the subject comprises higher level of protein lysine acetylation after administration of the first agent and the second agent, relative to a subject administered the first agent without the second agent.

[0056] In some embodiments, the first agent further comprises a tumor-targeting moiety. In some embodiments, the tumor-targeting moiety comprises a full antibody, an antigen binding domain thereof, or a functional fragment or variant that binds to a cancer antigen.

[0057] In some embodiments, the cancer antigen is selected from the group consisting of BCMA, CD 19, CD20, CD22, FcRH5, PDL1, CD47, gangloside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PMSA), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), Ron Kinase, c-Met, Immature laminin receptor, TAG-72, BING-4, Calcium-activated chloride channel 2, Cyclin-Bl, 9D7, Ep-CAM, EphA3, Her2 / neu, Telomerase, SAP-1, Survivin, NY-ESO-l / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gpl00 / pmell7, Tyrosinase, TRP-1 / -2, MC1R, P-catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, p53, Ras, TGF-B receptor, AFP, ETA, MAGE, MUC-1, CA- 125, BAGE, GAGE, NY-ESO-1, P-catenin, CDK4, CDC27, a actinin-4, TRPl / gp75, TRP2, gplOO, Melan-A / MARTl, gangliosides, WT1, EphA3, Epidermal growth factor receptor (EGFR), MART-2, MART-1, MUC1, MUC2, MUM1, MUM2, MUM3, MSLN, NA88-1, NPM, OA1, OGT, RCC, RUH,RUI2, SAGE, TRG, TSTA, Folate receptor alpha, LI -CAM, CAIX, gpA33, GD3, GM2, VEGFR, Intergrins, carbohydrates, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, TGF-beta, hyaluronic acid, collagen, tenascin C, and tenascin W.

[0058] In some embodiments, the condition or disease is cancer. In some embodiments, the cancer is a solid tumor, a hematological cancer, a metastatic cancer, a soft tissue tumor, or a combination thereof.

[0059] In some embodiments, the cancer is non-responsive to an immune checkpoint inhibitor therapy.

[0060] In some embodiments, the cancer is non-responsive to an anti-PDl therapy or an anti-PDLl therapy.

[0061] In some embodiments, the cancer is the solid tumor, and wherein the solid tumor is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, and prostate cancer, and a combination thereof.

[0062] In some embodiments, the solid tumor is the colorectal cancer and wherein the tumor comprises a KRAS mutation. In some embodiments, the KRAS mutation is a G12D mutation.

[0063] In some embodiments, the solid tumor is the breast cancer and wherein the breast cancer is Triple Negative Breast Cancer (TNBC).

[0064] In some embodiments, the cancer is the hematological cancer, and wherein the hematological cancer is selected from the group consisting of Hodgkin’s lymphoma, Non-Hodgkin’s lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia, myelodysplastic syndrome, multiple myeloma, T- cell lymphoma, acute lymphocytic leukemia, and a combination thereof.

[0065] In some embodiments, the Non-Hodgkin’s lymphoma is selected from the group consisting of B cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (B-CLL), mantle cell lymphoma, marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, and a combination thereof. In some embodiments, the T-cell lymphoma is peripheral T-cell lymphoma.

[0066] In some embodiments, the cancer is characterized by a cancer antigen present on the cancer. In some embodiments, the cancer antigen is a tumor antigen, a stromal antigen, or a hematological antigen.

[0067] In some embodiments, the cancer antigen is selected from the group consisting of BCMA, CD 19, CD20, CD22, FcRH5, PDL1, CD47, ganglioside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PMSA), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), Ron Kinase, c-Met, Immature laminin receptor, TAG-72, BING-4, Calcium-activated chloride channel 2, Cyclin-Bl, 9D7, Ep-CAM, EphA3, Her2 / neu, Telomerase, SAP-1, Survivin, NY-ESO-l / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gpl00 / pmell7, Tyrosinase, MC1R, -catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, p53, Ras, TGF-B receptor, AFP, ETA, MAGE, CA-125, BAGE, GAGE, CDC27, a actinin-4, TRPl / gp75, TRP2, gangliosides, WT1, Epi-dermal growth factor receptor (EGFR), MART-2, MUC1, MUC2, MUM1, MUM2, MUM3, MSLN, NA88-1, NPM, OA1, OGT, RCC, RUH, RU12, SAGE, TRG, TSTA, Folate receptor alpha, LI -CAM, CAIX, gpA33, GD3, GM2, VEGFR, Integrins, Carbohydrates, IGF1R, TRAILR1, TRAILR2, RANKL, FAP, TGF-beta, hyaluronic acid, collagen, tenascin C, and tenascin W.

[0068] In some embodiments, the composition is pharmaceutical composition. In some embodiments, the composition or kit further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0069] Also described herein is a method of treating a disease or condition in a subject in need thereof comprising: administering a first agent comprising a first domain that binds to a TCR P variable (TCR V) region; and administering a second agent comprising a histone deacetylase (HDAC) inhibitor; wherein a therapeutically effective amount of the first agent and the second agent is administered to the subject, thereby treating the disease or condition in the subject.

[0070] Also described herein is a method of treating a disease or condition in a subject in need thereof comprising administering to the subject a first agent comprising first domain that binds to a TCR variable (TCRpV) region, wherein the subject has been previously treated with a second agent comprising a histone deacetylase (HDAC) inhibitor, thereby treating the disease or condition in the subject.

[0071] Also described herein is a method of improving a therapeutic efficacy of a second agent comprising a histone deacetylase (HDAC) inhibitor in a subject in need thereof comprising: administering a first agent comprising a first domain that binds to a TCR P variable (TCRpV) region, thereby improving the therapeutic efficacy of the second agent in the subject.

[0072] Also described herein is a method of treating a disease or condition in a subject in need thereof comprising: administering a first agent comprising a first domain that binds to a TCR P variable (TCRpV) region; and administering a second agent comprising a histone deacetylase (HDAC) inhibitor; wherein the disease is non-responsive to an immune checkpoint inhibitor therapy, and wherein a therapeutically effective amount of the first agent and the second agent is administered to the subject, thereby treating the disease or condition in the subject.

[0073] Also described herein is a method of treating a disease or condition in a subject in need thereof comprising: administering a first agent comprising a first domain that binds to a TCR P variable (TCRpV) region; and administering a second agent comprising a histone deacetylase (HDAC) inhibitor; wherein a therapeutically effective amount of the first agent and the second agent is administered to the subject to promote immune response or immune infiltration, thereby treating the disease or condition in the subject.

[0074] Also described herein is a method of expanding and / or activating T-cells in a subject in need thereof comprising: administering a first agent comprising a first domain that binds to a TCR P variable (TCRpV) region; and administering a second agent comprising a histone deacetylase (HDAC) inhibitor; wherein a therapeutically effective amount of the first agent and the second agent is administered to the subject, thereby expanding and / or activating T-cells in the subject.

[0075] Also described herein is a combination therapy for treating a disease or condition in a subject in need thereof a first agent comprising a first domain that binds to a TCR P variable (TCRpV) region, and a second agent comprising a histone deacetylase (HDAC) inhibitor.

[0076] In some embodiments, administering the first agent and the second agent induces an improved therapeutic effect in the subject, relative to a subject administered the second agent without the first agent.

[0077] In some embodiments, administering the first agent and the second agent induces more activated and / or expanded T cells in the subject, relative to a subject administered the second agent without the first agent.

[0078] In some embodiments, administering the first agent and the second agent induces less exhausted T cells in the subject, relative to a subject administered the second agent without the first agent.

[0079] In some embodiments, administering the first agent and the second agent induces more antitumor efficacy in the subject, relative to a subject administered the second agent without the first agent.

[0080] In some embodiments, administering the first agent and the second agent induces more T cells that are more cytotoxic against tumor cells or cancer cells in the subject, relative to a subject administered the second agent without the first agent.

[0081] In some embodiments, administering the first agent and the second agent induces more cytotoxicity against tumor cells or cancer cells in the subject, relative to a subject administered the second agent without the first agent.

[0082] In some embodiments, administering the first agent and the second agent expands or activates more T cells that are cytotoxic against tumor cells or cancer cells in the subject, relative to a subject administered the second agent without the first agent.

[0083] In some embodiments, administering the first agent and the second agent induces more cytotoxic activity against tumor cells or cancer cells in the subject, relative to a subject administered the second agent without the first agent.

[0084] In some embodiments, the subject comprises more immune permissive tumor environment after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

[0085] In some embodiments, the subject exhibits an improved therapeutic effect after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

[0086] In some embodiments, the subject comprises more activated and / or expanded T cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

[0087] In some embodiments, the subject comprises less exhausted T cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

[0088] In some embodiments, the subject exhibits more anti -tumor efficacy after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

[0089] In some embodiments, the subject comprises T cells that are more cytotoxic against tumor cells or cancer cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

[0090] In some embodiments, the subject comprises more T cells that are cytotoxic against tumor cells or cancer cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

[0091] In some embodiments, the subject exhibits more cytotoxic activity against tumor cells or cancer cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

[0092] In some embodiments, the subject comprises tumors that are more immune infiltrated after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

[0093] In some embodiments, the subject comprises more immune permissive tumor environment after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

[0094] In some embodiments, the first agent and the second agent are not concomitantly administered to the subject.

[0095] In some embodiments, the first agent and the second agent are concomitantly administered to the subject.

[0096] In some embodiments, the method comprises administering the first agent to the subject at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, at least 1, 2, 3, 4, 5, 6, or 7 days, at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years after the subject has been treated with a dose of the second agent.

[0097] In some embodiments, the method comprises administering the first agent to the subject at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, at least 1, 2, 3, 4, 5, 6, or 7 days, at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years after the subject has been treated with a first dose of the second agent.

[0098] In some embodiments, the method comprises administering the first agent to the subject at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, at least 1, 2, 3, 4, 5, 6, or 7 days, at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years after the subject has been treated with a last dose of the second agent.

[0099] In some embodiments, the method comprises administering the first agent to the subject at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, at most 1, 2, 3, 4, 5, 6, or 7 days, at most 1, 2, 3, or 4 weeks, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at most 1, 2, or 3 years after being treated with the second agent.

[0100] In some embodiments, the method comprises administering the first agent to the subject at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, at most 1, 2, 3, 4, 5, 6, or 7 days, at most 1, 2, 3, or 4weeks, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at most 1, 2, or 3 years after being treated with a dose of the second agent.

[0101] In some embodiments, the method comprises administering the first agent to the subject at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, at most 1, 2, 3, 4, 5, 6, or 7 days, at most 1, 2, 3, or 4 weeks, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at most 1, 2, or 3 years after being treated with a first dose of the second agent.

[0102] In some embodiments, the method comprises administering the first agent to the subject at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, at most 1, 2, 3, 4, 5, 6, or 7 days, at most 1, 2, 3, or 4 weeks, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at most 1, 2, or 3 years after being treated with a last dose of the second agent.

[0103] In some embodiments, the method comprises administering the first agent to the subject from 1 hour to 24 hours after being treated with a dose of the second agent.

[0104] In some embodiments, the method comprises administering the first agent to the subject from 1 day to 7 days after being treated with a dose of the second agent.

[0105] In some embodiments, the method comprises administering the first agent to the subject from 1 week to 4 weeks after being treated with a dose of the second agent.

[0106] In some embodiments, the method comprises administering the first agent to the subject from 1 month to 12 months after being treated with a dose of the second agent.

[0107] In some embodiments, the method comprises administering the first agent to the subject from 1 year to 3 years after being treated with a dose of the second agent.

[0108] In some embodiments, the method comprises administering the first agent to the subject from 1 hour to 24 hours after being treated with a first dose of the second agent.

[0109] In some embodiments, the method comprises administering the first agent to the subject from 1 day to 7 days after being treated with a first dose of the second agent.

[0110] In some embodiments, the method comprises administering the first agent to the subject from 1 week to 4 weeks after being treated with a first dose of the second agent.[OHl] In some embodiments, the method comprises administering the first agent to the subject from 1 month to 12 months after being treated with a first dose of the second agent.

[0112] In some embodiments, the method comprises administering the first agent to the subject from 1 year to 3 years after being treated with a first dose of the second agent.

[0113] In some embodiments, the method comprises administering the first agent to the subject from 1 hour to 24 hours after being treated with a last dose of the second agent.

[0114] In some embodiments, the method comprises administering the first agent to the subject from 1 week to 4 weeks after being treated with a last dose of the second agent.

[0115] In some embodiments, the method comprises administering the first agent to the subject from 1 month to 12 months after being treated with a last dose of the second agent.

[0116] In some embodiments, the method comprises administering the first agent to the subject from 1 year to 3 years after being treated with a last dose of the second agent.

[0117] In some embodiments, the method comprises administering the first agent to the subject on the same day the subject had been previously treated with the second agent.

[0118] In some embodiments, the method is repeated twice or more.

[0119] In some embodiments, the administration of the first agent comprises one or more doses.

[0120] In some embodiments, the administration of the first agent comprises two or more doses.

[0121] In some embodiments, the administration of the first agent comprises one dose per week.

[0122] In some embodiments, the administration of the first agent comprises two or more doses per week.

[0123] In some embodiments, the first agent is administered to the subject once every two weeks.

[0124] In some embodiments, the first agent is administered to the subject once every three weeks.

[0125] In some embodiments, the first agent is administered to the subject once every week for at least1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years.

[0126] In some embodiments, the administration of the first agent comprises two or more doses per week for at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years.

[0127] In some embodiments, the first agent is administered to the subject once every two weeks for at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years.

[0128] In some embodiments, the first agent is administered to the subject once every three weeks for at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years.

[0129] In some embodiments, the administration of the second agent comprises one or more doses.

[0130] In some embodiments, the administration of the second agent comprises two or more doses.

[0131] In some embodiments, the administration of the second agent comprises one dose per week.

[0132] In some embodiments, the administration of the second agent comprises two or more doses per week.

[0133] In some embodiments, the second agent is administered to the subject once every two weeks.

[0134] In some embodiments, the second agent is administered to the subject once every three weeks.

[0135] In some embodiments, the second agent is administered to the subject once every week for at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years.

[0136] In some embodiments, the administration of the second agent comprises two or more doses per week for at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years.

[0137] In some embodiments, the second agent is administered to the subject once every two weeks for at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years.

[0138] In some embodiments, the second agent is administered to the subject once every three weeks for at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years.

[0139] In some embodiments, the method comprises administering the first agent to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, at least 1, 2, 3, 4, 5, 6, or 7 days, at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years prior to administering the second agent to the subject.

[0140] In some embodiments, the method comprises administering the first agent to the subject at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, at most 1, 2, 3, 4, 5, 6, or 7 days, at most 1, 2, 3, or 4 weeks, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at most 1, 2, or 3 years prior to administering the second agent to the subject.

[0141] In some embodiments, the method comprises administering the first agent to the subject from 1 hour to 24 hours prior to administering the second agent to the subject. In some embodiments, the method comprises administering the first agent to the subject from 1 day to 7 days prior to administering the second agent to the subject. In some embodiments, the method comprises administering the first agent to the subject from 1 week to 4 weeks prior to administering the second agent to the subject. In some embodiments, the method comprises administering the first agent to the subject from 1 month to 12 months prior to administering the second agent to the subject. In some embodiments, the method comprises administering the first agent to the subject from 1 year to 3 years prior to administering the second agent to the subject.

[0142] In some embodiments, the administration of the first agent comprises two or more doses, and each dose comprises a same amount of the first agent. In some embodiments, the administration of the first agent comprises two or more doses, and each dose comprises different amounts of the first agent.

[0143] In some embodiments, the administration of the second agent comprises two or more doses, and each dose comprises a same amount of the second agent. In some embodiments, the administration of the second agent comprises two or more doses, and each dose comprises different amounts of the second agent. In some embodiments, the first agent is administered to the subject at a dose of from about 0.001 mg / kg to about 20 mg / kg. In some embodiments, the second agent is administered to the subject at a dose of from about 0.001 mg / kg to about 20 mg / kg. In some embodiments, the method comprises administering the first agent and the second agent to the subject simultaneously or on the same day.

[0144] In some embodiments, the disease or condition in the subject is treated to a higher extent relative to a corresponding method in which the first agent is administered and the second agent is not administered. In some embodiments, the method is more effective to treat the disease or condition in the subject relative to a method in which the first agent is administered to the subject but not the second agent. In some embodiments, the method is more effective to treat the disease or condition in the subject relative to a method in which the second agent is administered to the subject but not the first agent.

[0145] In some embodiments, the TCRpV region is a human TCRpV region. In some embodiments, the first domain binds to one or more of a TCRpV subfamily selected from the group consisting of TCRP V6 subfamily or TCRP V10 subfamily. In some embodiments, the first domain binds to one or more of a TCRpV subfamily selected from the group consisting of: TCRP V6 subfamily comprising one or more selected from TCRP V6-l*01, TCRP V6-2*01, TCRP V6-3*01, TCRP V6-4*01, TCRP V6-4*02, TCRPV6-5*01, TCRp V6-6*01, TCR V6-6*02, TCRp V6-6*03, TCRp V6-6*04, TCRp V6-6*05, TCRp V6- 7*01, TCRp V6-8*01, and TCRp V6-9*01; or TCRp V10 subfamily comprising one or more selected from TCRP V10-l*01, TCRp V10-l*02, TCRp V10-l*03, TCRp V10-2*01, TCRp V10-2*02, TCRp V10-3*01, TCRP VI 0-3 *02, TCRp VI 0-3 *03, and TCRp VI 0-3 *04. In some embodiments, the first domain is a full antibody, an antigen binding domain thereof, or a functional fragment or variant thereof, a bivalent antibody, a bispecific antibody, or a biparatopic antibody that binds to a TCRpV region.

[0146] In some embodiments, the full antibody, an antigen binding domain thereof, or a functional fragment or variant thereof is a Fab, a Fab', a F(ab’)2, a F(ab)2, a variable fragment (Fv), a single domain antibody, a diabody, a nanobody, a camelid single domain antibody, or a single chain variable fragment (scFv). In some embodiments, the first agent comprises one or more heavy chain constant regions selected from the group consisting of IgGl heavy chain constant region or fragment thereof, IgG2 heavy chain constant region or fragment thereof, IgG3 heavy chain constant region or fragment thereof, IgGAl heavy chain constant region or fragment thereof, IgGA2 heavy chain constant region or fragment thereof, IgG4 heavy chain constant region or fragment thereof, IgJ heavy chain constant region or fragment thereof, IgM heavy chain constant region or fragment thereof, IgD heavy chain constant region or fragment thereof, and IgE heavy chain constant region or fragment thereof.

[0147] In some embodiments, the first agent comprises a kappa light chain constant region or fragment thereof, a lambda light chain constant region or fragment thereof, or a combination thereof.

[0148] In some embodiments, the first agent is a multifunctional molecule. In some embodiments, the first agent comprises at least two non-contiguous polypeptide chains; wherein a first polypeptide chain of the at least two non-contiguous polypeptide chains comprises a first Fc region, and a second polypeptide chain of the at least two non-contiguous polypeptide chains comprises a second Fc region.

[0149] In some embodiments, the first Fc region and the second Fc region comprise an Fc interface with a knob-in-a hole. In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises an Asn297Ala mutation, a Leu234Ala / Leu235Ala mutation, or a combination thereof.

[0150] In some embodiments, the first agent is a multispecific molecule that further comprises one or more of a tumor-targeting moiety, a cytokine molecule, a stromal modifying moiety, or an immune cell engager. In some embodiments, the first agent is a multispecific molecule that further comprises a cytokine molecule.

[0151] In some embodiments, the first agent comprises a dimerization module, comprising a first immunoglobulin chain constant region and a second immunoglobulin chain constant region.

[0152] In some embodiments, the first immunoglobulin chain constant region comprises the first fragment crystallizable region (Fc region) and the second first immunoglobulin chain constant region comprises the second fragment crystallizable region (Fc region).

[0153] In some embodiments, dimerization of the first Fc region and the second Fc region is enhanced by providing an Fc interface of the first Fc region and the second Fc region with one or more of a paired cavity-protuberance, an electrostatic interaction, or a strand-exchange, such that a greater ratio of heteromultimer to homomultimer forms relative to a non-engineered interface.

[0154] In some embodiments, the immune cell engager is selected from the group consisting of a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, a macrophage cell engager, and any combination thereof.

[0155] In some embodiments, the cytokine molecule is selected from the group consisting of inter- leukin-2 (IL-2) or functional variant thereof, interleukin-7 (IL-7) or functional variant thereof, in- terleukin-12 (IL-12) or functional variant thereof, interleukin- 15 (IL-15) or functional variant thereof, interleukin- 18 (IL- 18) or functional variant thereof, interleukin-21 (IL-21) or functional variant thereof, interferon gamma or functional variant thereof, and any combination thereof.

[0156] In some embodiments, the cytokine molecule comprises interleukin-2 (IL-2) or functional variant thereof. In some embodiments, the interleukin-2 (IL-2) or functional variant thereof comprises the sequence of SEQ ID NO : 2270 or SEQ ID NO : 2191.

[0157] In some embodiments, the second agent is a small molecule inhibitor.

[0158] In some embodiments, the second agent is a Class I HDAC inhibitor. In some embodiments, the second agent is a HDAC1 inhibitor. In some embodiments, the second agent is a HDAC3 inhibitor. In some embodiments, the second agent is a HDAC 1 and HDAC3 inhibitor. In some embodiments, the second agent is a benzamide HDAC inhibitor. In some embodiments, the second agent is selected from the group consisting of: entinostat, tacedinaline, mocetinostat, vorinostat, belinostat, LAQ824, and romidepsin panobinostat.

[0159] In some embodiments, the subject comprises higher level of protein lysine acetylation after administration of the first agent and the second agent, relative to a subject administered the first agent without the second agent.

[0160] In some embodiments, the first agent further comprises a tumor-targeting moiety.

[0161] In some embodiments, the tumor-targeting moiety comprises a full antibody, an antigen binding domain thereof, or a functional fragment or variant that binds to a cancer antigen.

[0162] In some embodiments, the cancer antigen is selected from the group consisting of BCMA, CD 19, CD20, CD22, FcRH5, PDL1, CD47, gangloside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PMSA), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), Ron Kinase, c-Met, Immature laminin receptor, TAG-72, BING-4, Calcium-activated chloride channel 2, Cyclin-Bl, 9D7, Ep-CAM, EphA3, Her2 / neu, Telomerase, SAP-1, Survivin, NY-ESO-l / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gpl00 / pmell7, Tyrosinase, TRP-1 / -2, MC1R, P-catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, p53, Ras, TGF-B receptor, AFP, ETA, MAGE, MUC-1, CA- 125, BAGE, GAGE, NY-ESO-1, P-catenin, CDK4, CDC27, a actinin-4, TRPl / gp75, TRP2, gplOO, Melan-A / MARTl, gangliosides, WT1, EphA3, Epidermal growth factor receptor (EGFR), MART-2, MART-1, MUC1, MUC2, MUM1, MUM2, MUM3, MSLN, NA88-1, NPM, OA1, OGT, RCC, RUI1, RUI2, SAGE, TRG, TSTA, Folate receptor alpha, LI -CAM, CAIX, gpA33, GD3, GM2, VEGFR, Intergrins, carbohydrates, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, TGF-beta, hyaluronic acid, collagen, tenascin C, and tenascin W.

[0163] In some embodiments, the condition or disease is cancer. In some embodiments, the cancer is a solid tumor, a hematological cancer, a metastatic cancer, a soft tissue tumor, or a combination thereof.

[0164] In some embodiments, the cancer is non-responsive to an immune checkpoint inhibitor therapy. In some embodiments, the cancer is non-responsive to an anti-PDl therapy or an anti-PDLl therapy.

[0165] In some embodiments, the cancer is the solid tumor, and wherein the solid tumor is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, prostate cancer, and a combination thereof.

[0166] In some embodiments, the solid tumor is the colorectal cancer and wherein the tumor comprises a KRAS mutation. In some embodiments, the KRAS mutation is a G12D mutation.

[0167] In some embodiments, the solid tumor is the breast cancer and wherein the breast cancer is Triple Negative Breast Cancer (TNBC).

[0168] In some embodiments, the cancer is the hematological cancer, and wherein the hematological cancer is selected from the group consisting of Hodgkin’s lymphoma, Non-Hodgkin’s lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia, myelodysplastic syndrome, multiple myeloma, T- cell lymphoma, acute lymphocytic leukemia, and a combination thereof.

[0169] In some embodiments, the Non-Hodgkin’s lymphoma is selected from the group consisting of B cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (B-CLL), mantle cell lymphoma, marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, and a combination thereof.

[0170] In some embodiments, the T-cell lymphoma is peripheral T-cell lymphoma.

[0171] In some embodiments, the cancer is characterized by a cancer antigen present on the cancer.

[0172] In some embodiments, the cancer antigen is a tumor antigen, a stromal antigen, or a hematological antigen.

[0173] In some embodiments, the cancer antigen is selected from the group consisting of BCMA, CD 19, CD20, CD22, FcRH5, PDL1, CD47, ganglioside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PMSA), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), Ron Kinase, c-Met, Immature laminin receptor, TAG-72, BING-4, Calcium-activated chloride channel 2, Cyclin-Bl, 9D7, Ep-CAM, EphA3, Her2 / neu, Telomerase, SAP-1, Survivin, NY-ESO-l / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gpl00 / pmell7, Tyrosinase, MC1R, -catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, p53, Ras, TGF-B receptor, AFP, ETA, MAGE, CA-I25, BAGE, GAGE, CDC27, a actinin-4, TRPl / gp75, TRP2, gangliosides, WT1, Epi-dermal growth factor receptor (EGFR), MART-2, MUC1, MUC2, MUM1, MUM2, MUM3, MSLN, NA88-1, NPM, OA1, OGT, RCC, RUH, RU12, SAGE, TRG, TSTA, Folate receptor alpha, LI -CAM, CAIX, gpA33, GD3, GM2, VEGFR, Integrins, Carbohydrates, IGF1R, TRAILR1, TRAILR2, RANKL, FAP, TGF-beta, hyaluronic acid, collagen, tenascin C, and tenascin W.

[0174] In some embodiments, the first agent is administered to the subject prior to, concurrently, or after administration of the second agent to the subject.

[0175] Also described herein is a kit comprising the composition described herein or the kit described herein, and instructions for administering the first agent and the second agent to a human subject in need thereof to treat a disease or condition in the human subject.

[0176] In some embodiments, the first agent comprises a multispecific molecule comprising a first polypeptide, a second polypeptide, and a third polypeptide; wherein the first polypeptide, the second polypeptide and the third polypeptide are non-contiguous, wherein the first polypeptide comprises a first portion of a dimerization module linked to a first portion of the TCRpV6-binding moiety comprising a VH of the TCRpV6-binding moiety; the second polypeptide comprises a second portion of the dimerization module, wherein the IL-2 or functional fragment or functional variant thereof is covalently linked to the second polypeptide; and the third polypeptide comprises a second portion of the TCRPV6- binding moiety comprising a VL of the TCRpV6-b inding moiety.

[0177] In some embodiments, the first polypeptide comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 3517, 4000, 4004, 4006, 4008, 4010, 4011, 4014, 4016 and 4018, the second polypeptide comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 3521, 4002, 4007, 4003, 4013 and 4015 and the third polypeptide comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 3518, 4005, 4009, 4012 and 4017.

[0178] In some embodiments, the first agent comprises a multispecific molecule comprising a first polypeptide and a second polypeptide; wherein the first polypeptide and the second polypeptide are noncontiguous, wherein the TCRpV6-binding moiety comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), or a single domain antibody, wherein the first polypeptide comprises a first portion of a dimerization module linked to the TCRpV6-binding moiety; and the second polypeptide comprises a second portion of the dimerization module, wherein the IL-2 or functional fragment or functional variant thereof is covalently linked to the second polypeptide.

[0179] In some embodiments, the first polypeptide comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 4019, 4021, 4023, 4025 and 4027, and the second polypeptide comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 4020, 4022, 4024, 4026 and 4028.

[0180] Also described herein is a method of activating and / or expanding T cells, wherein the method comprises: administering a first agent comprising a first domain that binds to a TCR P variable (TCRpV) region on the T cells; and administering a second agent comprising a histone deacetylase (HDAC) inhibitor, wherein the T cells are activated or expanded.

[0181] Also described herein is a method of preventing or reducing T cell exhaustion, wherein the method comprises: administering a first agent comprising a first domain that binds to a TCR P variable (TCRpV) region on a T cell; and administering a second agent comprising a histone deacetylase (HDAC) inhibitor, wherein the administrations prevent or reduced the T cells exhaustion.

[0182] Also described herein is a method of increasing T cell cytotoxicity, wherein the method comprises administering a first agent comprising a first domain that binds to a TCR P variable (TCRpV)region; and administering a second agent comprising a histone deacetylase (HDAC) inhibitor, wherein the administrations increase the T cell cytotoxicity.

[0183] Also described herein is a method of increasing tumor or cancer immune infdtration, wherein the method comprises: administering a first agent comprising a first domain that binds to a TCR P variable (TCR V) region; and administering a second agent comprising a histone deacetylase (HDAC) inhibitor, wherein the administrations increase the tumor or cancer immune infiltration.

[0184] Also described herein is a method of promoting immune permissive tumor environment, wherein the method comprises: administering a first agent comprising a first domain that binds to a TCR variable (TCRpV) region; and administering a second agent comprising a histone deacetylase (HDAC) inhibitor, wherein the administration promotes the immune permissive tumor environment.INCORPORATION BY REFERENCE

[0185] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0186] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0187] FIG. 1A depicts an exemplary embodiment of the multifunctional molecule as described herein. FIG. IB depicts a schematic of expansion of target CD4+ and CD8+ hVp6 / VpiO T cells by the multifunctional molecule as described herein. Note that Human Vp6 / Vpi0 (hVp6 / VP 10) correspond to murine Vpi3 (mVpi3).

[0188] FIGs. 2A-2B show tumor growth curves for anti-PDl and / or anti-PDLl treatment in CT26, MC38 B2m / _, EMT.6 B2mz- (FIG. 2A), RM-1, and CMT.64 tumor models (FIG. 2B). CT26, MC38 B2m / _, EMT.6 B2m / _, RM-1, and CMT.64 are anti-PDl / PDLl nonresponsive tumors.

[0189] FIG. 3 shows a schematic of Entinostat and anti-mVpi3-IL2 (mBKM0186) combination treatment.

[0190] FIGs. 4A-4B show tumor growth (FIG. 4A) and survival (FIG. 4B) curves of mice treated with vehicle, Entinostat, anti-mVpi3-IL2 (mBKM0186), or Entinostat+ anti-mVpi3-IL2 (mBKM0186) in a CT26 tumor model.

[0191] FIGs. 5A-5B show tumor growth (FIG. 5A) and survival (FIG. 5B) curves of mice treated with vehicle, Entinostat, anti-mVpi3-IL2 (mBKM0186), or Entinostat+ anti-mVpi3-IL2 (mBKM0186) in a EMT6 tumor model.

[0192] FIGs. 6A-6C show tumor growth curve (FIG. 6A), tumor volume (FIG. 6B), and survival curve (FIG. 6C) of mice treated with vehicle, Entinostat, anti-mVpi3-IL2 (mBKM0186), or Entinostat+ anti- mVpi3-IL2 (mBKM0186) in a EMT6 B2m KO tumor model.

[0193] FIGs. 7A-7B show tumor growth curve (FIG. 7A) and tumor volume (FIG. 7B) of mice treated with vehicle, Entinostat, anti-mVpi3-IL2 (mBKM0186), or Entinostat+ anti-mVpi3-IL2 (mBKM0186) in a EMT6 B2m KO tumor model.

[0194] FIGs. 8A-8E show study of Entinostat and anti-mVpi3-IL2 (mBKM0186) combination treatment in an EMT.6 tumor model. FIG. 8A shows study schematic. FIGs. 8B-C show respective tumor growth curves and tumor growth inhibition (%RGI) (FIG. 8B) and single tracks with cure rates (CR) (FIG. 8C) for each treatment group (vehicle, Entinostat, anti-mVpi3-IL2 (mBKM0186), or Entinostat+ anti-mVpi3-IL2 (mBKM0186)). FIG. 8D shows % survival for tumor bearing mice. Cured mice were rechallenged with EMT.6 and 4T1 cells. FIG. 8E shows tumor growth curves of rechallenged mice. One-way ANOVA with Turkey’s multiple comparisons were used for data presented in violin or scatter plots. Two-way ordinary ANOVA was used to analyze tumor growth curves. Survival was analyzed using Log-rank (Mantel-Cox) test. All tumor growth data are representative of 2 or 3 independent experiments. Statistical significance was set at *p<0.05, **p<0.005, ***p<0.001.

[0195] FIGs. 9A-9G show study of Entinostat and anti-mVpi3-IL2 (mBKM0186) combination treatment in a CT26 tumor model. FIG. 9A shows study schematic. FIGs. 9B-C show respective tumor growth curves and tumor growth inhibition (%RGI) (FIG. 9B) and single tracks with cure rates (CR) (FIG. 9C) for each treatment group (vehicle, Entinostat, anti-mVpi3-IL2 (mBKM0186), or Entinostat+ anti-mVpi3-IL2 (mBKM0186)). FIG. 9D shows % survival for tumor bearing mice. Cured mice were rechallenged with CT26 and 4T1 cells. FIG. 9E shows tumor growth curves of rechallenged mice. FIG.9F shows percentage of mVbl3-l / 13-2+ CD8 T cells. FIG. 9G shows percentage of mVbl3-l / 13- 2+ CD8 T cells expressing cytolytic granzyme B. One-way ANOVA with Turkey’s multiple comparisons were used for data presented in violin or scatter plots. Two-way ordinary ANOVA was used to analyze tumor growth curves. Survival was analyzed using Log-rank (Mantel-Cox) test. All tumor growth data are representative of 2 or 3 independent experiments. Statistical significance was set at *p<0.05, **p<0.005, ***p<0.001.

[0196] FIGs. 10A-F show a study of Entinostat and anti-mVpi3-IL2 (mBKM0186) combination treatment expands and activates target mVbl3-l / 13-2-specific CD8+ T cells in peripheral blood in a CT26 tumor model. FIG.10A shows study schematic. FIGs. 10B-C show CD8+ T cells as a frequency of CD45+ cells (FIG. 10B) and frequency of CD44hi CD8+ T cells (FIG. 10C). FIG 10D shows anti- mVbl3 target cells identified by the expression of Vbl3-l / 13-2 and CD8. FIGs. 10E-F show timeline of target mVbl3-l / 13-2+ cells as a frequency of CD8+ T cells (FIG. 10E) and frequency of mVbl3-l / 13- 2+ CD8+ T cells expressing Granzyme B (FIG. 10F). One-way ANOVA with Turkey’s multiple comparisons were used for data presented in violin or scatter plots. Two-way ordinary ANOVA was used to analyze tumor growth curves. Survival was analyzed using Log-rank (Mantel-Cox) test. All tumorgrowth data are representative of 2 or 3 independent experiments. Statistical significance was set at *p<0.05, **p<0.005, ***p<0.001.

[0197] FIGs. 11A-C show anti-tumor activity of Entinostat and anti-mVpi3-IL2 (mBKM0186) combination treatment is reversed by immune depletion. FIG.11A shows study schematic. FIG. 11B shows tumor growth in mice untreated, ortreated with Entinostat and anti-mVpi3-IL2 (mBKM0186) in the absence or presence of CD8 depletion, CD4 depletion, or NK depletion. FIG. 11C shows percent survival for EMT.6 tumor bearing mice with and without depletion. One-way ANOVA with Turkey’s multiple comparisons were used for data presented in violin or scatter plots. Two-way ordinary ANOVA was used to analyze tumor growth curves. Survival was analyzed using Log -rank (Mantel-Cox) test. All tumor growth data are representative of 2 or 3 independent experiments. Statistical significance was set at *p<0.05, **p<0.005, ***p<0.001.

[0198] FIGs. 12A-C show Entinostat and anti-mVpi3-IL2 (mBKM0186) combination treatment elicits significant tumor suppression and survival benefits in MC38 B2m KO tumor model. FIG.12A shows study schematic. FIG. 12B shows tumor volumes of individual mice at day 25 with %TGI. FIG.12C shows percental survival for tumor-bearing mice. One-way ANOVA with Turkey’s multiple comparisons were used for data presented in violin or scatter plots. Two-way ordinary ANOVA was used to analyze tumor growth curves. Survival was analyzed using Log-rank (Mantel-Cox) test. All tumor growth data are representative of 2 or 3 independent experiments. Statistical significance was set at *p<0.05, **p<0.005, ***p<0.001.

[0199] FIGs. 13A-C show Entinostat and anti-mVpi3-IL2 (mBKM0186) combination treatment elicits significant tumor suppression and survival benefits in EMT.6 B2m KO tumor model. FIG.13A shows study schematic. FIG. 13B shows tumor volumes of individual mice at day 25 with %TGI. FIG.13C shows percental survival for tumor-bearing mice. One-way ANOVA with Turkey’s multiple comparisons were used for data presented in violin or scatter plots. Two-way ordinary ANOVA was used to analyze tumor growth curves. Survival was analyzed using Log-rank (Mantel-Cox) test. All tumor growth data are representative of 2 or 3 independent experiments. Statistical significance was set at *p<0.05, **p<0.005, ***p<0.001.

[0200] FIGs. 14A-F show Entinostat and anti-mVpi3-IL2 (mBKM0186) combination treatment expands and activates peripheral CD8+ T cells and increases target mVbl3-l / 13-2+CD8+T cells in EMT.6 B2m / _tumor-bearing mice. FIG.14A shows study schematic. FIGs. 14B-C show CD8+ T cells as a % of CD45+ cells in peripheral blood (FIG. 14B) and spleen (FIG. 14C). FIG. 14E shows plots denoting frequency and gMFI of CD8+CD44hlT cells expressing IFNg and IFNg / TNFa upon CD3 / CD28 in vitro stimulation. FIG. 14F shows frequency of mVbl3-l / 13-2-specific CD8+T cells in peripheral blood.

[0201] FIGs. 15A-D show Entinostat and anti-mVpi3-IL2 (mBKM0186) combination treatment expands and promotes infiltration of activated and stem-like Cd8+ T cells in EMT.6 B2m / _tumors, promoting an inflammatory TME associated with mVbl3-l / 13-2-specific CD8+ T cells. FIG. 15A shows CD8+ T cells as frequency of tumor CD45+ cells and per tumor mg. FIG. 15B shows expression oftumor soluble analytes. FIG. 15C shows Seal expression as frequency of tumor CD8+ T cells and per tumor weight. FIG. 15D shows mVbl3-l / 13-2-specific CD8+ T cells as a percentage of tumor CD8+ T cells and by tumor weight.

[0202] FIGs. 16A-M show Entinostat and anti-mVpi3-IL2 (mBKM0186) combination treatment promoted a more immune permissive B2m / _tumor microenvironment. FIG. 16A shows macrophage count per tumor weight. FIG. 16B shows M1 / M2 tumor macrophage. FIG. 16C shows timeline of Vbl3-l / 13-2+ CD8 T cell expansion in peripheral blood. FIG. 16D shows quantification and representative spot assay of IFNg release. FIG. 16E shows percentage of polyfunctional CD8+ T cells (CD8+CD44hl). FIGs. 16F-G shows macrophage frequency of Ml-like (antitumor) (FIG. 16F) and M2- like (FIG. 16G) sub populations. FIG. 16H-I show frequency of Ml-like and M2-like macrophages expressing MHC-II (FIG. 16H) and PD-L1 (FIG. 161). FIGs. 16J-K show number of gMDSCs per tumor weight (FIG. 16J) and frequency of gMDSCs expressing MHC-II (FIG. 16K). FIGs. 16L-M show number if tumor-infiltrated CD8+ T cells (FIG. 16L) and amount of tumor soluble IFNg (FIG. 16M), indicative of higher T cell function.

[0203] FIGs. 17A-E show EMT.6 B2m / _tumor suppression elicited by Entinostat and anti-mVpi3-IL2 (mBKM0186) combination treatment is reversed by immune depletion. FIG. 17A shows study schematic. FIGs. 17B-D show depletion of CD8 (FIG. 17B), CD4 (FIG. 17C), and NK cells (FIG. 17D) in peripheral blood. FIG. 17E shows tumor growth in untreated mice and Entinostat and anti-mVpi3-IL2 (mBKM0186) treated mice in the absence, or presence of CD8 depletion, CD4 depletion, or NK depletion.

[0204] FIGs. 18A-C show Entinostat and anti-mVbl3-IL2 combination treatment in CMT.64 lung cancer tumors. FIG. 18A shows study schematic. FIGs. 18B-C show tumor volume (FIG. 18B) and survival curve (FIG. 18C).

[0205] FIGs. 19A-C show Entinostat and anti-mVbl3-IL2 combination treatment in RM1 prostate cancer tumors. FIG. 19A shows study schematic. FIGs. 19B-C show tumor volume (FIG. 19B) and survival curve (FIG. 19C).

[0206] FIGs. 20A-20D show Entinostat and anti-mVpi3-IL2 (mBKM0186) combination treatment in EMT6 breast cancer tumors. FIG. 20A shows study schematic. FIG. 20B shows expression of NXF1 in IFNg+ cells in the spleen, tumor-draining lymph nodes, or tumors. FIG. 20C shows expression of CD8+ tumor-infiltrating T cells. FIG. 20D shows expression of TRBV subfamilies.DETAILED DESCRIPTIONDEFINITION

[0207] Certain specific details of this description are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the present disclosure may be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring descriptions of the embodiments.

[0208] Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure.

[0209] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The use of the words “a” or “an” when used in conjunction with the term “comprising” herein may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0210] It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0211] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below.

[0212] The term “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. As used herein, “about” and “approximately” generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given range of values.

[0213] The term “acquire” or “acquiring” as the terms are used herein, refer to obtaining possession of a physical entity (e.g. , a sample, a polypeptide, a nucleic acid, or a sequence), or a value, e.g. , a numerical value, by “directly acquiring” or “indirectly acquiring” the physical entity or value. “Directly acquiring” means performing a process (e.g., performing a synthetic or analytical method) to obtain the physical entity or value. “Indirectly acquiring” refers to receiving the physical entity or value from another party or source (e.g. , a third party laboratory that directly acquired the physical entity or value). Directly acquiring a physical entity includes performing a process that includes a physical change in a physical substance, e.g., a starting material. Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance, e.g., performing an analytical process which includes a physical change in a substance, e.g., a sample.

[0214] “Antibody molecule” as used herein refers to a protein, e.g., an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain structure and / or sequence. An antibody molecule encompasses antibodies (e.g., full-length antibodies) and antibody fragments. In some embodiments, an antibody molecule comprises an antigen binding or functional fragment of a full length antibody, or a full length immunoglobulin chain. For example, a full-length antibody is an immunoglobulin (Ig) molecule (e.g., an IgG antibody) that is naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes). In embodiments, an antibody molecule refersto an immunologically active, antigen-binding portion of an immunoglobulin molecule, such as an antibody fragment. An antibody fragment, e.g., functional fragment, is a portion of an antibody, e.g., Fab, Fab', F(ab')2, F(ab)2, variable fragment (Fv), domain antibody (dAb), or single chain variable fragment (scFv). A functional antibody fragment binds to the same antigen as that recognized by the intact (e.g., full-length) antibody. The terms “antibody fragment” or “functional fragment” also include isolated fragments consisting of the variable regions, such as the “Fv” fragments consisting of the variable regions of the heavy and light chains or recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (“scFv proteins”). In some embodiments, an antibody fragment does not include portions of antibodies without antigen binding activity, such as Fc fragments or single amino acid residues. Exemplary antibody molecules include full length antibodies and antibody fragments, e.g, dAb (domain antibody), single chain, Fab, Fab’, and F(ab’)2 fragments, and single chain variable fragments (scFvs). In some embodiments, the antibody molecule is an antibody mimetic. In some embodiments, the antibody molecule is, or comprises, an antibody-like framework or scaffold, such as, fibronectins, ankyrin repeats (e.g., designed ankyrin repeat proteins (DARPins)), avimers, affibody affinity ligands, anti calins, or affilin molecules.

[0215] The term “human-like antibody molecule” as used herein refers to a humanized antibody molecule, human antibody molecule or an antibody molecule having at least 95% sequence identity with a non-murine germline framework region, e.g., FR1, FR2, FR3 and / or FR4. In some embodiments, the human-like antibody molecule comprises a framework region having at least 95% sequence identity to a human germline framework region, e.g., a FR1, FR2, FR3 and / or FR4 of a human germline framework region. In some embodiments, the human-like antibody molecule is a recombinant antibody. In some embodiments, the human-like antibody molecule is a humanized antibody molecule. In some embodiments, the human-like antibody molecule is human antibody molecule. In some embodiments, the human-like antibody molecule is a phage display or a yeast display antibody molecule. In some embodiments, the human-like antibody molecule is a chimeric antibody molecule. In some embodiments, the human-like antibody molecule is a CDR grafted antibody molecule.

[0216] As used herein, an “immunoglobulin variable domain sequence” refers to an amino acid sequence which can form the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally-occurring variable domain. For example, the sequence may or may not include one, two, or more N- or C-terminal amino acids, or may include other alterations that are compatible with formation of the protein structure.

[0217] In embodiments, an antibody molecule is monospecific, e.g., it comprises binding specificity for a single epitope. In some embodiments, an antibody molecule is multifunctional, e.g., it comprises a plurality of immunoglobulin variable domain sequences, where a first immunoglobulin variable domain sequence has binding specificity for a first epitope and a second immunoglobulin variable domain sequence has binding specificity for a second epitope. In some embodiments, an antibody molecule is a bispecific antibody molecule. “Bispecific antibody molecule” as used herein refers to an antibody molecule that has specificity for more than one (e.g. , two, three, four, or more) epitope and / or antigen.

[0218] “Antigen” (Ag) as used herein refers to a molecule that can provoke an immune response, e.g., involving activation of certain immune cells and / or antibody generation. Any macromolecule, including almost all proteins or peptides, can be an antigen. Antigens can also be derived from genomic recombinant or DNA. For example, any DNA comprising a nucleotide sequence or a partial nucleotide sequence that encodes a protein capable of eliciting an immune response encodes an “antigen.” In embodiments, an antigen does not need to be encoded solely by a full length nucleotide sequence of a gene, nor does an antigen need to be encoded by a gene at all. In embodiments, an antigen can be synthesized or can be derived from a biological sample, e.g. , a tissue sample, a tumor sample, a cell, or a fluid with other biological components. As used, herein a “tumor antigen” or interchangeably, a “cancer antigen” includes any molecule present on, or associated with, a cancer, e.g., a cancer cell or a tumor microenvironment that can provoke an immune response. As used, herein an “immune cell antigen” includes any molecule present on, or associated with, an immune cell that can provoke an immune response.

[0219] The “antigen-binding site,” or “binding portion” of an antibody molecule refers to the part of an antibody molecule, e.g., an immunoglobulin (Ig) molecule, that participates in antigen binding. In embodiments, the antigen binding site is formed by amino acid residues of the variable (V) regions of the heavy (H) and light (L) chains. Three highly divergent stretches within the variable regions of the heavy and light chains, referred to as hypervariable regions, are disposed between more conserved flanking stretches called “framework regions,” (FRs). FRs are amino acid sequences that are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In embodiments, in an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface, which is complementary to the three-dimensional surface of a bound antigen. The three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.” The framework region and CDRs have been defined and described, e.g., in Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917. Each variable chain (e.g., variable heavy chain and variable light chain) is typically made up of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the amino acid order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0220] As used herein, an “immune cell” refers to any of various cells that function in the immune system, e.g., to protect against agents of infection and foreign matter. In embodiments, this term includes leukocytes, e.g., neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Innate leukocytes include phagocytes (e.g., macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer cells. Innate leukocytes identify and eliminate pathogens, either by attacking larger pathogens through contact or by engulfing and then killing microorganisms, and are mediators in the activation of an adaptive immune response. The cells of the adaptive immune system are special types of leukocytes, called lymphocytes. B cells and T cells are important types of lymphocytes and arederived from hematopoietic stem cells in the bone marrow. B cells are involved in the humoral immune response, whereas T cells are involved in cell-mediated immune response. The term “immune cell” includes immune effector cells.

[0221] “Immune effector cell,” as that term is used herein, refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response. Examples of immune effector cells include, but are not limited to, T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NK T) cells, and mast cells.

[0222] The term “effector function” or “effector response” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.

[0223] The terms “polypeptide”, “peptide” and “protein” (if single chain) are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. The polypeptide can be isolated from natural sources, can be a produced by recombinant techniques from a eukaryotic or prokaryotic host, or can be a product of synthetic procedures.

[0224] The terms “nucleic acid,” “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence,” and “polynucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The polynucleotide may be either single-stranded or double -stranded, and if single -stranded may be the coding strand or noncoding (antisense) strand. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The nucleic acid may be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a non-natural arrangement.

[0225] The term “isolated,” as used herein, refers to material that is removed from its original or native environment (e.g., the natural environment if it is naturally occurring). For example, a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated by human intervention from some or all of the co-existing materials in the natural system, is isolated. Such polynucleotides could be part of a vector and / or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of the environment in which it is found in nature. An isolated polynucleotide (ribonucleic acid (RNA), deoxyribonucleic acid (DNA)), or polypeptide is free of the genes / nucleic acids or sequence s / amino acids that flank it in its naturally-occurring state.

[0226] The compositions and methods of the present invention encompass polypeptides and nucleic acids having the sequences specified, or sequences substantially identical or similar thereto, e.g.,sequences at least 80%, 85%, 90%, 95% identical or higher to the sequence specified. In the context of an amino acid sequence, the term “substantially identical” is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and / or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 80%, 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99%, 99.5%, 99.9%, or 100% sequence identity to a reference sequence, e.g., a sequence provided herein. In the context of nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99%, 99.5%, 99.9%, or 100% sequence identity to a reference sequence, e.g., a sequence provided herein.

[0227] The term “variant” refers to a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence, or is encoded by a substantially identical nucleotide sequence. In some embodiments, the variant is a functional variant. In some embodiments, a TCRpV variant can bind to TCRa and form a TCR a:P complex.

[0228] The term “functional variant” refers to a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence, or is encoded by a substantially identical nucleotide sequence, and is capable of having one or more activities of the reference amino acid sequence.

[0229] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g. , gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).

[0230] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0231] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0232] It is understood that the molecules of the present invention may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on their functions.

[0233] The term “amino acid” is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids. Exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing. As used herein the term “amino acid” includes both the D- or L- optical isomers and peptidomimetics.

[0234] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Eamilies of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0235] As used herein, the term “molecule” as used in, e.g., antibody molecule, cytokine molecule, receptor molecule, includes full-length, naturally-occurring molecules, as well as variants, e.g., functional variants (e.g., truncations, fragments, mutated (e.g., substantially similar sequences) or derivatized form thereof), so long as at least one function and / or activity of the unmodified (e.g., naturally-occurring) molecule remains.

[0236] As used herein, the term “mutation” refers to an alteration in the nucleotide sequence of the genome of an organism, virus, or extrachromosomal DNA. In some embodiments, the mutation may be a large-scale mutation, such as amplifications (or gene duplications) or repetitions of a chromosomal segment, deletions of large chromosomal regions, chromosomal rearrangements (e.g., chromosomal translocations, chromosomal inversions, non-homologous chromosomal crossover, and interstitial deletions), and loss of heterozygosity. In some embodiments, the mutation may be a small-scale mutation, such as insertions, deletions, and substitution mutations. As used herein, the term “substitution mutation” refers to the transition that exchange a single nucleotide for another.

[0237] “Interleukin-2” also known as IL2, IL-2, IL 2, TCGF, lymphokine, and interleukin 2, as referred to herein, includes any of the recombinant or naturally-occurring forms of IL-2 or variants or homologs thereof that have or maintain IL-2 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring IL-2. In some embodiments, IL-2 is substantially identical to the protein identified by the UniProt reference number P60568 or a variant or homolog having substantial identity thereto.Anti-TCRpV antibodiesHuman T cell receptor (TCR) complex

[0238] TCR is a disulfide-linked membrane -anchored heterodimeric protein normally consisting of the highly variable alpha (a) and beta (P) chains expressed as part of a complex with the invariant CD3 chain molecules. TCR on a T cells is formed by a heterodimer of one alpha chain and one beta chain. Each alpha or beta chain consists of a constant domain and a highly variable domain classified as the Immunoglobulin superfamily (IgSF) fold. The TCRpV chains can be further classified into 30 subfamilies (TRBV1-30). Despite their high structural and functional homology, the amino acid sequence homology in the TRBV genes is very low. Only 4 amino acids out of approximately 95 are identical while 10 additional amino acids are conserved among all subfamilies. Nevertheless, TCRs formed between alpha and beta chains of highly diverse sequences show a remarkable structural homology and elicit a similar function, e.g., activation of T cells.

[0239] T cell receptors (TCR) can be found on the surface of T cells. TCRs recognize antigens, e.g., peptides, presented on, e.g., bound to, major histocompatibility complex (MHC) molecules on the surfaceof cells, e.g, antigen-presenting cells. TCRs are heterodimeric molecules and can comprise an alpha chain, a beta chain, a gamma chain or a delta chain. TCRs comprising an alpha chain and a beta chain are also referred to as TCRap. The TCR beta chain consists of the following regions (also known as segments): variable (V), diversity (D), joining (J) and constant (C) (see Mayer G. and Nyland J. (2010) Chapter 10: Major Histocompatibility Complex and T-cell Receptors-Role in Immune Responses. In: Microbiology and Immunology on-line, University of South Carolina School of Medicine). The TCR alpha chain consists of V, J and C regions. The rearrangement of the T-cell receptor (TCR) through somatic recombination of V (variable), D (diversity), J (joining), and C (constant) regions is a defining event in the development and maturation of a T cell. TCR gene rearrangement takes place in the thymus.

[0240] TCRs can comprise a receptor complex, known as the TCR complex, which comprises a TCR heterodimer comprising of an alpha chain and a beta chain, and dimeric signaling molecules, e.g., CD3 co-receptors, e.g., CD3o / s. and / or CD3y / a.

[0241] As used herein, the term “T cell receptor beta variable chain” or “TCRpV,” refers to an extracellular region of the T cell receptor beta chain which comprises the antigen recognition domain of the T cell receptor. The term TCRpV includes isoforms, mammalian, e.g., human TCRpV, species homologs of human and analogs comprising at least one common epitope with TCRpV. Human TCRpV comprises a gene family comprising subfamilies including, but not limited to: a TCRP V6 subfamily, a TCRP V10 subfamily, a TCRP V12 subfamily, a TCRP V5 subfamily, a TCRP V7 subfamily, a TCRP VI 1 subfamily, a TCRP V14 subfamily, a TCRP V16 subfamily, a TCRP V18 subfamily, a TCRP V9 subfamily, a TCRP V13 subfamily, a TCRP V4 subfamily, a TCRP V3 subfamily, a TCRP V2 subfamily, a TCRP V15 subfamily, a TCRP V30 subfamily, a TCRP V19 subfamily, a TCRP V27 subfamily, a TCRP V28 subfamily, a TCRP V24 subfamily, a TCRP V20 subfamily, TCRP V25 subfamily, a TCRP V29 subfamily, a TCRP VI subfamily, a TCRP VI 7 subfamily, a TCRP V21 subfamily, a TCRP V23 subfamily, or a TCRP V26 subfamily, as well as family members of said subfamilies, and variants thereof (e.g., a structural or functional variant thereof). In some embodiments, the TCRP V6 subfamily comprises: TCRP V6-4*0I, TCRP V6-4*02, TCRP V6-9*0I, TCRP V6-8*0I, TCRP V6-5*0I, TCRP V6-6*02, TCRP V6-6*0I, TCRP V6-2*0I, TCRP V6-3*01 or TCRP V6-I*0I. In some embodiments, TCRpV comprises TCRP V6-5*01, or a variant thereof, e.g., a variant having 85%, 90%, 95%, 99% or more identity the naturally-occurring sequence. TCRP V6-5*01 is also known as TRBV65; TCRBV6S5; TCRBV13S1, TCRP VI 3.1, mTCRp VI 3.1, or mTRBV13-l. The amino acid sequence of TCRP V6- 5*01, e.g., human TCRP V6-5*0I, is known in that art, e.g., as provided by IMGT ID L36092. In some embodiments, TCRP V6-5*01 is encoded by the nucleic acid sequence of SEQ ID NO: 43, or a sequence having 85%, 90%, 95%, 99% or more identity thereof. In some embodiments, TCRP V6-5*01 comprises the amino acid sequence of SEQ ID NO: 44, or a sequence having 85%, 90%, 95%, 99% or more identity thereof.

[0242] SEQ ID NO: 43ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGC TGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGC TGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGC TACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTC CCAGACATCTGTGTACTTCTGTGCCAGCAGTTACTC

[0243] SEQ ID NO: 44MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMG LRLIHY-SVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYTCR beta V (TCR / 3V)

[0244] Diversity in the immune system enables protection against a huge array of pathogens. Since the germline genome is limited in size, diversity is achieved not only by the process of V(D)J recombination but also by junctional (junctions between V-D and D-J segments) deletion of nucleotides and addition of pseudo-random, non-templated nucleotides. The TCR beta gene undergoes gene arrangement to generate diversity.

[0245] The TCR V beta repertoire varies between individuals and populations because of, e.g., 7 frequently occurring inactivating polymorphisms in functional gene segments and a large insertion / deletion -related polymorphism encompassing 2 V beta gene segments.

[0246] Provided herein are, inter aha, antibody molecules and fragments thereof, that bind, e.g., specifically bind, to a human TCR beta V chain (TCRpV), e.g. , a TCRpV gene family (also referred to as a group), e.g. , a TCRpV subfamily (also referred to as a subgroup), e.g. , as described herein. TCR beta V families and subfamilies are known in the art, e.g., as described in Yassai et al., (2009) Immunogenetics 61(7)pp:493-502; Wei S. and Concannon P. (1994) Human Immunology 41(3) pp: 201-206. The antibodies described herein can be recombinant antibodies, e.g., recombinant non-murine antibodies, e.g., recombinant human or humanized antibodies.

[0247] The terms TCRBV, TCRVB, TRBV, TCRpV, TCRVP or TRpV are used interchangeably herein and refer to a TCR beta V chain, e.g., as described herein.

[0248] In some embodiments, provided herein is an anti -TCRpV antibody molecule that binds to human TCRPV, e.g. , a TCRpV family, e.g. , gene family or a variant thereof. In some embodiments a TCRBV gene family comprises one or more subfamilies, e.g., as described herein, e.g., in Table 8A or Table 8B. In some embodiments, the TCRpV gene family comprises: a TCRP V6 subfamily, or a TCRP V10 subfamily.

[0249] In some embodiments, TCRP V6 subfamily is also known as TCRP VI 3.1 or mTCRp VI 3.1. In some embodiments, the TCRP V6 subfamily comprises: TCRP V6-4*0I, TCRP V6-4*02, TCRP V6- 9*01, TCRP V6-8*0I, TCRP V6-5*0I, TCRP V6-6*02, TCRP V6-6*0I, TCRP V6-2*0I, TCRP V6- 3*01 or TCRP V6-l*01, or a variant thereof. In some embodiments, TCRP V6 comprises TCRP V6- 4*01, or a variant thereof. In some embodiments, TCRP V6 comprises TCRP V6-4*02, or a variant thereof. In some embodiments, TCRP V6 comprises TCRP V6-9*01, or a variant thereof. In some embodiments, TCRP V6 comprises TCRP V6-8*01, or a variant thereof. In some embodiments, TCRP V6 comprises TCRP V6-5*01, or a variant thereof. In some embodiments, TCRP V6 comprises TCRPV6-6*02, or a variant thereof. In some embodiments, TCRP V6 comprises TCRP V6-6*01, or a variant thereof. In some embodiments, TCRP V6 comprises TCRP V6-2*01, or a variant thereof. In some embodiments, TCRP V6 comprises TCRP V6-3*01, or a variant thereof. In some embodiments, TCRP V6 comprises TCRP V6-l*01, or a variant thereof.

[0250] In some embodiments, TCRP V6 comprises TCRP V6-5*01, or a variant thereof. In some embodiments, TCRP V6, e.g., TCRP V6-5*01, is recognized, e.g., bound, by SEQ ID NO: 1 and / or SEQ ID NO: 2. In some embodiments, TCRP V6, e.g., TCRP V6-5*01, is recognized, e.g., bound, by SEQ ID NO: 9 and / or SEQ ID NO: 10. In some embodiments, TCRP V6 is recognized, e.g., bound, by SEQ ID NO: 9 and / or SEQ ID NO: 11.

[0251] In some embodiments, the TCRP V10 subfamily comprises: TCRP V10-l*01, TCRP V10-l*02, TCRP V10-3*01 or TCRP V10-2*01, or a variant thereof. In some embodiments, TCRP V10 subfamily is also known as TCRP V13. In some embodiments, TCRP V10-1 is also known as TCRP V13.1, mTCRp VI 3.1, TRBV13-1 or mTRBVI3-l. In some embodiments, TCRP VI 0-2 is also known as TCRP V13.2, mTCRp V13.2, TRBV13-2, or mTRBV13-2.

[0252] Exemplary amino acid sequences for TCRpV subfamily members can be found on the ImMunoGeneTics Information System website: http: / / www.imgt.org / , or in a similar resource. Anti-TCRfiV antibodies

[0253] Current bispecific constructs designed to redirect T cells to promote tumor cell lysis for cancer immunotherapy typically utilize antibody fragments (Fab, scFv, VH, single domain antibody, etc.) that are derived from monoclonal antibodies (mAb) directed against the CD3e subunit of the T cell receptor (TCR). However, there are limitations to this approach which may prevent the full realization of the therapeutic potential for such bispecific constructs. Previous studies have shown that even low “activating” doses of anti-CD3e mAb can cause long-term T cell dysfunction and exert immunosuppressive effects. In addition, anti-CD3e mAbs have been associated with side effects that result from massive T cell activation. The large number of activated T cells secrete substantial amounts of cytokines, the most important of which is Interferon gamma (IFNy). This excess amount of IFNy in turn activates macrophages which then overproduce proinflammatory cytokines such as IL-lbeta, IL-6, IL- 10 and TNF-alpha, causing a “cytokine storm” known as the cytokine release syndrome (CRS) (Shimabukuro-Vomhagen et al., J Immunother Cancer. 2018 Jun 15 ;6( 1) :56, herein incorporated by reference in its entirety). Thus, the need exists for developing antibodies that are capable of binding and activating only a subset of effector T cells, e.g., to re-duce the CRS and / or neurotoxicity (NT).

[0254] Described herein are molecules targeting the TCRpV chain of TCR and methods thereof. Without wishing to be bound by theory, such molecules are capable of binding, activating, and / or expanding only a subset of T cells, avoiding or reducing CRS and / or NT and minimizing potential immunosuppressive effects of anti-CD3 mAbs.

[0255] Described herein is a class of antibodies, i.e., anti-TCRpV antibody molecules as described herein, which despite having low sequence similarity (e.g., low sequence identity among the different antibody molecules that recognize different TCRpV subfamilies), recognize a structurally conserved, yetsequence-wise variable, region, e.g., domain, on the TCRpV protein and have a similar function (e.g., activation of T cells and a similar cytokine profile as described herein). Thus, the anti -TCRpV antibody molecules as described herein share a structure -function relationship.

[0256] Without wishing to be bound by theory, in some embodiments, the anti-TCRpV antibody molecules as described herein bind to an outward facing epitope of a TCRpV protein when it is in a complex with a TCRalpha protein. In some embodiments, the anti-TCRpV antibody molecules as described herein recognize (e.g., bind to), a domain (e.g., an epitope) on the TCRpV protein that is: (1) structurally conserved among different TCRpV subfamilies; and (2) has minimal sequence identity among the different TCRpV subfamilies. TCRpV proteins from the different TCRBV subfamilies share minimal sequence similarity. However, TCRpV proteins which have minimal sequence similarity, share a similar 3D conformation and structure.

[0257] The alignment of TCRBV amino acid sequences underscores the diversity of TCR sequences. In particular, the TRBV sequences from different subfamilies are considerably different from each other.

[0258] In some embodiments, the anti-TCRpV antibody molecules as described herein do not recognize, e.g., bind to, an interface of a TCRpV: TCRalpha complex. In some embodiments, the anti-TCRpV antibody molecules as described herein do not recognize, e.g., bind to, a constant region of a TCRpV protein. An exemplary antibody that binds to a constant region of a TCRBV region is JOVI. 1 as described in Viney et al., (Hybridoma. 1992 Dec;l 1 (6) : 701 - 13) . In some embodiments, the anti-TCRpV antibody molecules as described herein do not recognize, e.g., bind to, one or more (e.g., all) of a complementarity determining region (e.g., CDR1, CDR2 and / or CDR3) of a TCRpV protein.

[0259] Provided herein are, inter alia, antibody molecules directed to the variable chain of the beta subunit of TCR (TCRpV) which bind and, e.g., activate a subset of T cells. The anti-TCRpV antibody molecules as described herein result in lesser or no production of cytokines associated with CRS, e.g., IL- 6, IL-lbeta, IL- 10 and TNF alpha; and enhanced and / or delayed production of IL-2 and IFNy. In some embodiments, the anti-TCRpV antibodies as described herein have a cytokine profile, e.g., as described herein, which differs from a cytokine profile of a T cell engager that binds to a receptor or molecule other than a TCRpV region (“a non-TCRpV-binding T cell engager”). In some embodiments, the non-TCRpV- binding T cell engager comprises an antibody that binds to a CD3 molecule (e.g., CD3 epsilon (CD3e) molecule); or a TCR alpha (TCRa) molecule. In some embodiments, the non-TCRpV-binding T cell engager is an OKT3 antibody or an SP34-2 antibody.

[0260] In some embodiments, the anti-TCRpV antibodies as described herein result in expansion of TCRPV+ T cells, e.g., a subset of memory effector T cells known as TEMRA. Without wishing to be bound by theory, it is believed that in some embodiments, TEMRA cells can promote tumor cell lysis but not CRS. Accordingly, provided herein are methods of making said anti-TCRpV antibody molecules and uses thereof. Also described herein are multifunctional molecules, e.g., bispecific molecules comprising said anti-TCRpV antibody molecules. In some embodiments, compositions comprising anti-TCRpV antibody molecules of the present disclosure, can be used, e.g., to: (1) activate and redirect T cells to promote tumor cell lysis for cancer immuno-therapy; and / or (2) expand TCRPV+ T cells. In someembodiments, compositions comprising anti-TCRpV antibody molecules as described herein limit the harmful side-effects of CRS and / or NT, e.g., CRS and / or NT associated with anti-CD3e targeting.

[0261] In some embodiments, the anti-TCRpV antibody molecule binds to one or more of TRBV6-1, TRBV6-2, TRBV6-3, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8 and TRBV6-9. In some embodiments, the anti-TCRpV antibody molecule is an anti-TRBV6-l, anti-TRBV6-2, anti-TRBV6-3, anti-TRBV6-4, anti-TRBV6-5, anti-TRBV6-6, anti-TRBV6-8, anti-TRBV6-9. Exemplary anti-TCRpV antibody molecules and the corresponding TCRpV subfamilies recognized by said anti-TCRpV antibody molecules are disclosed in Table 10A.

[0262] In some embodiments, the anti-TCRpV antibody molecule binds specifically to TRBV6-1, TRBV6-2, TRBV6-3, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8 or TRBV6-9. In some embodiments, the anti-TCRpV antibody molecule binds specifically to TRBV6-1. In some embodiments, the anti- TCRpV antibody molecule binds specifically to TRBV6-2. In some embodiments, the anti-TCRpV antibody molecule binds specifically to TRBV6-3. In some embodiments, the anti-TCRpV antibody molecule binds specifically to TRBV6-4. In some embodiments, the anti-TCRpV antibody molecule binds specifically to TRBV6-5. In some embodiments, the anti-TCRpV antibody molecule binds specifically to TRBV6-6. In some embodiments, the anti-TCRpV antibody molecule binds specifically to TRBV6-8. In some embodiments, the anti-TCRpV antibody molecule binds specifically to TRBV6-9.

[0263] In some embodiments, the light or the heavy chain variable framework (e.g., the region encompassing at least FR1, FR2, FR3, and optionally FR4) of the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule can be chosen from: (a) a light or heavy chain variable framework including at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or 100% of the amino acid residues from a human light or heavy chain variable framework, e.g. , a light or heavy chain variable framework residue from a human mature antibody, a human germline sequence, or a human consensus sequence; (b) a light or heavy chain variable framework including from 20% to 80%, 40% to 60%, 60% to 90%, or 70% to 95% of the amino acid residues from a human light or heavy chain variable framework, e.g., a light or heavy chain variable framework residue from a human mature antibody, a human germline sequence, or a human consensus sequence; (c) a non-human framework (e.g. , a rodent framework); or (d) a non-human framework that has been modified, e.g. , to remove antigenic or cytotoxic determinants, e.g, deimmunized, or partially humanized. In some embodiments, the light or heavy chain variable framework region (particularly FR1, FR2 and / or FR3) includes a light or heavy chain variable framework sequence at least 70, 75, 80, 85, 87, 88, 90, 92, 94, 95, 96, 97, 98, 99% identical or identical to the frameworks of a VL or VH segment of a human germline gene.

[0264] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, comprises a heavy chain variable domain having at least one, two, three, four, five, six, seven, ten, fifteen, twenty or more changes, e.g, amino acid substitutions or deletions, from an amino acid sequence of any one of A-H. 1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, e.g., the amino acid sequence of the FR region in the entire variable region, e.g., shown in SEQ ID NO: 9.

[0265] Alternatively, or in combination with the heavy chain substitutions described herein, the anti- TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, comprises a light chain variable domain having at least one, two, three, four, five, six, seven, ten, fifteen, twenty or more amino acid changes, e.g., amino acid substitutions or deletions, from an amino acid sequence of any one of A-H.1 to A-H.85, e.g., A-H. 1, A-H.2 or A-H.68, e.g. , the amino acid sequence of the FR region in the entire variable region, e.g., shown in SEQ ID NO: 10 or SEQ ID NO: 11.

[0266] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, comprises the light chain framework region 1 of A-H.l or A-H.2.

[0267] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, comprises the light chain framework region 2 of A-H.l or A-H.2.

[0268] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, comprises the light chain framework region 3 of A-H.l or A-H.2.

[0269] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, comprises the light chain framework region 4 of A-H.l or A-H.2.

[0270] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, comprises a light chain variable domain comprising a framework region, e.g., framework region 1 (FR1), comprising a change, e.g., a substitution (e.g., a conservative substitution) at position 10 according to Kabat numbering. In some embodiments, the FR1 comprises a Phenylalanine at position 10, e.g., a Serine to Phenyalanine substitution. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

[0271] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, comprises a light chain variable domain comprising a framework region, e.g., framework region 2 (FR2), comprising a change, e.g., a substitution (e.g., a conservative substitution) at a position as described herein according to Kabat numbering. In some embodiments, FR2 comprises a Histidine at position 36, e.g., a substitution at position 36 according to Kabat numbering, e.g., a Tyrosine to Histidine substitution. In some embodiments, FR2 comprises an Alanine at position 46, e.g., a substitution at position 46 according to Kabat numbering, e.g., an Arginine to Alanine substitution. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

[0272] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, comprises a light chain variable domain comprising a framework region, e.g., framework region 3 (FR3), comprising a change, e.g., a substitution (e.g., a conservative substitution) at a position as described herein according to Kabat numbering. In some embodiments, FR3 comprises a Phenyalanine at position 87, e.g., a substitution at position 87 according to Kabat numbering, e.g., a Tyrosine to Phenyalanine substitution. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

[0273] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, comprises a light chain variable domain comprising: (a) a frameworkregion 1 (FR1) comprising a Phenylalanine at position 10, e.g., a substitution at position 10 according to Kabat numbering, e.g., a Serine to Phenyalanine substitution; (b) a framework region 2 (FR2) comprising a Histidine at position 36, e.g., a substitution at position 36 according to Kabat numbering, e.g., a Tyrosine to Histidine substitution, and a Alanine at position 46, e.g., a substitution at position 46 according to Kabat numbering, e.g., a Arginine to Alanine substitution; and (c) a framework region 3 (FR3) comprising a Phenylalanine at position 87, e.g., a substitution at position 87 according to Kabat numbering, e.g., a Tyrosine to Phenyalanine substitution, e.g., as shown in the amino acid sequence of SEQ ID NO: 10. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

[0274] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, comprises a light chain variable domain comprising: (a) a framework region 2 (FR2) comprising a Histidine at position 36, e.g., a substitution at position 36 according to Kabat numbering, e.g., a Tyrosine to Histidine substitution, and a Alanine at position 46, e.g., a substitution at position 46 according to Kabat numbering, e.g., a Arginine to Alanine substitution; and (b) a framework region 3 (FR3) comprising a Phenylalanine at position 87, e.g., a substitution at position 87 according to Kabat numbering, e.g., a Tyrosine to Phenyalanine substitution, e.g., as shown in the amino acid sequence of SEQ ID NO: 11. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

[0275] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, comprises a light chain variable domain comprising: (a) a framework region 1 (FR1) comprising a change, e.g., a substitution (e.g., a conservative substitution) at one or more (e.g., all) positions as described herein according to Kabat numbering, ; (b) a framework region 2 (FR2) comprising a change, e.g., a substitution (e.g., a conservative substitution) at one or more (e.g., all) position as described herein according to Kabat numbering and (c) a framework region 3 (FR3) comprising a change, e.g., a substitution (e.g., a conservative substitution) at one or more (e.g., all) position as described herein according to Kabat numbering. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

[0276] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, comprises the heavy chain framework region 1 of A-H.l or A-H.2. In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, comprises the heavy chain framework region 2 of A-H.1 or A-H.2. In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, comprises the heavy chain framework region 3 of A-H.1 or A-H.2. In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, comprises the heavy chain framework region 4 of A-H.1 or A-H.2.

[0277] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, comprises a heavy chain variable domain comprising a framework region, e.g., framework region 3 (FR3), comprising a change, e.g., a substitution (e.g., a conservativesubstitution) at a position as described herein according to Kabat numbering. In some embodiments, FR3 comprises a Threonine at position 73, e.g., a substitution at position 73 according to Kabat numbering, e.g., a Glutamic Acid to Threonine substitution. In some embodiments, FR3 comprises a Glycine at position 94, e.g., a substitution at position 94 according to Kabat numbering, e.g., an Arginine to Glycine substitution. In some embodiments, the substitution is relative to a human germline heavy chain framework region sequence.

[0278] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, comprises a heavy chain variable domain comprising a framework region 3 (FR3) comprising a Threonine at position 73, e.g., a substitution at position 73 according to Kabat numbering, e.g., a Glutamic Acid to Threonine substitution, and a Glycine at position 94, e.g., a substitution at position 94 according to Kabat numbering, e.g., a Arginine to Glycine substitution, e.g., as shown in the amino acid sequence of SEQ ID NO: 10.

[0279] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, comprises the heavy chain framework regions 1-4 of A-H.1 or A-H.2, e.g., SEQ ID NO: 9. In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, comprises the light chain framework regions 1-4 of A-H.l, e.g., SEQ ID NO: 10. In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, comprises the light chain framework regions 1-4 of A-H.2, e.g., SEQ ID NO: 11. In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, comprises the heavy chain framework regions 1-4 of A-H.l, e.g., SEQ ID NO: 9; and the light chain framework regions 1-4 of A-H.1, e.g., SEQ ID NO: 10. In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, comprises the heavy chain framework regions 1-4 of A-H.2, e.g., SEQ ID NO: 9; and the light chain framework regions 1-4 of A-H.2, e.g., SEQ ID NO: 11.

[0280] In some embodiments, the heavy or light chain variable domain, or both, of the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, includes an amino acid sequence, which is substantially identical to an amino acid as described herein, e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical to a variable region of an antibody described herein, e.g. , an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or as described in Table 1, or encoded by the nucleotide sequence in Table 1; or which differs at least 1 or 5 residues, but less than 40, 30, 20, or 10 residues, from a variable region of an antibody described herein.

[0281] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, comprises at least one, two, three, or four antigen-binding regions, e.g., variable regions, having an amino acid sequence as set forth in Table 1, or a sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, or which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the sequences shown in Table 1. In another embodiment, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule includes a VH and / or VL domain encoded by a nucleic acid having a nucleotidesequence as set forth in Table 1, or a sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, or which differs by no more than 3, 6, 15, 30, or 45 nucleotides from the sequences shown in Table 1.

[0282] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 9, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 9; and / or a VL domain comprising the amino acid sequence of SEQ ID NO: 10, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 10.

[0283] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 9, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 9; and / or a VL domain comprising the amino acid sequence of SEQ ID NO: 11, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 11.

[0284] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule is a full antibody or fragment thereof (e.g., a Fab, F(ab')2, Fv, single domain antibody, or a single chain Fv fragment (scFv)). In embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule is a monoclonal antibody or an antibody with single specificity. In some embodiments, the anti-TCRpV antibody molecule, e.g., anti- TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, can also be a humanized, chimeric, camelid, shark, or an in w / ro-gcncratcd antibody molecule. In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, is a humanized antibody molecule. The heavy and light chains of the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, can be full-length (e.g, an antibody can include at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains) or can include an antigen-binding fragment (e.g, a Fab, F(ab')2, Fv, a single chain Fv fragment, a single domain antibody, a diabody (dAb), a bivalent antibody, or bispecific antibody or fragment thereof, a single domain variant thereof, or a camelid antibody).

[0285] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, is in the form of a multifunctional molecule, e.g., a bispecific molecule, e.g., as described herein.

[0286] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, has a heavy chain constant region (Fc) chosen from, e.g., the heavy chainconstant regions of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE. In some embodiments, the Fc region is chosen from the heavy chain constant regions of IgGl, IgG2, IgG3, and IgG4. In some embodiments, the Fc region is chosen from the heavy chain constant region of IgGl or IgG2 (e.g., human IgGl, or IgG2). In some embodiments, the heavy chain constant region is human IgGl. In some embodiments, the Fc region comprises a Fc region variant, e.g., as described herein.

[0287] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, has a light chain constant region chosen from, e.g., the light chain constant regions of kappa or lambda, preferably kappa (e.g., human kappa). In some embodiments, the constant region is altered, e.g., mutated, to modify the properties of the anti-TCRpV antibody molecule, e.g., anti- TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule (e.g, to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function). For example, the constant region is mutated at positions 296 (M to Y), 298 (S to T), 300 (T to E), 477 (H to K) and 478 (N to F) to alter Fc receptor binding (e.g. , the mutated positions correspond to positions 132 (M to Y), 134 (S to T), 136 (T to E), 313 (H to K) and 314 (N to F) of SEQ ID NOs: 212 or 214; or positions 135 (M to Y), 137 (S to T), 139 (T to E), 316 (H to K) and 317 (N to F) of SEQ ID NOs: 215, 216, 217 or 218), e.g., relative to human IgGl.

[0288] Antibody A-H.1 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3278 and a light chain comprising the amino acid sequence of SEQ ID NO: 72. Antibody A-H.2 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3278 and a light chain comprising the amino acid sequence of SEQ ID NO: 3279. Antibody A-H.68 comprises the amino acid sequence of SEQ ID NO: 1337, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity thereto. Antibody A-H.69 comprises the amino acid sequence of SEQ ID NO: 1500, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity thereto.

[0289] Additional exemplary humanized anti-TCRB V6 antibodies are provided in Table 1. In some embodiments, the anti-TCRp V6 is antibody A, e.g., humanized antibody A (antibody A-H), as provided in Table 1 . In some embodiments, the anti-TCRpV antibody comprises one or more (e.g., all three) of a LC CDR1, LC CDR2, and LC CDR3 provided in Table 1; and / or one or more (e.g., all three) of a HC CDR1, HC CDR2, and HC CDR3 provided in Table 1, or a sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity thereto. In some embodiments, antibody A comprises a variable heavy chain (VH) and / or a variable light chain (VL) provided in Table 1, or a sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity thereto.

[0290] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule comprises a VH of A-H. 1, A-H.2, A-H.3, A-H.4, A-H.5, A-H.6, A-H.7, A- H.8, A-H.9, A-H.10, A-H. l l, A-H.12, A-H.13, A-H.14, A-H.15, A-H.16, A-H.17, A-H.18, A-H.19, A- H.20, A-H.21, A-H.22, A-H.23, A-H.24, A-H.25, A-H.26, A-H.27, A-H.28, A-H.29, A-H.30, A-H.31, A- H.32, A-H.33, A-H.34, A-H.35, A-H.36, A-H.37, A-H.38, A-H.39, A-H.40, A-H. l, A-H.42, A-H.43, A-H.44, A-H.45, A-H.46, A-H.47, A-H.48, A-H.49, A-H.50, A-H.51, A-H.52, A-H.53, A-H.54, A-H.55, A- H.56, A-H.57, A-H.58, A-H.59, A-H.60, A-H.61, A-H.62, A-H.63, A-H.64, A-H.65, A-H.66, A-H.67, A- H.68, A-H.69, A-H.70, A-H.71, A-H.72, A-H.73, A-H.74, A-H.75, A-H.76, A-H.77, A-H.78, A-H.79, A- H.80, A-H.81, A-H.82, A-H.83, A-H.84, or A-H.85, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0291] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule comprises a VL of A-H.1, A-H.2, A-H.3, A-H.4, A-H.5, A-H.6, A-H.7, A- H.8, A-H.9, A-H.10, A-H.l l, A-H.12, A-H.13, A-H.14, A-H.15, A-H.16, A-H.17, A-H.18, A-H.19, A- H.20, A-H.21, A-H.22, A-H.23, A-H.24, A-H.25, A-H.26, A-H.27, A-H.28, A-H.29, A-H.30, A-H.31, A- H.32, A-H.33, A-H.34, A-H.35, A-H.36, A-H.37, A-H.38, A-H.39, A-H.40, A-H.l, A-H.42, A-H.43, A- H.44, A-H.45, A-H.46, A-H.47, A-H.48, A-H.49, A-H.50, A-H.51, A-H.52, A-H.53, A-H.54, A-H.55, A- H.56, A-H.57, A-H.58, A-H.59, A-H.60, A-H.61, A-H.62, A-H.63, A-H.64, A-H.65, A-H.66, A-H.67, A- H.68, A-H.69, A-H.70, A-H.71, A-H.72, A-H.73, A-H.74, A-H.75, A-H.76, A-H.77, A-H.78, A-H.79, A- H.80, A-H.81, A-H.82, A-H.83, A-H.84, or A-H.85, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0292] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule comprises a VH of A-H.l, A-H.2, A-H.3, A-H.4, A-H.5, A-H.6, A-H.7, A- H.8, A-H.9, A-H.10, A-H.l l, A-H.12, A-H.13, A-H.14, A-H.15, A-H.16, A-H.17, A-H.18, A-H.19, A- H.20, A-H.21, A-H.22, A-H.23, A-H.24, A-H.25, A-H.26, A-H.27, A-H.28, A-H.29, A-H.30, A-H.31, A- H.32, A-H.33, A-H.34, A-H.35, A-H.36, A-H.37, A-H.38, A-H.39, A-H.40, A-H.l, A-H.42, A-H.43, A- H.44, A-H.45, A-H.46, A-H.47, A-H.48, A-H.49, A-H.50, A-H.51, A-H.52, A-H.53, A-H.54, A-H.55, A- H.56, A-H.57, A-H.58, A-H.59, A-H.60, A-H.61, A-H.62, A-H.63, A-H.64, A-H.65, A-H.66, A-H.67, A- H.68, A-H.69, A-H.70, A-H.71, A-H.72, A-H.73, A-H.74, A-H.75, A-H.76, A-H.77, A-H.78, A-H.79, A- H.80, A-H.81, A-H.82, A-H.83, A-H.84, or A-H.85, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto; and a VL of A-H.l, A-H.2, A-H.3, A-H.4, A-H.5, A-H.6, A-H.7, A-H.8, A-H.9, A-H.10, A-H.l l, A-H.12, A-H.13, A-H.14, A-H.15, A-H.16, A-H.17, A-H.18, A- H.19, A-H.20, A-H.21, A-H.22, A-H.23, A-H.24, A-H.25, A-H.26, A-H.27, A-H.28, A-H.29, A-H.30, A- H.31, A-H.32, A-H.33, A-H.34, A-H.35, A-H.36, A-H.37, A-H.38, A-H.39, A-H.40, A-H.l, A-H.42, A- H.43, A-H.44, A-H.45, A-H.46, A-H.47, A-H.48, A-H.49, A-H.50, A-H.51, A-H.52, A-H.53, A-H.54, A- H.55, A-H.56, A-H.57, A-H.58, A-H.59, A-H.60, A-H.61, A-H.62, A-H.63, A-H.64, A-H.65, A-H.66, A- H.67, A-H.68, A-H.69, A-H.70, A-H.71, A-H.72, A-H.73, A-H.74, A-H.75, A-H.76, A-H.77, A-H.78, A- H.79, A-H.80, A-H.81, A-H.82, A-H.83, A-H.84, or A-H.85, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0293] Exemplary anti-TCRpV antibody molecules and the corresponding TCRpV subfamilies recognized by said anti-TCRpV antibody molecules are disclosed in Table 10A.

[0294] The various TCRpV subfamilies and / or subfamily members can be expressed at different levels in individuals, e.g., healthy individuals, as disclosed in Kitaura K. et al (2016), BMC Immunology vol17: 38, the entire contents of which are hereby incorporated by reference. For example, TCRp V6-5 is represented in approximately 3-6% healthy donors.

[0295] The representation of various TCRBV subfamilies and / or subfamily members can also be different in cancer cells. For example, TCRpV is present in about 3-6% of tumor infdtrating T cells irrespective of tumor type (see Li B. et al., Nature Genetics, 2016, vol:48(7):725-32 the entire contents of which are hereby incorporated by references). Li et al., also disclose that TCRp V6-5 is present at a high frequency in tumor cells.Anti-TCRp V6 antibodies

[0296] In one aspect, provided herein is an anti-TCRpV antibody molecule that binds to human TCRp V6, e.g., a TCRP V6 subfamily comprising: TCRp V6-4*01, TCRp V6-4*02, TCRp V6-9*01, TCRp V6-8*01, TCRP V6-5*01, TCRp V6-6*02, TCRp V6-6*01, TCRp V6-2*01, TCRp V6-3*01 or TCRp V6-l*01. In some embodiments the TCRP V6 subfamily comprises TCRP V6-5*01 or a variant thereof. In some embodiments, TCRp V6 comprises TCRp V6-4*01, or a variant thereof. In some embodiments, TCRP V6 comprises TCRP V6-4*02, or a variant thereof. In some embodiments, TCRP V6 comprises TCRp V6-9*01, or a variant thereof. In some embodiments, TCRp V6 comprises TCRp V6-8*01, or a variant thereof. In some embodiments, TCRp V6 comprises TCRp V6-5*01, or a variant thereof. In some embodiments, TCRP V6 comprises TCRP V6-6*02, or a variant thereof. In some embodiments, TCRP V6 comprises TCRp V6-6*01, or a variant thereof. In some embodiments, TCRp V6 comprises TCRp V6-2*01, or a variant thereof. In some embodiments, TCRp V6 comprises TCRp V6-3*01, or a variant thereof. In some embodiments, TCRp V6 comprises TCRp V6-l*01, or a variant thereof.

[0297] In some embodiments, the human TCRP V6 subfamily corresponds to the murine TCRP V13-1 or murine TCRP VI 3-2 subfamily.

[0298] In some embodiments, TCRp V6-5*01 is encoded by the nucleic acid sequence of SEQ ID NO: 43, or a sequence having 85%, 90%, 95%, 99% or more identity thereof. In some embodiments, TCRP V6-5*01 comprises the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having 85%, 90%, 95%, 99% or more identity thereof.

[0299] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, is a non-murine antibody molecule, e.g., a human or humanized antibody molecule. In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti- TCRp V6-5*01) antibody molecule is a human antibody molecule. In some embodiments, the anti- TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule is a humanized antibody molecule.

[0300] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, is isolated or recombinant.

[0301] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, comprises at least one antigen-binding region, e.g., a variable region or an antigen-binding fragment thereof, from an antibody described herein, e.g., an antibody chosen from anyone of A-H.l to A-H.85, e.g., A-H. 1, A-H.2 or A-H.68, or an antibody described in Table 1, or encoded by a nucleotide sequence in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

[0302] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, comprises at least one, two, three or four variable regions from an antibody described herein, e.g, an antibody chosen from any one of A-H.l to A-H.85, e.g., A-H.l, A-H.2 or A-H.68, or an antibody described in Table 1, or encoded by a nucleotide sequence in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

[0303] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, comprises at least one or two heavy chain variable regions from an antibody described herein, e.g., an antibody chosen from any one of A-H. l to A-H.85, e.g., A-H.l, A-H.2 or A-H.68, or an antibody molecule described in Table 1, or encoded by a nucleotide sequence in TableI, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

[0304] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, comprises at least one or two light chain variable regions from an antibody described herein, e.g, an antibody chosen from any one of A-H.l to A-H.85, e.g., A-H.l, A-H.2 or A- H.68, or an antibody described in Table 1, or encoded by a nucleotide sequence in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

[0305] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, comprises a heavy chain constant region for an IgG4, e.g., a human IgG4. In still another embodiment, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule includes a heavy chain constant region for an IgGl, e.g., a human IgGl. In some embodiments, the heavy chain constant region comprises an amino sequence set forth in Table 3, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) thereto.

[0306] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, includes a kappa light chain constant region, e.g., a human kappa light chain constant region. In some embodiments, the light chain constant region comprises an amino sequence set forth in Table 3, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) thereto.

[0307] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, includes at least one, two, or three complementarity determining regions (CDRs) from a heavy chain variable region (VH) of an antibody described herein, e.g., an antibody chosen from any one of A-H.l to A-H.85, e.g., A-H.l, A-H.2 or A-H.68, or an antibody described inTable 1, or encoded by a nucleotide sequence in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

[0308] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, includes at least one, two, or three CDRs (or collectively all of the CDRs) from a heavy chain variable region comprising an amino acid sequence shown in Table 1, or encoded by a nucleotide sequence shown in Table 1. In some embodiments, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 1, or encoded by a nucleotide sequence shown in Table 1.

[0309] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, includes at least one, two, or three complementarity determining regions (CDRs) from a light chain variable region of an antibody described herein, e.g., an antibody chosen from any one of A-H.l to A-H.85, e.g., A-H.l, A-H.2 or A-H.68, or an antibody described in Table 1, or encoded by a nucleotide sequence in Table 1, or a sequence substantially identical (e.g, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

[0310] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, includes at least one, two, or three CDRs (or collectively all of the CDRs) from a light chain variable region comprising an amino acid sequence shown in Table 1, or encoded by a nucleotide sequence shown in Table 1. In some embodiments, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 1, or encoded by a nucleotide sequence shown in Table 1.

[0311] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, includes at least one, two, three, four, five or six CDRs (or collectively all of the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in Table 1, or encoded by a nucleotide sequence shown in Table 1. In some embodiments, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 1, or encoded by a nucleotide sequence shown in Table 1.

[0312] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, molecule includes all six CDRs from an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H. 1, A-H.2 or A-H.68, or an antibody described in Table 1, or encoded by a nucleotide sequence in Table 1, or closely related CDRs, e.g, CDRs which are identical or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions). In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, may include any CDR described herein.

[0313] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule includes at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs according to the Kabat definition as set out in Table 1) from a heavy chain variable region of an antibody described herein, e.g. , an antibody chosen from any one of A-H. 1 to A- H.85, e.g., A-H.l, A-H.2 or A-H.68, or an antibody described in Table 1, or a sequence substantially identical (e.g, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g, substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Kabat et al. shown in Table 1.

[0314] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule includes at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs according to the Kabat definition as set out in Table 1) from a light chain variable region of an antibody described herein, e.g. , an antibody chosen from any one of A-H. 1 to A- H.85, e.g., A-H.l, A-H.2 or A-H.68, or an antibody described in Table 1, or a sequence substantially identical (e.g, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g, substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Kabat et al. shown in Table 1.

[0315] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, includes at least one, two, three, four, five, or six CDRs according to Kabat et al. (e.g., at least one, two, three, four, five, or six CDRs according to the Kabat definition as set out in Table 1) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H. 1, A-H.2 or A-H.68, or an antibody described in Table 1, or encoded by a nucleotide sequence in Table 1; or a sequence substantially identical (e.g, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g, substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, three, four, five, or six CDRs according to Kabat et al. shown in Table 1.

[0316] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, includes all six CDRs according to Kabat et al. (e.g., all six CDRs according to the Kabat definition as set out in Table 1) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody chosen from any one of A-H.l to A-H.85, e.g., A-H.l, A- H.2 or A-H.68, or an antibody described in Table 1, or encoded by a nucleotide sequence in Table 1; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to all six CDRs according to Kabat et al. shown in Table 1. In some embodiments,the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, may include any CDR described herein.

[0317] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, includes at least one, two, or three hypervariable loops that have the same canonical structures as the corresponding hypervariable loop of an antibody described herein, e.g., an antibody chosen from chosen from any one of A-H.l to A-H.85, e.g., A-H.l, A-H.2 or A-H.68, e.g., the same canonical structures as at least loop 1 and / or loop 2 of the heavy and / or light chain variable domains of an antibody described herein. See, e.g, Chothia et al., (1992) J. Mol. Biol. 227:799-817; Tomlinson et al., (1992) J. Mol. Biol. 227:776-798 for descriptions of hypervariable loop canonical structures. These structures can be determined by inspection of the tables described in these references.

[0318] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule includes at least one, two, or three CDRs according to Chothia et al. (e.g., at least one, two, or three CDRs according to the Chothia definition as set out in Table 1) from a heavy chain variable region of an antibody described herein, e.g., an antibody chosen from any one of A-H. 1 to A-H.85, e.g., A-H.l, A-H.2 or A-H.68, or as described in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Chothia et al. shown in Table 1.

[0319] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule includes at least one, two, or three CDRs according to Chothia et al. (e.g., at least one, two, or three CDRs according to the Chothia definition as set out in Table 1) from a light chain variable region of an antibody described herein, e.g. , an antibody chosen from any one of A-H. 1 to A- H.85, e.g., A-H.l, A-H.2 or A-H.68, or an antibody described in Table 1, or a sequence substantially identical (e.g, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g, substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Chothia et al. shown in Table 1.

[0320] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule, includes at least one, two, three, four, five, or six CDRs according to Chothia et al. (e.g., at least one, two, three, four, five, or six CDRs according to the Chothia definition as set out in Table 1) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody chosen from any one of A-H. 1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or an antibody described in Table 1, or encoded by the nucleotide sequence in Table 1; or a sequence substantially identical (e.g, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g, substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, three, four, five, or six CDRs according to Chothia et al. shown in Table 1.

[0321] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, includes all six CDRs according to Chothia et al. (e.g., all six CDRs according to the Chothia definition as set out in Table 1) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or an antibody described in Table 1, or encoded by a nucleotide sequence in Table 1; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to all six CDRs according to Chothia et al. shown in Table 1. In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, may include any CDR described herein.

[0322] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, molecule includes a combination of CDRs or hypervariable loops defined according to Kabat et al., Chothia et al., or as described in Table 1.

[0323] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, can contain any combination of CDRs or hypervariable loops according to the Kabat and Chothia definitions.

[0324] In some embodiments, a combined CDR as set out in Table 1 is a CDR that comprises a Kabat CDR and a Chothia CDR.

[0325] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, molecule includes a combination of CDRs or hypervariable loops identified as combined CDRs in Table 1. In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule, can contain any combination of CDRs or hypervariable loops according the “combined” CDRs are described in Table 1.

[0326] In some embodiments, e.g., an embodiment comprising a variable region, a CDR (e.g., a combined CDR, Chothia CDR or Kabat CDR), or other sequence referred to herein, e.g., in Table 1, the antibody molecule is a monospecific antibody molecule, a bispecific antibody molecule, a bivalent antibody molecule, a biparatopic antibody molecule, or an antibody molecule that comprises an antigen binding fragment of an antibody, e.g., a half antibody or antigen binding fragment of a half antibody. In certain embodiments the antibody molecule comprises a multifunctional molecule, e.g., a bispecific molecule, e.g., as described herein.

[0327] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule includes: (i) one, two or all of a light chain complementarity determining region 1 (LC CDR1), a light chain complementarity determining region 2 (LC CDR2), and a light chain complementarity determining region 3 (LC CDR3) of SEQ ID NO: 2, SEQ ID NO: 10 or SEQ ID NO: 11, and / or (ii) one, two or all of a heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and a heavy chain complementarity determining region 3 (HC CDR3) of SEQ ID NO: 1 or SEQ ID NO: 9.

[0328] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule comprises a LC CDR1, LC CDR2, and LC CDR3 of SEQ ID NO: 2, and a HC CDR1, HC CDR2, and HC CDR3 of SEQ ID NO: 1.

[0329] In some embodiments the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g., anti-TCRp V6-5*01) antibody molecule comprises a LC CDR1, LC CDR2, and LC CDR3 of SEQ ID NO: 10, and a HC CDR1, HC CDR2, and HC CDR3 of SEQ ID NO: 9.

[0330] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule comprises a LC CDR1, LC CDR2, and LC CDR3 of SEQ ID NO: 11, and a HC CDR1, HC CDR2, and HC CDR3 of SEQ ID NO: 9.

[0331] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule comprises: (i) a LC CDR1 amino acid sequence of SEQ ID NO: 6, a LC CDR2 amino acid sequence of SEQ ID NO: 7, or a LC CDR3 amino acid sequence of SEQ ID NO: 8; and / or (ii) a HC CDR1 amino acid sequence of SEQ ID NO: 3, a HC CDR2 amino acid sequence of SEQ ID NO: 4, or a HC CDR3 amino acid sequence of SEQ ID NO: 5.

[0332] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule comprises: (i) a light chain variable region (VL) comprising a LC CDR1 amino acid sequence of SEQ ID NO: 6, a LC CDR2 amino acid sequence of SEQ ID NO: 7, or a LC CDR3 amino acid sequence of SEQ ID NO: 8; and / or (ii) a heavy chain variable region (VH) comprising a HC CDR1 amino acid sequence of SEQ ID NO: 3, a HC CDR2 amino acid sequence of SEQ ID NO: 4, or a HC CDR3 amino acid sequence of SEQ ID NO: 5.

[0333] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule comprises: (i) a LC CDR1 amino acid sequence of SEQ ID NO: 51, a LC CDR2 amino acid sequence of SEQ ID NO: 52, or a LC CDR3 amino acid sequence of SEQ ID NO: 53; and / or (ii) a HC CDR1 amino acid sequence of SEQ ID NO: 45, a HC CDR2 amino acid sequence of SEQ ID NO: 46, or a HC CDR3 amino acid sequence of SEQ ID NO: 47.

[0334] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule comprises: (i) a light chain variable region (VL) comprising a LC CDR1 amino acid sequence of SEQ ID NO: 51, a LC CDR2 amino acid sequence of SEQ ID NO: 52, or a LC CDR3 amino acid sequence of SEQ ID NO: 53; and / or (ii) a heavy chain variable region (VH) comprising a HC CDR1 amino acid sequence of SEQ ID NO: 45, a HC CDR2 amino acid sequence of SEQ ID NO: 46, or a HC CDR3 amino acid sequence of SEQ ID NO: 47.

[0335] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule comprises: (i) a LC CDR1 amino acid sequence of SEQ ID NO: 54, a LC CDR2 amino acid sequence of SEQ ID NO: 55, or a LC CDR3 amino acid sequence of SEQ ID NO: 56; and / or (ii) a HC CDR1 amino acid sequence of SEQ ID NO: 48, a HC CDR2 amino acid sequence of SEQ ID NO: 49, or a HC CDR3 amino acid sequence of SEQ ID NO: 50.

[0336] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule comprises: (i) a light chain variable region (VL) comprising a LC CDR1amino acid sequence of SEQ ID NO: 54, a LC CDR2 amino acid sequence of SEQ ID NO: 55, or a LC CDR3 amino acid sequence of SEQ ID NO: 56; and / or (ii) a heavy chain variable region (VH) comprising a HC CDR1 amino acid sequence of SEQ ID NO: 48, a HC CDR2 amino acid sequence of SEQ ID NO: 49, or a HC CDR3 amino acid sequence of SEQ ID NO: 50.

[0337] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule comprises a VH and / or a VL of an antibody described in Table 1, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0338] In some embodiments, the anti-TCRpV antibody molecule, e.g., anti-TCRp V6 (e.g, anti-TCRp V6-5*01) antibody molecule comprises a VH and a VL of an antibody described in Table 1, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0339] In some embodiments, an anti-TCRVb antibody as described herein has an antigen binding domain having a VL having a consensus sequence of SEQ ID NO: 230, wherein position 30 is G, E, A or D; position 31 is N or D; position 32 is R or K; position 36 is Y or H; and / or position 56 is K or S.

[0340] In some embodiments, an anti-TCRVb antibody as described herein has an antigen binding domain having a VH having a consensus sequence of SEQ ID NO: 231, wherein: position 27 is H or T or G or Y; position 28 is D or T or S; position 30 is H or R or D or K or T; position 31 is L or D or K or T or N; position 32 is W or F or T or I or Y or G; position 49 is R or W; position 50 is V or I or F; position 51 is F or S or Y; position 52 is A or P; position 56 is N or S; position 57 is T or V or Y or I; position 58 is K or R; position 97 is G or V; position 99 is Y or I; position 102 is Y or A; and / or position 103 is D or G.Anti-TCRp VI 0 antibodies

[0341] In one aspect, provided herein is an anti-TCRpV antibody molecule that binds to a human TCRp V10 subfamily member. In some embodiments, TCRP V10 subfamily is also known as TCRP VI 2. In some embodiments, the TCRp V10 subfamily comprises: TCRp V10-I*0I, TCRp V10-I*02, TCRp V10-3*01 or TCRP VI 0-2*01, or a variant thereof.

[0342] In some embodiments, the human TCRP VI 0 subfamily corresponds to the murine TCRP V13 subfamily. In some embodiments, the human TCRP V10-1 corresponds to murine TCRP V13-1 (mTRBV13-l). In some embodiments, the human TCRP V10-2 corresponds to murine TCRP V13-2 (mTRBV13-2).

[0343] Exemplary anti-TCRp V10 antibodies are provided in Table 12. In some embodiments, the anti- TCRp V10 is antibody D, e.g., humanized antibody D (antibody D-H), as provided in Table 12. In some embodiments, antibody D comprises one or more (e.g., three) light chain CDRs and / or one or more (e.g., three) heavy chain CDRs provided in Table 12, or a sequence with at least 95% sequence identity thereto. In some embodiments, antibody D comprises a variable heavy chain (VH) and / or a variable light chain (VL) provided in Table 12, or a sequence with at least 95% sequence identity thereto.

[0344] In some embodiments, the anti-TCRp V10 antibody molecule comprises a VH or a VL of an antibody described in Table 12, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0345] In some embodiments, the anti-TCRp V10 antibody molecule comprises a VH and a VL of an antibody described in Table 12, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.Antibody-like Frameworks or Scaffolds

[0346] A wide variety of antibody / immunoglobulin frameworks or scaffolds can be employed in the anti-TCRvb antibody molecules as described herein or multifunctional formats thereof so long as the resulting polypeptide includes at least one binding region which specifically binds to the target antigen, e.g., a TCRvb, a tumor antigen, among others. Such frameworks or scaffolds include the 5 main idiotypes of human immunoglobulins, or fragments thereof, and include immunoglobulins of other animal species, preferably having humanized aspects. Novel frameworks, scaffolds and fragments continue to be discovered and developed by those skilled in the art.

[0347] In some embodiments, the anti-TCRvb antibody molecules as described herein or multifunctional formats thereof include non-immunoglobulin based antibodies using non- immunoglobulin scaffolds onto which CDRs can be grafted. Any non-immunoglobulin frameworks and scaffolds may be employed, as long as they comprise a binding region specific for the target antigen (e.g., TCRvb or a tumor antigen). Exemplary non-immunoglobulin frameworks or scaffolds include, but are not limited to, fibronectin (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, MA, and Ablynx nv, Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG, Freising, Germany), small modular immuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), maxybodies (Avidia, Inc., Mountain View, CA), Protein A (Affibody AG, Sweden), and affilin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

[0348] Fibronectin scaffolds are typically based on fibronectin type III domain (e.g., the tenth module of the fibronectin type III (10 Fn3 domain)). The fibronectin type III domain has 7 or 8 beta strands which are distributed between two beta sheets, which themselves pack against each other to form the core of the protein, and further containing loops (analogous to CDRs) which connect the beta strands to each other and are solvent exposed. There are at least three such loops at each edge of the beta sheet sandwich, where the edge is the boundary of the protein perpendicular to the direction of the beta strands (see US 6,818,418). Because of this structure, the non-immunoglobulin antibody mimics antigen binding properties that are similar in nature and affinity to those of antibodies. These scaffolds can be used in a loop randomization and shuffling strategy in vitro that is similar to the process of affinity maturation of antibodies in vivo. These fibronectin-based molecules can be used as scaffolds where the loop regions of the molecule can be replaced with CDRs of the invention using standard cloning techniques.

[0349] The ankyrin technology is based on using proteins with ankyrin derived repeat modules as scaffolds for bearing variable regions which can be used for binding to different targets. The ankyrin repeat module typically is a about 33 amino acid polypeptide consisting of two anti-parallel a-helices and a P-tum. Binding of the variable regions can be optimized by using ribosome display.

[0350] Avimers are used by nature for protein-protein interactions and in human over 250 proteins are structurally based on A-domains. Avimers consist of a number of different “A-domain” monomers (2-10) linked via amino acid linkers. Avimers can be created that can bind to the target antigen using the methodology described in, for example, U.S. Patent Application Publication Nos. 20040175756; 20050053973; 20050048512; and 20060008844.

[0351] Affibody affinity ligands are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This scaffold domain consists of 58 amino acids, 13 of which are randomized to generate affibody libraries with a large number of ligand variants (See e.g., US 5,831,012). Affibody molecules mimic antibodies, they have a molecular weight of 6 kDa, compared to the molecular weight of antibodies, which is 150 kDa. In spite of its small size, the binding site of affibody molecules is similar to that of an antibody.

[0352] Anticalins are known commercially, e.g., Pieris ProteoUab AG. They are derived from lipocalins, a widespread group of small and robust proteins that are usually involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins occur in human tissues or body liquids. The protein architecture is reminiscent of immunoglobulins, with hypervariable loops on top of a rigid framework. However, in contrast with antibodies or their recombinant fragments, lipocalins are composed of a single polypeptide chain with 160 to 180 amino acid residues, being just marginally bigger than a single immunoglobulin domain. The set of four loops, which makes up the binding pocket, shows pronounced structural plasticity and tolerates a variety of side chains. The binding site can thus be reshaped in a proprietary process in order to recognize prescribed target molecules of different shape with high affinity and specificity. One protein of lipocalin family, the bilin-binding protein (BBP) of Pieris Brassicae has been used to develop anticalins by mutagenizing the set of four loops. One example of a patent application describing anticalins is in PCT Publication No. WO 199916873.

[0353] Affilin molecules are small non-immunoglobulin proteins which are designed for specific affinities towards proteins and small molecules. New affilin molecules can be very quickly selected from two libraries, each of which is based on a different human derived scaffold protein. Affilin molecules do not show any structural homology to immunoglobulin proteins. Currently, two affilin scaffolds are employed, one of which is gamma crystalline, a human structural eye lens protein and the other is “ubiquitin” superfamily proteins. Both human scaffolds are very small, show high temperature stability and are almost resistant to pH changes and denaturing agents. This high stability is mainly due to the expanded beta sheet structure of the proteins. Examples of gamma crystalline derived proteins are described in W0200104144 and examples of “ubiquitin-like” proteins are described in W02004106368.

[0354] Protein epitope mimetics (PEM) are medium-sized, cyclic, peptide-like molecules (MW l-2kDa) mimicking beta-hairpin secondary structures of proteins, the major secondary structure involved in protein-protein interactions.

[0355] Domain antibodies (dAbs) can be used in the anti-TCRvb antibody molecules as described herein or multifunctional formats thereof are small functional binding fragments of antibodies, corresponding to the variable regions of either the heavy or light chains of antibodies. Domain antibodies are well expressed in bacterial, yeast, and mammalian cell systems. Further details of domain antibodies and methods of production thereof are known in the art (see, for example, U.S. Pat. Nos. 6,291,158;6,582,915; 6,593,081; 6,172,197; 6,696,245; European Patents 0368684 & 0616640; WO05 / 035572, W004 / 101790, W004 / 081026, W004 / 058821, W004 / 003019 and W003 / 002609. Nanobodies are derived from the heavy chains of an antibody.

[0356] A nanobody typically comprises a single variable domain and two constant domains (CH2 and CH3) and retains antigen-binding capacity of the original antibody. Nanobodies can be prepared by methods known in the art (See e.g., U.S. Pat. No. 6,765,087, U.S. Pat. No. 6,838,254, WO 06 / 079372). Unibodies consist of one light chain and one heavy chain of an IgG4 antibody. Unibodies may be made by the removal of the hinge region of IgG4 antibodies. Further details of unibodies and methods of preparing them may be found in W02007 / 059782.Anti-TCRVP antibody effector function and Fc variants

[0357] In some embodiments, an anti-TCRVp antibody as described herein comprises an Fc region, e.g., as described herein. In some embodiments, the Fc region is a wildtype Fc region, e.g., a wildtype human Fc region. In some embodiments, the Fc region comprises a variant, e.g., an Fc region comprising an addition, substitution, or deletion of at least one amino acid residue in the Fc region which results in, e.g., reduced or ablated affinity for at least one Fc receptor.

[0358] The Fc region of an antibody interacts with a number of receptors or ligands including Fc Receptors (e.g., FcyRI, FcyRIIA, FcyRIIIA), the complement protein Clq, and other molecules such as proteins A and G. These interactions are essential for a variety of effector functions and downstream signaling events including: antibody dependent cell-mediated cytotoxicity (ADCC), Antibody-dependent cellular phagocytosis (ADCP) and complement dependent cytotoxicity (CDC).

[0359] In some embodiments, an anti-TCRVp antibody comprising a variant Fc region has reduced, e.g., ablated, affinity for an Fc receptor, e.g., an Fc receptor described herein. In some embodiments, the reduced affinity is compared to an otherwise similar antibody with a wildtype Fc region.

[0360] In some embodiments, an anti-TCRVp antibody comprising a variant Fc region has one or more of the following properties: (1) reduced effector function (e.g., reduced ADCC, ADCP and / or CDC); (2) reduced binding to one or more Fc receptors; and / or (3) reduced binding to Clq complement. In some embodiments, the reduction in any one, or all of properties ( l)-(3) is compared to an otherwise similar antibody with a wildtype Fc region.

[0361] In some embodiments, an anti-TCRVP antibody comprising a variant Fc region has reduced affinity to a human Fc receptor, e.g., FcyR I, FcyR II and / or FcyR III. In some embodiments, the anti- TCRVP antibody comprising a variant Fc region comprises a human IgGl region or a human IgG4 region.

[0362] In some embodiments, an anti-TCRVP antibody comprising a variant Fc region activates and / or expands T cells, e.g., as described herein. In some embodiments, an anti-TCRVP antibody comprising a variant Fc region has a cytokine profile described herein, e.g., a cytokine profile that differs from a cytokine profile of a T cell engager that binds to a receptor or molecule other than a TCRpV region (“a non-TCRpV-binding T cell engager”). In some embodiments, the non-TCRpV-binding T cell engager comprises an antibody that binds to a CD3 molecule (e.g., CD3 epsilon (CD3e) molecule); or a TCR alpha (TCRa) molecule.

[0363] Exemplary Fc region variants are provided in Table 14 and also disclosed in Saunders O, (2019) Frontiers in Immunology; vol 10, article 1296, the entire contents of which is hereby incorporated by reference.

[0364] In some embodiments, an anti-TCRVP antibody as described herein comprises any one or all, or any combination of Fc region variants disclosed in Table 14.

[0365] In some embodiments, an anti-TCRVP antibody as described herein comprises any one or all, or any combination of Fc region variants, e.g., mutations, disclosed in Table 14. In some embodiments, an anti-TCRVP antibody as described herein comprise an Asn297Ala (N297A) mutation. In some embodiments, an anti-TCRVP antibody as described herein comprise a Leu234Ala / Leu235Ala (LALA) mutation.Multifunctional Molecules

[0366] The terms “multifunctional molecule” and “multispecific molecule,” as used herein interchangeably, refer to a molecule, e.g., a polypeptide, that has two or more functionalities, e.g., two or more binding specificities. In some embodiments, the functionalities can include one or more immune cell engagers, one or more tumor binding molecules, one or more cytokine molecules, one or more stromal modifiers, and other moieties described herein. In some embodiments, the multifunctional molecule is a multifunctional antibody molecule, e.g., a bispecific antibody molecule. In some embodiments, the multifunctional molecule includes an anti-TCRVb antibody molecule as described herein.

[0367] Described herein, in certain embodiments, is a multifunctional molecule comprising a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, and at least one cytokine polypeptide or a functional fragment or a functional variant thereof, wherein the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide are non-contiguous, wherein: (i) the first polypeptide comprising a first portion of a first T cell receptor variable beta (TCRpV)-binding moiety and a first dimerization module linked to the first portion of the first TCRpV -binding moiety; (ii) the second polypeptide comprising a second portion of the first TCRpV-binding moiety; (iii) the third polypeptide comprising a first portion of a second TCRpV-binding moiety and a second dimerizationmodule linked to the first portion of the second TCRpV-binding moiety; and (iv) the fourth polypeptide comprising a second portion of the second TCRpV-binding moiety; and wherein the at least one cytokine polypeptide or a functional fragment or a functional variant thereof is covalently linked to the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof.

[0368] Described herein, in certain embodiments, is a multifunctional molecule comprising a first polypeptide, a second polypeptide, a third polypeptide, and at least one cytokine polypeptide or a functional fragment or a functional variant thereof, wherein the first polypeptide, the second polypeptide, and the third polypeptide are non-contiguous, wherein: (i) the first polypeptide comprising a first portion of a first TCRpV-binding moiety and a first dimerization module linked to the first portion of the first TCRpV-binding moiety; (ii) the second polypeptide comprising a second portion of the first TCRpV- binding moiety; and (iii) the third polypeptide comprising a second dimerization module; and wherein the at least one cytokine polypeptide or a functional fragment or a functional variant thereof is covalently linked to the first polypeptide, the second polypeptide, the third polypeptide, or a combination thereof.

[0369] Described herein, in certain embodiments, is a multifunctional molecule comprising a first polypeptide, a second polypeptide, a third polypeptide, and at least one cytokine polypeptide or a functional fragment or a functional variant thereof, wherein the first polypeptide, the second polypeptide, and the third polypeptide are non-contiguous, wherein: (i) the first polypeptide comprising a first portion of a first TCRpV-binding moiety and a first dimerization module linked to the first portion of the first TCRpV-binding moiety; (ii) the second polypeptide comprising a second portion of the first TCRpV- binding moiety; and (iii) the third polypeptide comprising a second dimerization module; wherein the at least one cytokine polypeptide or a functional fragment or a functional variant thereof is covalently linked to the first polypeptide, the second polypeptide, the third polypeptide, or a combination thereof; and wherein the multifunctional molecule does not comprise an additional TCRpV-binding moiety except the first TCRpV-binding moiety.

[0370] In some embodiments, the first portion of the first TCRpV-binding moiety comprises a first heavy chain variable domain (VH) and a first heavy chain constant domain 1 (CHI) linked to the first VH. In some embodiments, the first CHI is linked to the C-terminus of the first VH. In some embodiments, the second portion of the first TCRpV-binding moiety comprises a first light chain variable domain (VL) and a first light chain constant domain (CL) linked to the first VL. In some embodiments, first CL is linked to the C-terminus of the first VL. In some embodiments, wherein the first dimerization module is linked to the first portion of the first TCRpV-binding moiety. In some embodiments, the first dimerization module is linked to the C-terminus of the first portion of the first TCRpV-binding moiety. In some embodiments, wherein the first portion of the second TCRpV-binding moiety comprises a second VH and a second CHI linked to the second VH. In some embodiments, the second CHI is linked to the C-terminus of the second VH. In some embodiments, the second portion of the second TCRpV-binding moiety comprises a second VL and a second CL linked to the second VL. In some embodiments, the second CL is linked to the C-terminus of the second VL. In some embodiments,the second dimerization module is linked to the first portion of the second TCRpV-binding moiety. In some embodiments, the second dimerization module is linked to the C-terminus of the first portion of the second TCRpV-binding moiety.

[0371] In some embodiments, (a) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; (b) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; (c) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; (d) the N-terminus of the fourth polypeptide is linked to a seventh cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the fourth polypeptide is linked to an eighth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; or (e) a combination thereof.

[0372] In some embodiments, (a-1) the N-terminus of the first polypeptide is linked to the first cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the first polypeptide is linked to the second cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; and (a-2) the N-terminus of the second polypeptide is linked to the third cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the second polypeptide is linked to the fourth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; (b-1) the N-terminus of the first polypeptide is linked to the first cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the first polypeptide is linked to the second cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; and (b-2) the N-terminus of the third polypeptide is linked to the fifth cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the third polypeptide is linked to the sixth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; (c-1) the N-terminus of the first polypeptide is linked to the first cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the first polypeptide is linked to the second cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; and (c-2) the N-terminus of the fourth polypeptide is linked to the seventh cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the fourth polypeptide is linked to the eighth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; (d-1) the N-terminus of the second polypeptide is linked to the third cytokine polypeptide or a functional fragment or a functional variant thereof; the C- terminus of the second polypeptide is linked to the fourth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; and (d-2) the N-terminus of the thirdpolypeptide is linked to the fifth cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the third polypeptide is linked to the sixth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; (e-1) the N-terminus of the second polypeptide is linked to the third cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the second polypeptide is linked to the fourth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; and (e-2) the N-terminus of the fourth polypeptide is linked to the seventh cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the fourth polypeptide is linked to the eighth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; or (f-1) the N-terminus of the third polypeptide is linked to the fifth cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the third polypeptide is linked to the sixth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; and (f-2) the N-terminus of the fourth polypeptide is linked to the seventh cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the fourth polypeptide is linked to the eighth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof.

[0373] In some embodiments, (a-1) the N-terminus of the first polypeptide is linked to the first cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the first polypeptide is linked to the second cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; (a-2) the N-terminus of the second polypeptide is linked to the third cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the second polypeptide is linked to the fourth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; and (a-3) the N-terminus of the third polypeptide is linked to the fifth cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the third polypeptide is linked to the sixth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; (b-1) the N-terminus of the first polypeptide is linked to the first cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the first polypeptide is linked to the second cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; (b-2) the N-terminus of the second polypeptide is linked to the third cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the second polypeptide is linked to the fourth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; and (b-3) the N-terminus of the fourth polypeptide is linked to the seventh cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the fourth polypeptide is linked to the eighth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; or (c-1) the N-terminus of the second polypeptide is linked to the third cytokine polypeptide or a functional fragment or a functional variant thereof; the C- terminus of the second polypeptide is linked to the fourth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; (c-2) the N-terminus of the third polypeptide is linked to the fifth cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the third polypeptide is linked to the sixth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; and (c-3) the N-terminus of the fourth polypeptide is linked to the seventh cytokine polypeptide or a functional fragment or a functional variant thereof; the C- terminus of the fourth polypeptide is linked to the eighth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof.

[0374] In some embodiments, (1) the N-terminus of the first polypeptide is linked to the first cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the first polypeptide is linked to the second cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; (2) the N-terminus of the second polypeptide is linked to the third cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the second polypeptide is linked to the fourth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; (3) the N-terminus of the third polypeptide is linked to the fifth cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the third polypeptide is linked to the sixth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; and (4) the N-terminus of the fourth polypeptide is linked to the seventh cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the fourth polypeptide is linked to the eighth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof.

[0375] In some embodiments, the first cytokine polypeptide, the second cytokine polypeptide, or a combination thereof is within a single contiguous polypeptide chain of the first polypeptide, the third cytokine polypeptide, the fourth cytokine polypeptide, or a combination thereof is within a single contiguous polypeptide chain of the second polypeptide, the fifth cytokine polypeptide, the sixth cytokine polypeptide, or a combination thereof is within a single contiguous polypeptide chain of the third polypeptide, the seventh cytokine polypeptide, the eighth cytokine polypeptide, or a combination thereof is within a single contiguous polypeptide chain of the fourth polypeptide, or a combination thereof.

[0376] In some embodiments, (a) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; (b) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; (c) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; or (d) a combination thereof.

[0377] In some embodiments, (a-1) the N-terminus of the first polypeptide is linked to the first cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the first polypeptide is linked to the second cytokine polypeptide or a functional fragment or a functional variantthereof; or a combination thereof; and (a-2) the N-terminus of the second polypeptide is linked to the third cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the second polypeptide is linked to the fourth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; (b-1) the N-terminus of the first polypeptide is linked to the first cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the first polypeptide is linked to the second cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; and (b-2) the N-terminus of the third polypeptide is linked to the fifth cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the third polypeptide is linked to the sixth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; or (c-1) the N-terminus of the second polypeptide is linked to the third cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the second polypeptide is linked to the fourth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; and (c-2) the N-terminus of the third polypeptide is linked to the fifth cytokine polypeptide or a functional fragment or a functional variant thereof; the C- terminus of the third polypeptide is linked to the sixth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof.

[0378] In some embodiments, (1) the N-terminus of the first polypeptide is linked to the first cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the first polypeptide is linked to the second cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; (2) the N-terminus of the second polypeptide is linked to the third cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the second polypeptide is linked to the fourth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; and (3) the N-terminus of the third polypeptide is linked to the fifth cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the third polypeptide is linked to the sixth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof.

[0379] In some embodiments, the first cytokine polypeptide, the second cytokine polypeptide, or a combination thereof is within a single contiguous polypeptide chain of the first polypeptide, the third cytokine polypeptide, the fourth cytokine polypeptide, or a combination thereof is within a single contiguous polypeptide chain of the second polypeptide, the fifth cytokine polypeptide, the sixth cytokine polypeptide, or a combination thereof is within a single contiguous polypeptide chain of the third polypeptide, or a combination thereof.

[0380] In some embodiments, the multifunctional molecule as described herein further comprises a linker between the first portion of the first TCRpV -binding moiety and the first dimerization module, a linker between the first portion of the second TCRpV-binding moiety and the second dimerization module, a linker between the first VH and the first CHI, a linker between the first VL and the first CL, a linker between the second VH and the second CHI, a linker between the second VL and the second CL, a linker between the at least one cytokine polypeptide or a functional fragment or a functional variantthereof and the first polypeptide, a linker between the at least one cytokine polypeptide or a functional fragment or a functional variant thereof and the second polypeptide, a linker between the at least one cytokine polypeptide or a functional fragment or a functional variant thereof and the third polypeptide, a linker between the at least one cytokine polypeptide or a functional fragment or a functional variant thereof and the fourth polypeptide, or a combination thereof.

[0381] In some embodiments, the multifunctional molecule as described herein further comprises comprising a linker between the first portion of the first TCRpV-binding moiety and the first dimerization module, a linker between the first VH and the first CHI, a linker between the first VL and the first CL, a linker between the at least one cytokine polypeptide or a functional fragment or a functional variant thereof and the first polypeptide, a linker between the at least one cytokine polypeptide or a functional fragment or a functional variant thereof and the second polypeptide, a linker between the at least one cytokine polypeptide or a functional fragment or a functional variant thereof and the third polypeptide, or a combination thereof. In some embodiments, linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker. In some embodiments, the linker is the peptide linker and wherein the linker is a GS linker. In some embodiments, the linker is the peptide linker and wherein the linker comprises the sequence of SEQ ID NO: 3308 or SEQ ID NO: 3643.

[0382] Described herein, in certain embodiments, is a multifunctional molecule comprising a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a first cytokine polypeptide or a functional fragment or a functional variant thereof, and a second cytokine polypeptide or a functional fragment or a functional variant thereof, wherein the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide are non-contiguous, wherein: (i) the first polypeptide comprising a first portion of a first TCRpV-binding moiety and a first dimerization module linked to the first portion of the first TCRpV-binding moiety; (ii) the second polypeptide comprising a second portion of the first TCRpV-binding moiety; (iii) the third polypeptide comprising a first portion of a second TCRpV-binding moiety and a second dimerization module linked to the first portion of the second TCRpV-binding moiety; and (iv) the fourth polypeptide comprising a second portion of the second TCRpV-binding moiety; and wherein the first cytokine polypeptide or a functional fragment or a functional variant thereof is covalently linked to the C-terminus of the second polypeptide, and the second cytokine polypeptide or a functional fragment or a functional variant thereof is covalently linked to the C-terminus of the fourth polypeptide.

[0383] Described herein, in certain embodiments, is a multifunctional molecule comprising a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a cytokine polypeptide or a functional fragment or a functional variant thereof, wherein the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide are non-contiguous, wherein: (i) the first polypeptide comprising a first portion of a first TCRpV-binding moiety and a first dimerization module linked to the first portion of the first TCRpV-binding moiety; (ii) the second polypeptide comprising a second portion of the first TCRpV-binding moiety; (iii) the third polypeptide comprising a first portion of a secondTCRpV-binding moiety and a second dimerization module linked to the first portion of the second TCRpV-binding moiety; and (iv) the fourth polypeptide comprising a second portion of the second TCRpV-binding moiety; and wherein the cytokine polypeptide or a functional fragment or a functional variant thereof is covalently linked to the C-terminus of the second polypeptide or the C-terminus of the fourth polypeptide.

[0384] Described herein, in certain embodiments, is a multifunctional molecule comprising a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a cytokine polypeptide or a functional fragment or a functional variant thereof, wherein the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide are non-contiguous, wherein: (i) the first polypeptide comprising a first portion of a first TCRpV-binding moiety and a first dimerization module linked to the first portion of the first TCRpV-binding moiety; (ii) the second polypeptide comprising a second portion of the first TCRpV-binding moiety; (iii) the third polypeptide comprising a first portion of a second TCRpV-binding moiety and a second dimerization module linked to the first portion of the second TCRpV-binding moiety; and (iv) the fourth polypeptide comprising a second portion of the second TCRpV-binding moiety; and wherein the cytokine polypeptide or a functional fragment or a functional variant thereof is covalently linked to the C-terminus of the first polypeptide or the C-terminus of the third polypeptide.

[0385] Described herein, in certain embodiments, is a multifunctional molecule comprising a first polypeptide, a second polypeptide, a third polypeptide, and a cytokine polypeptide or a functional fragment or a functional variant thereof, wherein the first polypeptide, the second polypeptide, and the third polypeptide are non-contiguous, wherein: (i) the first polypeptide comprising a first portion of a first TCRpV-binding moiety and a first dimerization module linked to the first portion of the first TCRpV-binding moiety; (ii) the second polypeptide comprising a second portion of the first TCRpV- binding moiety; and (iii) the third polypeptide comprising a second dimerization module; wherein the at least one cytokine polypeptide or a functional fragment or a functional variant thereof is covalently linked to the N terminus of the third polypeptide; and wherein the multifunctional molecule does not comprise an additional TCRpV-binding moiety except the first TCRpV-binding moiety.

[0386] In some embodiments, the first portion of the first TCRpV-binding moiety comprises a first VH and a first CHI linked to the first VH. In some embodiments, the first CHI is linked to the C-terminus of the first VH.

[0387] In some embodiments, the second portion of the first TCRpV-binding moiety comprises a first VL and a first CL linked to the first VL. In some embodiments, first CL is linked to the C-terminus of the first VL.

[0388] In some embodiments, the first dimerization module is linked to the first portion of the first TCRpV-binding moiety. In some embodiments, the first dimerization module is linked to the C-terminus of the first portion of the first TCRpV-binding moiety. In some embodiments, the first portion of the second TCRpV-binding moiety comprises a second VH and a second CHI linked to the second VH. In some embodiments, the second CHI is linked to the C-terminus of the second VH. In someembodiments, the second portion of the second TCRpV -binding moiety comprises a second VL and a second CL linked to the second VL. In some embodiments, the second CL is linked to the C-terminus of the second VL. In some embodiments, the second dimerization module is linked to the first portion of the second TCRpV -binding moiety. In some embodiments, the second dimerization module is linked to the C-terminus of the first portion of the second TCRpV-binding moiety.

[0389] In some embodiments, the multifunctional molecule as described herein further comprises a linker between the first portion of the first TCRpV-binding moiety and the first dimerization module, a linker between the first portion of the second TCRpV-binding moiety and the second dimerization module, a linker between the first VH and the first CHI, a linker between the first VL and the first CL, a linker between the second VH and the second CHI, a linker between the second VL and the second CL, a linker between the at least one cytokine polypeptide or a functional fragment or a functional variant thereof and the first polypeptide, a linker between the at least one cytokine polypeptide or a functional fragment or a functional variant thereof and the second polypeptide, a linker between the at least one cytokine polypeptide or a functional fragment or a functional variant thereof and the third polypeptide, a linker between the at least one cytokine polypeptide or a functional fragment or a functional variant thereof and the fourth polypeptide, or a combination thereof. In some embodiments, the multifunctional molecule as described herein further comprises a linker between the first portion of the first TCRpV- binding moiety and the first dimerization module, a linker between the first VH and the first CHI, a linker between the first VL and the first CL, a linker between the at least one cytokine polypeptide or a functional fragment or a functional variant thereof and the third polypeptide, or a combination thereof. In some embodiments, linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker. In some embodiments, the linker is the peptide linker and wherein the linker is a GS linker. In some embodiments, the linker is the peptide linker and wherein the linker comprises the sequence of SEQ ID NO: 3308 or SEQ ID NO: 3643.

[0390] In some embodiments, the first TCRpV-binding moiety, the second TCRpV-binding moiety, or a combination thereof comprises any one selected from the group consisting of a Fab, F(ab')2, Fv, a single chain Fv (scFv), a single domain antibody, a diabody (dAb), a camelid antibody and a combination thereof. In some embodiments, the first TCRpV-binding moiety, the second TCRpV-binding moiety, or a combination thereof comprises a scFv or a Fab.

[0391] In some embodiments, the multifunctional molecule does not comprise an additional antigenbinding moiety except the TCRpV-binding moiety. In some embodiments, the multifunctional molecule further comprise an additional antigen-binding moiety that is not the TCRpV-binding moiety.

[0392] Described herein, in certain embodiments, is a multifunctional molecule comprising a first polypeptide, a second polypeptide, and at least one cytokine polypeptide or a functional fragment or a functional variant thereof, wherein the first polypeptide and the second polypeptide are non-contiguous, wherein: (i) the first polypeptide comprising a first TCRpV-binding moiety and a first dimerization module linked to the C-terminus of the first TCRpV-binding moiety, wherein the first TCRpV-bindingmoiety comprises a first VL and a first VH; and (ii) the second polypeptide comprising a second TCRpV- binding moiety and a second dimerization module linked to the C-terminus of the second TCRpV- binding moiety; wherein the at least one cytokine polypeptide or a functional fragment or a functional variant thereof is covalently linked to the first polypeptide, the second polypeptide, or a combination thereof; wherein the first TCRpV -binding moiety, the second TCRpV -binding moiety, or a combination thereof comprises a scFv; and wherein the multifunctional molecule does not comprise an additional antigen-binding moiety except the first TCRpV-binding moiety and the second TCRpV-binding moiety.

[0393] Described herein, in certain embodiments, is a multifunctional molecule comprising a first polypeptide, a second polypeptide, and at least one cytokine polypeptide or a functional fragment or a functional variant thereof, wherein the first polypeptide and the second polypeptide are non-contiguous, wherein: (i) the first polypeptide comprising a first TCRpV-binding moiety and a first dimerization module linked to the C-terminus of the first TCRpV-binding moiety, wherein the first TCRpV-binding moiety comprises a first VL and a first VH; and (ii) the second polypeptide comprising a second dimerization module; wherein the at least one cytokine polypeptide or a functional fragment or a functional variant thereof is covalently linked to the first polypeptide, the second polypeptide, or a combination thereof; wherein the first TCRpV-binding moiety comprises a scFv; wherein the multifunctional molecule does not comprise an additional antigen-binding moiety except the first TCRpV-binding moiety; and wherein the multifunctional molecule does not comprise an additional TCRpV-binding moiety except the first TCRpV-binding moiety.

[0394] In some embodiments, (a) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; (b) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or a functional fragment or a functional variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or a functional fragment or a functional variant thereof; or a combination thereof; or (e) a combination thereof.

[0395] In some embodiments, the first cytokine polypeptide, the second cytokine polypeptide, or a combination thereof is within a single contiguous polypeptide chain of the first polypeptide, the third cytokine polypeptide, the fourth cytokine polypeptide, or a combination thereof is within a single contiguous polypeptide chain of the second polypeptide, or a combination thereof.

[0396] In some embodiments, the multifunctional molecule as described herein further comprises a linker between the first TCRpV-binding moiety and the first dimerization module, a linker between the second TCRpV-binding moiety and the second dimerization module, a linker between the at least one cytokine polypeptide or a functional fragment or a functional variant thereof and the first polypeptide, a linker between the at least one cytokine polypeptide or a functional fragment or a functional variant thereof and the second polypeptide, or a combination thereof.

[0397] In some embodiments, the multifunctional molecule as described herein further comprises a linker between the first TCRpV-binding moiety and the first dimerization module, a linker between the atleast one cytokine polypeptide or a functional fragment or a functional variant thereof and the first polypeptide, a linker between the at least one cytokine polypeptide or a functional fragment or a functional variant thereof and the second polypeptide, or a combination thereof. In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker. In some embodiments, the linker is the peptide linker and wherein the linker is a GS linker. In some embodiments, the linker is the peptide linker and wherein the linker comprises the sequence of SEQ ID NO: 3308 or SEQ ID NO: 3643.

[0398] In some embodiments, the multifunctional molecule comprises at least two of the cytokine polypeptide. In some embodiments, the multifunctional molecule comprises at least three of the cytokine polypeptide. In some embodiments, the multifunctional molecule comprises at least four of the cytokine polypeptide. In some embodiments, the multifunctional molecule comprises at least five of the cytokine polypeptide. In some embodiments, the multifunctional molecule comprises at least six of the cytokine polypeptide. In some embodiments, the multifunctional molecule comprises at least seven of the cytokine polypeptide. In some embodiments, the multifunctional molecule comprises at least eight of the cytokine polypeptide. In some embodiments, the multifunctional molecule comprises two of the cytokine polypeptide. In some embodiments, the multifunctional molecule comprises three of the cytokine polypeptide. In some embodiments, the multifunctional molecule comprises four of the cytokine polypeptide. In some embodiments, the multifunctional molecule comprises five of the cytokine polypeptide. In some embodiments, the multifunctional molecule comprises six of the cytokine polypeptide. In some embodiments, the multifunctional molecule comprises seven of the cytokine polypeptide. In some embodiments, the multifunctional molecule comprises eight of the cytokine polypeptide. In some embodiments, the multifunctional molecule comprises two of the cytokine polypeptide, each of which is linked to the first polypeptide and the second polypeptide; the first polypeptide and the third polypeptide;, the first polypeptide and the fourth polypeptide; the second and the third polypeptide; the second polypeptide and the fourth polypeptide; or the third polypeptide and the fourth polypeptide, respectively. In some embodiments, the multifunctional molecule comprises three of the cytokine polypeptide, each of which is linked to the first polypeptide, the second polypeptide, and the third polypeptide; the first polypeptide, the second polypeptide, and the fourth polypeptide; the first polypeptide, the third polypeptide, and the fourth polypeptide; or the second polypeptide, the third polypeptide, and the fourth polypeptide, respectively. In some embodiments, the multifunctional molecule comprises four of the cytokine polypeptide, each of which is linked to the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide, respectively. In some embodiments, the cytokine polypeptide is not linked to the polypeptides that comprise the first TCRpV- binding moiety.

[0399] In some embodiments, the at least one cytokine polypeptide is selected from the group consisting of interleukin-2 (IL-2) or a fragment or a functional fragment or a functional variant thereof, or a combination thereof.

[0400] In some embodiments, the at least one cytokine polypeptide comprises interleukin-2 (IL-2) or a fragment thereof. In some embodiments, the at least one cytokine polypeptide is interleukin-2 (IL-2) or a fragment thereof. In some embodiments, the at least one cytokine polypeptide comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 2191. In some embodiments, the at least one cytokine polypeptide comprises the sequence of SEQ ID NO: 2191. In some embodiments, the sequence of the at least one cytokine polypeptide is a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 2191. In some embodiments, the sequence of the at least one cytokine polypeptide is the sequence of SEQ ID NO: 2191.

[0401] In some embodiments, the variant of the at least one cytokine polypeptide comprises an IL-2 variant comprising a mutation. In some embodiments, the mutation comprises an insertion mutation, a deletion mutation, or a substitution mutation. In some embodiments, the mutation comprises the substitution mutation. In some embodiments, the variant comprises an IL-2 variant comprising C125A mutation. In some embodiments, the variant of the at least one cytokine polypeptide is an IL-2 variant comprising a mutation. In some embodiments, the mutation is an insertion mutation, a deletion mutation, or a substitution mutation. In some embodiments, the mutation is the substitution mutation. In some embodiments, the variant is an IL-2 variant comprising C125A mutation. In some embodiments, the variant comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 2270. In some embodiments, the variant comprises the sequence of SEQ ID NO: 2270. In some embodiments, the sequence of the variant is a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 2270. In some embodiments, the sequence of the variant is the sequence of SEQ ID NO: 2270.

[0402] In some embodiments, the first dimerization module comprises a first immunoglobulin constant regions (Fc regions) and the second dimerization module comprises a second Fc region. In some embodiments, the first dimerization module is a first immunoglobulin constant regions (Fc regions) and the second dimerization module is a second Fc region.

[0403] In some embodiments, the first Fc region, the second Fc region, or a combination thereof is selected from an IgGl Fc region or a fragment thereof, an IgG2 Fc region or a fragment thereof, an IgG3 Fc region or a fragment thereof, an IgGAl Fc region or a fragment thereof, an IgGA2 Fc region or a fragment thereof, an IgG4 Fc region or a fragment thereof, an IgJ Fc region or a fragment thereof, an IgM Fc region or a fragment thereof, an IgD Fc region or a fragment thereof, and an IgE Fc region or a fragment thereof.

[0404] In some embodiments, the first Fc region, the second Fc region, or a combination thereof is selected from a human IgGl Fc region or a fragment thereof, a human IgG2 Fc region or a fragment thereof, and a human IgG4 Fc region or a fragment thereof.

[0405] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises an Fc interface with one or more of: a paired cavity-protuberance, an electrostatic interaction,or a strand-exchange, wherein the dimerization of the first Fc region and the second Fc region is enhanced as indicated by a greater ratio of heteromultimer:homomultimer forms relative to a dimerization of Fc regions with a non-engineered interface. In some embodiments, the dimerization of the first Fc region and the second Fc region is enhanced at least by 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold, 75 fold, 80 fold, 85 fold, 90 fold, 95 fold, 100 fold, 150 fold, 200 fold, 250 fold, 300 fold, 250 fold, 400 fold, 450 fold, 500 fold, 550 fold, 600 fold, 650 fold, 700 fold, 750 fold, 800 fold, 850 fold, 900 fold, 950 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, or 10000 fold relative to a dimerization of Fc regions with a non-engineered interface. In some embodiments, the dimerization of the first Fc region and the second Fc region is enhanced at most by 1. 1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold, 75 fold, 80 fold, 85 fold, 90 fold, 95 fold, 100 fold, 150 fold, 200 fold, 250 fold, 300 fold, 250 fold, 400 fold, 450 fold, 500 fold, 550 fold, 600 fold, 650 fold, 700 fold, 750 fold, 800 fold, 850 fold, 900 fold, 950 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, or 10000 fold relative to a dimerization of Fc regions with a nonengineered interface. In some embodiments, the dimerization of the first Fc region and the second Fc region is enhanced by 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold, 75 fold, 80 fold, 85 fold, 90 fold, 95 fold, 100 fold, 150 fold, 200 fold, 250 fold, 300 fold, 250 fold, 400 fold, 450 fold, 500 fold, 550 fold, 600 fold, 650 fold, 700 fold, 750 fold, 800 fold, 850 fold, 900 fold, 950 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, or 10000 fold relative to a dimerization of Fc regions with a non-engineered interface.

[0406] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises an amino acid substitution listed in Table 14.

[0407] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises an Asn297Ala (N297A) mutation or a Leu234Ala / Leu235Ala (LALA) mutation.

[0408] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 3645, SEQ ID NO: 3646, SEQ ID NO: 3647, SEQ ID NO:3648, or SEQ ID NO: 3649. In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises the sequence of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 3645, SEQ ID NO: 3646, SEQ ID NO: 3647, SEQ ID NO:3648, or SEQ ID NO: 3649.

[0409] In some embodiments, the sequence of the first Fc region, the second Fc region, or a combination thereof is a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequenceidentity to the sequence of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 3645, SEQ ID NO: 3646, SEQ ID NO: 3647, SEQ ID NO:3648, or SEQ ID NO: 3649. In some embodiments, the sequence of the first Fc region, the second Fc region, or a combination thereof is the sequence of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 3645, SEQ ID NO: 3646, SEQ ID NO: 3647, SEQ ID NO:3648, or SEQ ID NO: 3649.

[0410] In some embodiments, the first TCRpV -binding moiety, the second TCRpV-binding moiety, or a combination thereof binds to one or more of a TCRpV subfamily selected from the group consisting of: (i) the TCRP V6 subfamily comprising one or more selected from TCRP V6-4*0I, TCRP V6-4*02, TCRP V6-9*0I, TCRP V6-8*0I, TCRP V6-5*0I, TCRP V6-6*02, TCRP V6-6*0I, TCRP V6-2*0I, TCRP V6-3*0I, and TCRP V6-I*0I, and (ii) TCRP V10 subfamily comprising one or more selected from TCRP V10-I*0I, TCRP V10-I*02, TCRP V10-3*0I, and TCRP V10-2*0I.

[0411] In some embodiments, the first TCRpV-binding moiety and the second TCRpV-binding moiety are same. In some embodiments, the first TCRpV-binding moiety and the second TCRpV-binding moiety are different.

[0412] In some embodiments, the first TCRpV-binding moiety and the second TCRpV-binding moiety binds one or more of a TCRP V6 subfamily member and one or more of a TCRP V 10 subfamily member, respectively.

[0413] In some embodiments, the first TCRpV-binding moiety, the second TCRpV-binding moiety, or a combination thereof comprises: (i) a HC CDR1, a HC CDR2 and a HC CDR3 of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the CDR1, CDR2, and CDR3 sequences listed in Table 1; (ii) a LC CDR1, a LC CDR2, and a LC CDR3 of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the CDR1, CDR2, and CDR3 the sequences listed in Table 1; or (iii) a combination thereof. In some embodiments, the first TCRpV-binding moiety, the second TCRpV- binding moiety, or a combination thereof comprises: (i) a HC CDR1, a HC CDR2 and a HC CDR3 having any one of the CDR1, CDR2, and CDR3 sequences listed in Table 1; (ii) a LC CDR1, a LC CDR2, and a LC CDR3 having any one of the CDR1, CDR2, and CDR3 the sequences listed in Table 1; or (iii) a combination thereof.

[0414] In some embodiments, the first TCRpV-binding moiety, the second TCRpV-binding moiety, or a combination thereof comprises: (i) a HC CDR1, a HC CDR2 and a HC CDR3 of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the CDR1, CDR2, and CDR3 sequences listed in Table 1, respectively; (ii) a LC CDR1, a LC CDR2, and a LC CDR3 of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the CDR1, CDR2, and CDR3 the sequences listed in Table 1, respectively; or (iii) a combination thereof. In some embodiments, the first TCRpV-binding moiety, the second TCRpV-binding moiety, or a combination thereof comprises: (i) a HC CDR1, a HC CDR2 and a HC CDR3 having any one of the CDR1, CDR2, and CDR3 sequences listed in Table 1, respectively; (ii)a LC CDR1, a LC CDR2, and a LC CDR3 having any one of the CDR1, CDR2, and CDR3 the sequences listed in Table 1, respectively; or (iii) a combination thereof.

[0415] In some embodiments, the first TCRpV -binding moiety, the second TCRpV-binding moiety, or a combination thereof comprises: (i) a VH comprising a framework region (FR) comprising a framework 1 (FR1), a framework region 2 (FR2), a framework region 3 (FR3), and a framework region 4 (FR4) that have at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity with a nonmurine germline FR1, a non-murine germline FR2, a non-murine germline FR3, and a non-murine germline FR4; (ii) a VL comprising a FR comprising a FR1, a FR2, a FR3, and a FR4 that have at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity with anon-murine germline FR1, a non-murine germline FR2, a non-murine germline FR3, and a non-murine germline FR4; or (iii) a combination thereof. In some embodiments, the first TCRpV-binding moiety, the second TCRpV- binding moiety, or a combination thereof comprises: (i) a VH comprising a FR comprising a FR1, a FR2, a FR3, and a FR4 having the sequences of a non-murine germline FR1, a non-murine germline FR2, a non-murine germline FR3, and a non-murine germline FR4; (ii) a VL comprising a FR comprising a FR1, a FR2, a FR3, and a FR4 having the sequences of a non-murine germline FR1, a non-murine germline FR2, a non-murine germline FR3, and a non-murine germline FR4; or (iii) a combination thereof.

[0416] In some embodiments, the first TCRpV-binding moiety, the second TCRpV-binding moiety, or a combination thereof comprises: (i) a VH comprising a FR1, a FR2, a FR3, and a FR4 that have at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity with anon-murine germline FR1, a non-murine germline FR2, a non-murine germline FR3, and a non-murine germline FR4, respectively; (ii) a VL comprising a FR comprising a FR1, a FR2, a FR3, and a FR4 that have at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity with anon-murine germline FR1, a non-murine germline FR2, a non-murine germline FR3, and a non-murine germline FR4, respectively; or (iii) a combination thereof. In some embodiments, the first TCRpV-binding moiety, the second TCRpV-binding moiety, or a combination thereof comprises: (i) a VH comprising a FR comprising a FR1, a FR2, a FR3, and a FR4 having the sequences of a non-murine germline FR1, a non- murine germline FR2, a non-murine germline FR3, and a non-murine germline FR4, respectively; (ii) a VL comprising a FR comprising a FR1, a FR2, a FR3, and a FR4 having the sequences of a non-murine germline FR1, a non-murine germline FR2, a non-murine germline FR3, and a non-murine germline FR4, respectively; or (iii) a combination thereof.

[0417] In some embodiments, the VH comprises the FR3 comprising (i) a Threonine at position 73 according to Kabat numbering; (ii) a Glycine a position 94 according to Kabat numbering; or (iii) a combination thereof. In some embodiments, the VL comprises the FR1 comprising a Phenyalanine at position 10 according to Kabat numbering. In some embodiments, the VL comprises the FR2 comprising (i) a Histidine at position 36 according to Kabat numbering; (ii) an Alanine at position 46 according to Kabat numbering; or (iii) a combination thereof. In some embodiments, the VL comprises the FR3 comprising a Phenyalanine at position 87 according to Kabat numbering.

[0418] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region having a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences listed in Table 3 or a combination thereof. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region having any one of the sequences listed in Table 3 or a combination thereof. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region of which sequence is a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences listed in Table 3 or a combination thereof. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region having any one of the heavy chain constant region sequences listed in Table 3 or a combination thereof. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region of an IgM or a fragment thereof. In some embodiments, the heavy chain constant region of the IgM comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 73. In some embodiments, the heavy chain constant region of the IgM comprises the sequence of SEQ ID NO: 73. In some embodiments, the sequence of the heavy chain constant region of the IgM is the sequence of SEQ ID NO: 73.

[0419] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region of an IgJ or a fragment thereof. In some embodiments, the heavy chain constant region of the IgJ comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 76. In some embodiments, the heavy chain constant region of the IgJ comprises the sequence of SEQ ID NO: 76. In some embodiments, the sequence of the heavy chain constant region of the IgJ is the sequence of SEQ ID NO: 76.

[0420] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region of an IgGAl or a fragment thereof. In some embodiments, the heavy chain constant region of the IgGAl comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 74. In some embodiments, the heavy chain constant region of the IgGAl comprises the sequence of SEQ ID NO: 74. In some embodiments, the sequence of the heavy chain constant region of the IgGAl is the sequence of SEQ ID NO: 74.

[0421] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region of an IgGA2 or a fragment thereof. In some embodiments, the heavy chain constant region of the IgGA2 comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity tothe sequence of SEQ ID NO: 75. In some embodiments, the heavy chain constant region of the IgGA2 comprises the sequence of SEQ ID NO: 75. In some embodiments, the sequence of the heavy chain constant region of the IgGA2 is the sequence of SEQ ID NO: 75.

[0422] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region of an IgGl or a fragment thereof. In some embodiments, the heavy chain constant region of the IgGl comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 41. In some embodiments, the heavy chain constant region of the IgGl comprises the sequence of SEQ ID NO: 41. In some embodiments, the sequence of the heavy chain constant region of the IgGl is the sequence of SEQ ID NO: 41. In some embodiments, the heavy chain constant region of the IgGl comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 3645. In some embodiments, the heavy chain constant region of the IgGl comprises the sequence of SEQ ID NO: 3645. In some embodiments, the sequence of the heavy chain constant region of the IgGl is the sequence of SEQ ID NO: 3645.

[0423] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a light chain constant region having a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences listed in Table 3 or a combination thereof. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a light chain constant region having any one of the sequences listed in Table 3 or a combination thereof. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a light chain constant region having any one of the light chain constant region sequences listed in Table 3 or a combination thereof.

[0424] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a light chain constant region of a kappa chain or a fragment thereof. In some embodiments, the light chain constant region of a kappa chain comprises a light chain constant region sequence listed in Table 3.

[0425] In some embodiments, the light chain constant region of a kappa chain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 39 or SEQ ID NO: 3644. In some embodiments, the light chain constant region of a kappa chain comprises the sequence of SEQ ID NO: 39 or SEQ ID NO: 3644. In some embodiments, the sequence of the light chain constant region of a kappa chain is a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 39 or SEQ ID NO: 3644. In some embodiments, the sequence of the light chain constant region of a kappa chain is the sequence of SEQ ID NO: 39 or SEQ ID NO: 3644.

[0426] In some embodiments, the first TCRpV -binding moiety, the second TCRpV-binding moiety, or a combination thereof comprises a light chain comprising a FR1 comprising: (i) an Aspartic Acid atposition 1 according to Kabat numbering; (ii) an Asparagine at position 2 according to Kabat numbering; (iii) a Leucine at position 4 according to Kabat numbering; or (iv) a combination thereof.

[0427] In some embodiments, the first TCRpV -binding moiety, the second TCRpV-binding moiety, or a combination thereof comprises a light chain comprising a FR3 comprising: (i) a Glycine at position 66 according to Kabat numbering; (ii) an Asparagine at position 69 according to Kabat numbering; (iii) a Tyrosine at position 71 according to Kabat numbering; or (iv) a combination thereof

[0428] In some embodiments, the first TCRpV-binding moiety, the second TCRpV-binding moiety, or a combination thereof binds to an outward facing region on a TCRpV protein. In some embodiments, the outward facing region on the TCRpV protein comprises a structurally conserved region of TCRpV having a similar structure across one or more TCRpV subfamilies.Cytokine Molecules

[0429] In some embodiments, the multifunctional molecule includes a cytokine molecule. As used herein, a “cytokine molecule” or a “cytokine polypeptide” as interchangeably used herein, refers to full length, a fragment or a variant of a cytokine; a cytokine further comprising a receptor domain, e.g. , a cytokine receptor dimerizing domain; or an agonist of a cytokine receptor, e.g., an antibody molecule (e.g., an agonistic antibody) to a cytokine receptor, that elicits at least one activity of a naturally- occurring cytokine. In some embodiments the cytokine molecule is interleukin-2 (IL-2), or a fragment or variant thereof, or a combination. The cytokine molecule can be a monomer or a dimer. In embodiments, the cytokine molecule can further include a cytokine receptor dimerizing domain. In other embodiments, the cytokine molecule is an agonist of a cytokine receptor.

[0430] Cytokines are generally polypeptides that influence cellular activity, for example, through signal transduction pathways. Accordingly, a cytokine of the multispecific or multifunctional is useful and can be associated with receptor-mediated signaling that transmits a signal from outside the cell membrane to modulate a response within the cell. Cytokines are proteinaceous signaling compounds that are mediators of the immune response. They control many different cellular functions including proliferation, differentiation and cell survival / apoptosis; cytokines are also involved in several pathophysiological processes including viral infections and autoimmune diseases. Cytokines are synthesized under various stimuli by a variety of cells of both the innate (monocytes, macrophages, dendritic cells) and adaptive (T- and B-cells) immune systems. Cytokines can be classified into two groups: pro- and anti-inflammatory. Pro-inflammatory cytokines, including IFNy, IL-1, IL-6 and TNF -alpha, are predominantly derived from the innate immune cells and Thl cells. Anti-inflammatory cytokines, including IL- 10, IL-4, IL- 13 and IL-5, are synthesized from Th2 immune cells.

[0431] Provided herein are, inter alia, multispecific (e.g. , bi-, tri-, quad- specific) or multifunctional molecules, that include, e.g, are engineered to contain, one or more cytokine molecules, e.g., immunomodulatory (e.g., proinflammatory) cytokines and variants, e.g., functional variants, thereof. Accordingly, in some embodiments, the cytokine molecule is an interleukin or a variant, e.g., a functional variant thereof. In some embodiments the interleukin is a proinflammatory interleukin. In someembodiments the interleukin is interleukin-2 (IL-2). In some embodiments, the cytokine molecule is a proinflammatory cytokine.

[0432] In certain embodiments, the cytokine is a single chain cytokine. In certain embodiments, the cytokine is a multichain cytokine (e.g., the cytokine comprises 2 or more (e.g., 2) polypeptide chains.

[0433] Examples of useful cytokines include, but are not limited to, IL-2. In some embodiments the cytokine of the multispecific or multifunctional molecule is IL-2. In certain embodiments the cytokine is mutated to remove N- and / or O-glycosylation sites. Elimination of glycosylation increases homogeneity of the product obtainable in recombinant production.

[0434] In some embodiments, the cytokine of the multispecific or multifunctional molecule is IL-2. In a specific embodiment, the IL-2 cytokine can elicit one or more of the cellular responses selected from the group consisting of: proliferation in an activated T lymphocyte cell, differentiation in an activated T lymphocyte cell, cytotoxic T cell (CTL) activity, proliferation in an activated B cell, differentiation in an activated B cell, proliferation in a natural killer (NK) cell, differentiation in a NK cell, cytokine secretion by an activated T cell or an NK cell, and NK / lymphocyte activated killer (LAK) antitumor cytotoxicity. In another particular embodiment the IL-2 cytokine is a mutant IL-2 cytokine having reduced binding affinity to the .alpha. -subunit of the IL-2 receptor. Together with the .beta.- and .gamma. -subunits (also known as CD 122 and CD 132, respectively), the .alpha. -subunit (also known as CD25) forms the heterotrimeric high-affinity IL-2 receptor, while the dimeric receptor consisting only of the - and y- subunits is termed the intermediate-affinity IL-2 receptor. As described in PCT patent application number PCT / EP2012 / 051991, which is incorporated herein by reference in its entirety, a mutant IL-2 polypeptide with reduced binding to the .alpha. -subunit of the IL-2 receptor has a reduced ability to induce IL-2 signaling in regulatory T cells, induces less activation-induced cell death (AICD) in T cells, and has a reduced toxicity profile in vivo, compared to a wild-type IL-2 polypeptide. The use of such an cytokine with reduced toxicity is particularly advantageous in a multispecific or multifunctional molecule according to the invention, having a long serum half-life due to the presence of an Pc domain. In some embodiments, the mutant IL-2 cytokine of the multispecific or multifunctional molecule according to the invention comprises at least one amino acid mutation that reduces or abolishes the affinity of the mutant IL-2 cytokine to the .alpha. -subunit of the IL-2 receptor (CD25) but preserves the affinity of the mutant IL-2 cytokine to the intermediate-affinity IL-2 receptor (consisting of the P and y subunits of the IL-2 receptor), compared to the non-mutated IL-2 cytokine. In some embodiments the one or more amino acid mutations are amino acid substitutions. In a specific embodiment, the mutant IL-2 cytokine comprises one, two or three amino acid substitutions at one, two or three position(s) selected from the positions corresponding to residue 42, 45, and 72 of human IL-2. In a more specific embodiment, the mutant IL-2 cytokine comprises three amino acid substitutions at the positions corresponding to residue 42, 45 and 72 of human IL-2. In an even more specific embodiment, the mutant IL-2 cytokine is human IL-2 comprising the amino acid substitutions F42A, Y45A and L72G. In some embodiments the mutant IL-2 cytokine additionally comprises an amino acid mutation at a position corresponding to position 3 of human IL-2, which eliminates the O-glycosylation site of IL-2. Particularly, said additional amino acidmutation is an amino acid substitution replacing a threonine residue by an alanine residue. A particular mutant IL-2 cytokine useful in the invention comprises four amino acid substitutions at positions corresponding to residues 3, 42, 45 and 72 of human IL-2. Specific amino acid substitutions are T3A, F42A, Y45A and L72G. As demonstrated in PCT patent application number PCT / EP2012 / 051991 and in the appended Examples, said quadruple mutant IL-2 polypeptide (IL-2 qm) exhibits no detectable binding to CD25, reduced ability to induce apoptosis in T cells, reduced ability to induce IL-2 signaling in T. sub. reg cells, and a reduced toxicity profile in vivo. However, it retains ability to activate IL-2 signaling in effector cells, to induce proliferation of effector cells, and to generate IFN-y as a secondary cytokine by NK cells.

[0435] The IL-2 or mutant IL-2 cytokine according to any of the above embodiments may comprise additional mutations that provide further advantages such as increased expression or stability. For example, the cysteine at position 125 may be replaced with a neutral amino acid such as alanine, to avoid the formation of disulfide-bridged IL-2 dimers. Thus, in certain embodiments the IL-2 or mutant IL-2 cytokine of the multispecific or multifunctional molecule according to the invention comprises an additional amino acid mutation at a position corresponding to residue 125 of human IL-2. In some embodiments said additional amino acid mutation is the amino acid substitution C125A.

[0436] In a specific embodiment the IL-2 cytokine of the multispecific or multifunctional molecule comprises the polypeptide sequence of SEQ ID NO: 2270 [APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK PLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTL T],

[0437] In another specific embodiment the IL-2 cytokine of the multispecific or multifunctional molecule comprises the polypeptide sequence of SEQ ID NO: 2280 [APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEEL KPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIIS TLT],

[0438] Mutant cytokine molecules useful as effector moieties in the multispecific or multifunctional molecule can be prepared by deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing. Substitution or insertion may involve natural as well as non-natural amino acid residues. Amino acid modification includes well known methods of chemical modification such as the addition or removal of glycosylation sites or carbohydrate attachments, and the like.

[0439] In some embodiments, the multispecific or multifunctional molecule of the invention binds to an cytokine receptor with a dissociation constant (KD) that is at least about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 times greater than that for a control cytokine. In another embodiment, the multispecific or multifunctional molecule binds to an cytokine receptor with a KD that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater than that for a corresponding multispecific ormultifunctional molecule comprising two or more effector moieties. In another embodiment, the multispecific or multifunctional molecule binds to an cytokine receptor with a dissociation constant KD that is about 10 times greater than that for a corresponding the multispecific or multifunctional molecule comprising two or more cytokines.

[0440] In some embodiments, the multispecific molecules as described herein include a cytokine molecule. In embodiments, the cytokine molecule includes a full length, a fragment or a variant of a cytokine; a cytokine receptor domain, e.g., a cytokine receptor dimerizing domain; or an agonist of a cytokine receptor, e.g., an antibody molecule (e.g., an agonistic antibody) to a cytokine receptor.

[0441] In other embodiments, the cytokine molecule is IL-2, e.g., human IL-2 (e.g., comprising the amino acid sequence: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK PLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL T (SEQ ID NO: 2191), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO:2191).Immune Cell Engagers

[0442] In some embodiments, the multifunctional molecule further includes an immune cell engager. “An immune cell engager” refers to one or more binding specificities that bind and / or activate an immune cell, e.g., a cell involved in an immune response. In embodiments, the immune cell is chosen from a T cell, an NK cell, a B cell, a dendritic cell, and / or the macrophage cell. The immune cell engager can be an antibody molecule, a receptor molecule (e.g., a full length receptor, receptor fragment, or fusion thereof (e.g., a receptor-Fc fusion)), or a ligand molecule (e.g., a full length ligand, ligand fragment, or fusion thereof (e.g., a ligand-Fc fusion)) that binds to the immune cell antigen (e.g., the T cell, the NK cell antigen, the B cell antigen, the dendritic cell antigen, and / or the macrophage cell antigen). In embodiments, the immune cell engager specifically binds to the target immune cell, e.g., binds preferentially to the target immune cell. For example, when the immune cell engager is an antibody molecule, it binds to an immune cell antigen (e.g., a T cell antigen, an NK cell antigen, a B cell antigen, a dendritic cell antigen, and / or a macrophage cell antigen) with a dissociation constant of less than about 10 nM.

[0443] The immune cell engagers, e.g., first and / or second immune cell engager, of the multispecific or multifunctional molecules as described herein can mediate binding to, and / or activation of, an immune cell, e.g., an immune effector cell. In some embodiments, the immune cell is chosen from a T cell, an NK cell, a B cell, a dendritic cell, or a macrophage cell engager, or a combination thereof. In some embodiments, the immune cell engager is chosen from one, two, three, or all of a T cell engager, NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager, or a combination thereof. The immune cell engager can be an agonist of the immune system. In some embodiments, theimmune cell engager can be an antibody molecule, a ligand molecule (e.g., a ligand that further comprises an immunoglobulin constant region, e.g., an Fc region), a small molecule, a nucleotide molecule.Antibody Molecules

[0444] In some embodiments, the antibody molecule binds to a cancer antigen, e.g., a tumor antigen or a stromal antigen. In some embodiments, the cancer antigen is, e.g., a mammalian, e.g., a human, cancer antigen. In other embodiments, the antibody molecule binds to an immune cell antigen, e.g., a mammalian, e.g., a human, immune cell antigen. For example, the antibody molecule binds specifically to an epitope, e.g., linear or conformational epitope, on the cancer antigen or the immune cell antigen.

[0445] In some embodiments, an antibody molecule is a monospecific antibody molecule and binds a single epitope. E.g. , a monospecific antibody molecule having a plurality of immunoglobulin variable domain sequences, each of which binds the same epitope.

[0446] In some embodiments, an antibody molecule is a multispecific or multifunctional antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domains sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In some embodiments, a multifunctional antibody molecule comprises a third, fourth or fifth immunoglobulin variable domain. In some embodiments, a multifunctional antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0447] In some embodiments, a multifunctional antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In some embodiments, a bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In some embodiments, a bispecific antibody moleculecomprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a scFv or a Fab, or fragment thereof, have binding specificity for a first epitope and a scFv or a Fab, or fragment thereof, have binding specificity for a second epitope.

[0448] In some embodiments, an antibody molecule comprises a diabody, and a single-chain molecule, as well as an antigen-binding fragment of an antibody (e.g., Fab, F(ab’)2, and Fv). For example, an antibody molecule can include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL). In some embodiments, an antibody molecule comprises or consists of a heavy chain and a light chain (referred to herein as a half antibody. In another example, an antibody molecule includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequence, thereby forming two antigen binding sites, such as Fab, Fab’, F(ab’)2, Fc, Fd, Fd’, Fv, single chain antibodies (scFv for example), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to selectively bind with their respective antigen or receptor. Antibodies and antibody fragments can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgGl, IgG2, IgG3, and IgG4) of antibodies. The preparation of antibody molecules can be monoclonal or polyclonal. An antibody molecule can also be a human, humanized, CDR-grafted, or in vitro generated antibody. The antibody can have a heavy chain constant region chosen from, e.g., IgGl, IgG2, IgG3, or IgG4. The antibody can also have a light chain chosen from, e.g, kappa or lambda. The term “immunoglobulin” (Ig) is used interchangeably with the term “antibody” herein.

[0449] Examples of antigen-binding fragments of an antibody molecule include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv), see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (viii) a single domain antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0450] Antibody molecules include intact molecules as well as functional fragments thereof. Constant regions of the antibody molecules can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function).

[0451] Antibody molecules can also be single domain antibodies. Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, singledomain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine. According to another aspect of the invention, a single domain antibody is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in WO 9404678, for example. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the invention.

[0452] The VH and VL regions can be subdivided into regions of hypervariability, termed “complementarity determining regions” (CDR), interspersed with regions that are more conserved, termed “framework regions” (FR or FW).

[0453] The extent of the framework region and CDRs has been precisely defined by a number of methods (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg).

[0454] The terms “complementarity determining region,” and “CDR,” as used herein refer to the sequences of amino acids within antibody variable regions which confer antigen specificity and binding affinity. In general, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3).

[0455] The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273,1927-948 (“Chothia” numbering scheme). As used herein, the CDRs defined according the “Chothia” number scheme are also sometimes referred to as “hypervariable loops.”

[0456] For example, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89- 97 (LCDR3). Under Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50- 52 (LCDR2), and 91-96 (LCDR3).

[0457] Each VH and VL typically includes three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0458] The antibody molecule can be a polyclonal or a monoclonal antibody.

[0459] The terms “monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. A monoclonal antibody can be made by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods).

[0460] The antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods, or by yeast display.

[0461] Phage display and combinatorial methods for generating antibodies are known in the art (as described in, e.g., Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No. WO 92 / 18619; Dower et al. International Publication No. WO 91 / 17271; Winter et al. International Publication WO 92 / 20791; Markland et al. International Publication No. WO 92 / 15679; Breitling et al. International Publication WO 93 / 01288; McCafferty et al. International Publication No. WO 92 / 01047; Garrard et al. International Publication No. WO 92 / 09690; Ladner et al. International Publication No. WO 90 / 02809; Fuchs et al. (1991) Bio / T echnology 9: 1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246: 1275-1281; Griffths et al. (1993) EMBO J 12:725- 734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio / Technology 9: 1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contents of all of which are incorporated by reference herein).

[0462] The yeast display method for generating or identifying antibodies is known in the art, e.g., as described in Chao et al. (2006) Nature Protocols l(2):755-68, the entire contents of which is incorporated by reference herein.

[0463] In some embodiments, the antibody is a fully human antibody (e.g., an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g, monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Methods of producing rodent antibodies are known in the art.

[0464] Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91 / 00906, Kucherlapati et al. PCT publication WO 91 / 10741; Lonberg et al. International Application WO 92 / 03918; Kay et al. International Application 92 / 03917; Lonberg, N. et al. 1994 Nature 368:856- 859; Green, L.L. et al. 1994 Nature Genet. 7: 13-21; Morrison, S.L. et al. 1994 Proc. Natl. Acad. Sci.USA 81 :6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720- 3724; Bruggeman et al. 1991 Eur J Immunol 21: 1323-1326).

[0465] An antibody molecule can be one in which the variable region, or a portion thereof, e.g. , the CDRs, are generated in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the invention. Antibody molecules generated in a non-human organism, e.g. , a rat or mouse, and then modified, e.g. , in the variable framework or constant region, to decrease antigenicity in a human are within the invention.

[0466] An “effectively human” protein is a protein that does substantially not evoke a neutralizing antibody response, e.g., the human anti -murine antibody (HAMA) response. HAMA can be problematic in a number of circumstances, e.g., if the antibody molecule is administered repeatedly, e.g., in treatment of a chronic or recurrent disease condition. A HAMA response can make repeated antibody administration potentially ineffective because of an increased antibody clearance from the serum (see, e.g., Saleh etal.^ Cancer Immunol. Immunother., 32: 180-190 (1990)) and also because of potential allergic reactions (see, e.g., LoBuglio et al., Hybridoma, 5:5117-5123 (1986)).

[0467] Chimeric antibodies can be produced by recombinant DNA techniques known in the art (see Robinson et al., International Patent Publication PCT / US86 / 02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et a , International Application WO 86 / 01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al. , European Patent Application 125,023; Better et al. (1988 Science 240: 1041-1043); Liu et al. (1987) AAS 84:3439-3443; Liu et a / ., 1987, J. Immunol. 139:3521-3526;Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Cane. Res. 47:999-1005; Wood et a / . (1985) Nature 314:446-449; and Shaw et a / ., 1988, J. Natl Cancer Inst. 80: 1553-1559).

[0468] A humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDRs (of heavy and or light immuoglobulin chains) replaced with a donor CDR. The antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding to the antigen. Preferably, the donor will be a rodent antibody, e.g., a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDRs is called the “donor” and the immunoglobulin providing the framework is called the “acceptor.” In some embodiments, the donor immunoglobulin is a non-human (e.g., rodent). The acceptor framework is a naturally-occurring (e.g., a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto.

[0469] As used herein, the term “consensus sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in theconsensus sequence. A “consensus framework” refers to the framework region in the consensus immunoglobulin sequence.

[0470] An antibody molecule can be humanized by methods known in the art (see e.g., Morrison, S. L., 1985, Science 229: 1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. US 5,585,089, US 5,693,761 and US 5,693,762, the contents of all of which are hereby incorporated by reference).

[0471] Humanized or CDR-grafted antibody molecules can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDRs of an immunoglobulin chain can be replaced. See e.g., U.S. Patent 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239: 1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter US 5,225,539, the contents of all of which are hereby expressly incorporated by reference. Winter describes a CDR-grafting method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on March 26, 1987; Winter US 5,225,539), the contents of which is expressly incorporated by reference.

[0472] Also within the scope of the invention are humanized antibody molecules in which specific amino acids have been substituted, deleted or added. Criteria for selecting amino acids from the donor are described in US 5,585,089, e.g., columns 12-16 of US 5,585,089, e.g., columns 12-16 of US 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 Al, published on December 23, 1992.

[0473] The antibody molecule can be a single chain antibody. A single-chain antibody (scFV) may be engineered (see, for example, Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target protein.

[0474] In yet other embodiments, the antibody molecule has a heavy chain constant region chosen from, e.g., the heavy chain constant regions of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE; particularly, chosen from, e.g., the (e.g., human) heavy chain constant regions of IgGl, IgG2, IgG3, and IgG4. In another embodiment, the antibody molecule has a light chain constant region chosen from, e.g., the (e.g., human) light chain constant regions of kappa or lambda. The constant region can be altered, e.g. , mutated, to modify the properties of the antibody (e.g. , to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, and / or complement function). In some embodiments the antibody has: effector function; and can fix complement. In other embodiments the antibody does not; recruit effector cells; or fix complement. In another embodiment, the antibody has reduced or no ability to bind an Fc receptor. For example, it is a isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.

[0475] Methods for altering an antibody constant region are known in the art. Antibodies with altered function, e.g. altered affinity for an effector ligand, such as FcR on a cell, or the Cl component of complement can be produced by replacing at least one amino acid residue in the constant portion of theantibody with a different residue (see e.g., EP 388,151 Al, U.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260, the contents of all of which are hereby incorporated by reference). Similar type of alterations could be described which if applied to the murine, or other species immunoglobulin would reduce or eliminate these functions.

[0476] An antibody molecule can be derivatized or linked to another functional molecule (e.g., another peptide or protein). As used herein, a “derivatized” antibody molecule is one that has been modified. Methods of derivatization include but are not limited to the addition of a fluorescent moiety, a radionucleotide, a toxin, an enzyme or an affinity ligand such as biotin. Accordingly, the antibody molecules of the invention are intended to include derivatized and otherwise modified forms of the antibodies described herein, including immunoadhesion molecules. For example, an antibody molecule can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0477] One type of derivatized antibody molecule is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifiinctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.CDR-grafted scaffolds

[0478] In some embodiments, the antibody molecule is a CDR-grafted scaffold domain. In some embodiments, the scaffold domain is based on a fibronectin domain, e.g., fibronectin type III domain. The overall fold of the fibronectin type III (Fn3) domain is closely related to that of the smallest functional antibody fragment, the variable domain of the antibody heavy chain. There are three loops at the end of Fn3; the positions of BC, DE and FG loops approximately correspond to those of CDR1, 2 and 3 of the VH domain of an antibody. Fn3 does not have disulfide bonds; and therefore Fn3 is stable under reducing conditions, unlike antibodies and their fragments (see, e g., WO 98 / 56915; WO 01 / 64942; WO 00 / 34784). An Fn3 domain can be modified (e.g, using CDRs or hypervariable loops described herein) or varied, e.g., to select domains that bind to an antigen / marker / cell described herein.

[0479] In some embodiments, a scaffold domain, e.g., a folded domain, is based on an antibody, e.g., a “minibody” scaffold created by deleting three beta strands from a heavy chain variable domain of a monoclonal antibody (see, e.g., Tramontane et al., 1994, J Mol. Recognit. 7:9; and Martin et al., 1994, EMBO J. 13:5303-5309). The “minibody” can be used to present two hypervariable loops. In some embodiments, the scaffold domain is a V-like domain (see, e.g., Coia et al. WO 99 / 45110) or a domain derived from tendamistatin, which is a 74 residue, six-strand beta sheet sandwich held together by two disulfide bonds (see, e.g, McConnell and Hoess, 1995, J Mol. Biol. 250:460). For example, the loops of-n-tendamistatin can be modified (e.g, using CDRs or hypervariable loops) or varied, e.g.. to select domains that bind to a marker / antigen / cell described herein. Another exemplary scaffold domain is a betasandwich structure derived from the extracellular domain of CTLA-4 (see, e.g., WO 00 / 60070).

[0480] Other exemplary scaffold domains include but are not limited to T-cell receptors; MHC proteins; extracellular domains (e.g, fibronectin Type III repeats, EGF repeats); protease inhibitors (e.g., Kunitz domains, ecotin, BPTI, and so forth); TPR repeats; trifoil structures; zinc finger domains; DNA-binding proteins; particularly monomeric DNA binding proteins; RNA binding proteins; enzymes, e.g., proteases (particularly inactivated proteases), RNase; chaperones, e.g., thioredoxin, and heat shock proteins; and intracellular signaling domains (such as SH2 and SH3 domains). See, e.g., US 20040009530 and US 7,501,121, incorporated herein by reference.

[0481] In some embodiments, a scaffold domain is evaluated and chosen, e.g. , by one or more of the following criteria: (1) amino acid sequence, (2) sequences of several homologous domains, (3) 3- dimensional structure, and / or (4) stability data over a range of pH, temperature, salinity, organic solvent, oxidant concentration. In some embodiments, the scaffold domain is a small, stable protein domain, e.g., a protein of less than 100, 70, 50, 40 or 30 amino acids. The domain may include one or more disulfide bonds or may chelate a metal, e.g., zinc.Antibody-Based Fusions

[0482] A variety of formats can be generated which contain additional binding entities attached to the N or C terminus of antibodies. These fusions with single chain or disulfide stabilized Fvs or Fabs result in the generation of tetravalent molecules with bivalent binding specificity for each antigen. Combinations of scFvs and scFabs with IgGs enable the production of molecules which can recognize three or more different antigens.Antibody-Fab Fusion

[0483] Antibody -Fab fusions are bispecific antibodies comprising a traditional antibody to a first target and a Fab to a second target fused to the C terminus of the antibody heavy chain. Commonly the antibody and the Fab will have a common light chain. Antibody fusions can be produced by (1 ) engineering the DNA sequence of the target fusion, and (2) transfecting the target DNA into a suitable host cell to express the fusion protein. It seems like the antibody-scFv fusion may be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv, as described by Coloma, J. et al. (1997) Nature Biotech 15: 159.Antibody-scFv Fusion

[0484] Antibody-scFv Fusions are bispecific antibodies comprising a traditional antibody and a scFv of unique specificity fused to the C terminus of the antibody heavy chain. The scFv can be fused to the C terminus through the Heavy Chain of the scFv either directly or through a linker peptide. Antibody fusions can be produced by (1 ) engineering the DNA sequence of the target fusion, and (2) transfectingthe target DNA into a suitable host cell to express the fusion protein. It seems like the antibody-scFv fusion may be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N- terminus of the scFv, as described by Coloma, J. et al. (1997) Nature Biotech 15: 159.Variable Domain Immunoglobulin DVD.

[0485] A related format is the dual variable domain immunoglobulin (DVD), which are composed of VH and VL domains of a second specificity place upon the N termini of the V domains by shorter linker sequences.

[0486] Other exemplary multifunctional antibody formats include, e.g., those described in the following US20I60I I4057AI, US20I30243775AI, US20140051833, US20130022601, US20I500I7I87AI, US20120201746A1, US20I50I33638AI, US20I30266568AI, US20I60I45340AI, WO20I5I27I58AI, US20I5020359IAI, US20140322221A1, US20130303396A1, US20110293613, US20130017200A1, US20I60I02I35AI, WO2015197598A2, WO20I5I97582AI, US9359437, US20150018529, WO2016115274AI, WO2016087416A1, US20080069820A1, US9145588B, US7919257, and US20150232560A1. Exemplary multifunctional molecules utilizing a full antibody-Fab / scFab format include those described in the following, US9382323B2, US20I4007258IAI, US20I40308285AI, US20I30I65638AI, US20130267686A1, US20140377269A1, US7741446B2, and WO 1995009917A I. Exemplary multifunctional molecules utilizing a domain exchange format include those described in the following, US20150315296A1, W02016087650A1, US20160075785A1, WO2016016299A1, US20160130347A1, US20150166670, US8703132B2, US20100316645, US8227577B2, US20130078249.Fc-containing multifunctional molecules

[0487] In some embodiments, the multifunctional molecules as described herein includes an immunoglobulin constant region (e.g., an Fc region). Exemplary Fc regions can be chosen from the heavy chain constant regions of IgGl, IgG2, IgG3 or IgG4; more particularly, the heavy chain constant region of human IgGl, IgG2, IgG3, or IgG4.

[0488] In some embodiments, the immunoglobulin chain constant region (e.g., the Fc region) is altered, e.g. , mutated, to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function.

[0489] In other embodiments, an interface of a first and second immunoglobulin chain constant regions (e.g., a first and a second Fc region) is altered, e.g., mutated, to increase or decrease dimerization, e.g., relative to a non-engineered interface, e.g., a naturally-occurring interface. For example, dimerization of the immunoglobulin chain constant region (e.g., the Fc region) can be enhanced by providing an Fc interface of a first and a second Fc region with one or more of: a paired protuberance-cavity (“knob-in-a hole”), an electrostatic interaction, or a strand-exchange, such that a greater ratio of heteromultimer to homomultimer forms, e.g., relative to a non-engineered interface.

[0490] In some embodiments, the multifunctional molecules include a paired amino acid substitution at a position chosen from one or more of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399,405, 407, or 409, e.g., of the Fc region of human IgGl For example, the immunoglobulin chain constant region (e.g., Fc region) can include a paired an amino acid substitution chosen from: T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole), and T366W (e.g., corresponding to a protuberance or knob).

[0491] In other embodiments, the multifunctional molecule includes a half-life extender, e.g., a human serum albumin or an antibody molecule to human serum albumin.

[0492] In some embodiments, Fc contains exemplary Fc modifications listed in Table 14.

[0493] Heterodimerized Antibody Molecules & Methods of Making

[0494] Various methods of producing multifunctional antibodies have been disclosed to address the problem of incorrect heavy chain pairing. Exemplary methods are described below. Exemplary multifunctional antibody formats and methods of making said multifunctional antibodies are also disclosed in e.g., Speiss et al. Molecular Immunology 67 (2015) 95-106; and Klein et al mAbs 4:6, 653- 663; November / December 2012; the entire contents of each of which are incorporated by reference herein.

[0495] Heterodimerized bispecific antibodies are based on the natural IgG structure, wherein the two binding arms recognize different antigens. IgG derived formats that enable defined monovalent (and simultaneous) antigen binding are generated by forced heavy chain heterodimerization, combined with technologies that minimize light chain mispairing (e.g., common light chain). Forced heavy chain heterodimerization can be obtained using, e.g., knob-in-hole OR strand exchange engineered domains (SEED).

[0496] Knob-in-Hole

[0497] Knob-in-Hole as described in US 5,731,116, US 7,476,724 and Ridgway, J. et al. (1996) Prot. Engineering 9(7): 617-621, broadly involves: (1 ) mutating the CH3 domain of one or both antibodies to promote heterodimerization; and (2) combining the mutated antibodies under conditions that promote heterodimerization. “Knobs” or “protuberances” are typically created by replacing a small amino acid in a parental antibody with a larger amino acid (e.g., T366Y or T366W); “Holes” or “cavities” are created by replacing a larger residue in a parental antibody with a smaller amino acid (e.g. , Y407T, T366S, L368A and / or Y407V).

[0498] For bispecific antibodies including an Fc domain, introduction of specific mutations into the constant region of the heavy chains to promote the correct heterodimerization of the Fc portion can be utilized. Several such techniques are reviewed in Klein et al. (mAbs (2012) 4:6, 1-11), the contents of which are incorporated herein by reference in their entirety. These techniques include the “knobs-into- holes” (KiH) approach which involves the introduction of a bulky residue into one of the CH3 domains of one of the antibody heavy chains. This bulky residue fits into a complementary “hole” in the other CH3 domain of the paired heavy chain so as to promote correct pairing of heavy chains (see e.g., US7642228).

[0499] Exemplary KiH mutations include S354C, T366W in the “knob” heavy chain and Y349C, T366S, L368A, Y407V in the “hole” heavy chain. Other exemplary KiH mutations are provided in Table 4, with additional optional stabilizing Fc cysteine mutations.

[0500] Other Fc mutations are provided by Igawa and Tsunoda who identified 3 negatively charged residues in the CH3 domain of one chain that pair with three positively charged residues in the CH3 domain of the other chain. These specific charged residue pairs are: E356-K439, E357-K370, D399- K409 and vice versa. By introducing at least two of the following three mutations in chain A: E356K, E357K and D399K, as well as K370E, K409D, K439E in chain B, alone or in combination with newly identified disulfide bridges, they were able to favor very efficient heterodimerization while suppressing homodimerization at the same time (Martens T et al. A novel one-armed antic- Met antibody inhibits glioblastoma growth in vivo. Clin Cancer Res 2006; 12:6144-52; PMID: 17062691). Xencor defined 41 variant pairs based on combining structural calculations and sequence information that were subsequently screened for maximal heterodimerization, defining the combination of S364H, F405A (HA) on chain A and Y349T, T394F on chain B (TF) (Moore GL et al. A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens. MAbs 2011; 3:546-57; PMID: 22123055).

[0501] Other exemplary Fc mutations to promote heterodimerization of multifunctional antibodies include those described in the following references, the contents of each of which is incorporated by reference herein, WO2016071377A1, US20140079689A1, US20160194389A1, US20160257763, WO2016071376A2, W02015107026A1, W02015107025A1, W02015107015A1, US20150353636A1, US20140199294A1, US7750128B2, US20160229915A1, US20150344570A1, US8003774A1, US20150337049A1, US20150175707A1, US20140242075A1, US20130195849A1, US20120149876A1, US20140200331A1, US9309311B2, US8586713, US20140037621A1, US20130178605A1, US20140363426A1, US20140051835A1 and US20110054151A1.

[0502] Stabilizing cysteine mutations have also been used in combination with KiH and other Fc heterodimerization promoting variants, see e.g., US7183076. Other exemplary cysteine modifications include, e.g., those disclosed in US20140348839A1, US7855275B2, and US9000130B2.Strand Exchange Engineered Domains (SEED)

[0503] Heterodimeric Fc platform that support the design of bispecific and asymmetric fusion proteins by devising strand-exchange engineered domain (SEED) C(H)3 heterodimers are known. These derivatives of human IgG and IgA C(H)3 domains create complementary human SEED C(H)3 heterodimers that are composed of alternating segments of human IgA and IgG C(H)3 sequences. The resulting pair of SEED C(H)3 domains preferentially associates to form heterodimers when expressed in mammalian cells. SEEDbody (Sb) fusion proteins consist of [IgGl hinge]-C(H)2-[SEED C(H)3], that may be genetically linked to one or more fusion partners (see e.g., Davis JH et al. SEEDbodies: fusion proteins based on strand exchange engineered domain (SEED) CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies. Protein Eng Des Sei 2010;23: 195-202; PMID:20299542 and US8871912. The contents of each of which are incorporated by reference herein).Fc-containing entities (mini-antibodies)

[0504] Fc-containing entities, also known as mini-antibodies, can be generated by fusing scFv to the C- termini of constant heavy region domain 3 (CH3-scFv) and / or to the hinge region (scFv-hinge-Fc) of an antibody with a different specificity. Trivalent entities can also be made which have disulfide stabilized variable domains (without peptide linker) fused to the C-terminus of CH3 domains of IgGs.Duobody

[0505] “Duobody” technology to produce bispecific antibodies with correct heavy chain pairing are known. The DuoBody technology involves three basic steps to generate stable bispecific human IgGl antibodies in a post-production exchange reaction. In a first step, two IgGls, each containing single matched mutations in the third constant (CH3) domain, are produced separately using standard mammalian recombinant cell lines. Subsequently, these IgGl antibodies are purified according to standard processes for recovery and purification. After production and purification (post-production), the two antibodies are recombined under tailored laboratory conditions resulting in a bispecific antibody product with a very high yield (typically >95%) (see e.g, Labrijn et al, PNAS 2013; 110( 13):5145-5150 and Labrijn et al. Nature Protocols 2014;9(10):2450-63, the contents of each of which are incorporated by reference herein).Electrostatic Interactions

[0506] Methods of making multifunctional antibodies using CH3 amino acid changes with charged amino acids such that homodimer formation is electrostatically unfavorable are disclosed. EP 1870459 and WO 2009089004 describe other strategies for favoring heterodimer formation upon co-expression of different antibody domains in a host cell. In these methods, one or more residues that make up the heavy chain constant domain 3 (CH3), CH3-CH3 interfaces in both CH3 domains are replaced with a charged amino acid such that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable. Additional methods of making multifunctional molecules using electrostatic interactions are described in the following references, the contents of each of which is incorporated by reference herein, include US20100015133, US8592562B2, US9200060B2, US20I40I54254AI, and US9358286A1.Common Light Chain

[0507] Light chain mispairing needs to be avoided to generate homogenous preparations of bispecific IgGs. One way to achieve this is through the use of the common light chain principle, i.e. combining two binders that share one light chain but still have separate specificities. An exemplary method of enhancing the formation of a desired bispecific antibody from a mixture of monomers is by providing a commonvariable light chain to interact with each of the heteromeric variable heavy chain regions of the bispecific antibody. Compositions and methods of producing bispecific antibodies with a common light chain as disclosed in, e.g., US7183076B2, US20110177073A1, EP2847231A1, WO2016079081 Al, and EP3055329A1, the contents of each of which is incorporated by reference herein.CrossMab

[0508] Another option to reduce light chain mispairing is the CrossMab technology which avoids nonspecific L chain mispairing by exchanging CHI and CL domains in the Fab of one half of the bispecific antibody. Such crossover variants retain binding specificity and affinity, but make the two arms so different that L chain mispairing is prevented. The CrossMab technology (as reviewed in Klein et al. Supra) involves domain swapping between heavy and light chains so as to promote the formation of the correct pairings. Briefly, to construct a bispecific IgG-like CrossMab antibody that could bind to two antigens by using two distinct light chain-heavy chain pairs, a two-step modification process is applied. First, a dimerization interface is engineered into the C-terminus of each heavy chain using a heterodimerization approach, e.g., Knob-into-hole (KiH) technology, to ensure that only a heterodimer of two distinct heavy chains from one antibody (e.g., Antibody A) and a second antibody (e.g., Antibody B) is efficiently formed. Next, the constant heavy 1 (CHI) and constant light (CL) domains of one antibody are exchanged (Antibody A), keeping the variable heavy (VH) and variable light (VL) domains consistent. The exchange of the CHI and CL domains ensured that the modified antibody (Antibody A) light chain would only efficiently dimerize with the modified antibody (antibody A) heavy chain, while the unmodified antibody (Antibody B) light chain would only efficiently dimerize with the unmodified antibody (Antibody B) heavy chain; and thus only the desired bispecific CrossMab would be efficiently formed (see e.g., Cain, C. SciBX 4(28); doi: 10.1038 / scibx.2011.783, the contents of which are incorporated by reference herein).Common Heavy Chain

[0509] An exemplary method of enhancing the formation of a desired bispecific antibody from a mixture of monomers is by providing a common variable heavy chain to interact with each of the heteromeric variable light chain regions of the bispecific antibody. Compositions and methods of producing bispecific antibodies with a common heavy chain are disclosed in, e.g., US20120184716, US20130317200, and US20160264685A1, the contents of each of which is incorporated by reference herein.Amino Acid Modifications

[0510] Alternative compositions and methods of producing multifunctional antibodies with correct light chain pairing include various amino acid modifications. For example, Zymeworks describes heterodimers with one or more amino acid modifications in the CHI and / or CL domains, one or more amino acid modifications in the VH and / or VL domains, or a combination thereof, which are part of the interface between the light chain and heavy chain and create preferential pairing between each heavy chain and adesired light chain such that when the two heavy chains and two light chains of the heterodimer pair are co-expressed in a cell, the heavy chain of the first heterodimer preferentially pairs with one of the light chains rather than the other (see e.g., W02015181805). Other exemplary methods are described in WO2016026943 (Argen-X), US20150211001, US20140072581A1, US20160039947A1, and US20150368352.Lambda / Kappa Formats

[0511] Multifunctional molecules (e.g., multispecific antibody molecules) that include the lambda light chain polypeptide and a kappa light chain polypeptides, can be used to allow for heterodimerization. Methods for generating bispecific antibody molecules comprising the lambda light chain polypeptide and a kappa light chain polypeptides are disclosed in PCT / US17 / 53053 filed on September 22, 2017 and designated publication number WO 2018 / 057955, incorporated herein by reference in its entirety.

[0512] In some embodiments, the multifunctional molecule includes a multispecific antibody molecule, e.g., an antibody molecule comprising two binding specificities, e.g., a bispecific antibody molecule. The multispecific antibody molecule includes: a lambda light chain polypeptide 1 (LLCP1) specific for a first epitope; a heavy chain polypeptide 1 (HCP1) specific for the first epitope; a kappa light chain polypeptide 2 (KLCP2) specific for a second epitope; and a heavy chain polypeptide 2 (HCP2) specific for the second epitope.

[0513] “Lambda light chain polypeptide 1 (LLCP1)”, as that term is used herein, refers to a polypeptide comprising sufficient light chain (LC) sequence, such that when combined with a cognate heavy chain variable region, can mediate specific binding to its epitope and complex with an HCP1. In some embodiments, it comprises all or a fragment of a CHI region. In some embodiments, an LLCP1 comprises LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3, FR4, and CHI, or sufficient sequence therefrom to mediate specific binding of its epitope and complex with an HCP1. LLCP1, together with its HCP1, provide specificity for a first epitope (while KLCP2, together with its HCP2, provide specificity for a second epitope). As described elsewhere herein, LLCP1 has a higher affinity for HCP1 than for HCP2.

[0514] “Kappa light chain polypeptide 2 (KLCP2)”, as that term is used herein, refers to a polypeptide comprising sufficient light chain (LC) sequence, such that when combined with a cognate heavy chain variable region, can mediate specific binding to its epitope and complex with an HCP2. In some embodiments, it comprises all or a fragment of a CHI region. In some embodiments, a KLCP2 comprises LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3, FR4, and CHI, or sufficient sequence therefrom to mediate specific binding of its epitope and complex with an HCP2. KLCP2, together with its HCP2, provide specificity for a second epitope (while LLCP1, together with its HCP1, provide specificity for a first epitope).

[0515] “Heavy chain polypeptide 1 (HCP1)”, as that term is used herein, refers to a polypeptide comprising sufficient heavy chain (HC) sequence, e.g., HC variable region sequence, such that whencombined with a cognate LLCP1, can mediate specific binding to its epitope and complex with an HCP1. In some embodiments, it comprises all or a fragment of a CHlregion. In some embodiments, it comprises all or a fragment of a CH2 and / or CH3 region. In some embodiments, an HCP1 comprises HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CHI, CH2, and CH3, or sufficient sequence therefrom to: (i) mediate specific binding of its epitope and complex with an LLCP1, (ii) to complex preferentially, as described herein to LLCP1 as opposed to KLCP2; and (iii) to complex preferentially, as described herein, to an HCP2, as opposed to another molecule of HCP1. HCP1, together with its LLCP1, provide specificity for a first epitope (while KLCP2, together with its HCP2, provide specificity for a second epitope).

[0516] “Heavy chain polypeptide 2 (HCP2)”, as that term is used herein, refers to a polypeptide comprising sufficient heavy chain (HC) sequence, e.g., HC variable region sequence, such that when combined with a cognate LLCP1, can mediate specific binding to its epitope and complex with an HCP1. In some embodiments, it comprises all or a fragment of a CHlregion. In some embodiments, it comprises all or a fragment of a CH2 and / or CH3 region. In some embodiments, an HCP1 comprises HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CHI, CH2, and CH3, or sufficient sequence therefrom to: (i) mediate specific binding of its epitope and complex with an KLCP2, (ii) to complex preferentially, as described herein to KLCP2 as opposed to LLCP1; and (iii) to complex preferentially, as described herein, to an HCP1, as opposed to another molecule of HCP2. HCP2, together with its KLCP2, provide specificity for a second epitope (while LLCP1, together with its HCP1, provide specificity for a first epitope).

[0517] In some embodiments, in the multifunctional molecule as described herein:LLCP1 has a higher affinity for HCP1 than for HCP2; and / or KLCP2 has a higher affinity for HCP2 than for HCP 1.

[0518] In some embodiments, the affinity of LLCP1 for HCP1 is sufficiently greater than its affinity for HCP2, such that under preselected conditions, e.g., in aqueous buffer, e.g., at pH 7, in saline, e.g., at pH 7, or under physiological conditions, at least 75, 80, 90, 95, 98, 99, 99.5, or 99.9 % of the multispecific antibody molecule molecules have a LLCP1 complexed, or interfaced with, a HCP1.

[0519] In some embodiments, in the multifunctional molecule as described herein: the HCP1 has a greater affinity for HCP2, than for a second molecule of HCP 1; and / or the HCP2 has a greater affinity for HCP1, than for a second molecule of HCP2.

[0520] In some embodiments, the affinity of HCP 1 for HCP2 is sufficiently greater than its affinity for a second molecule of HCP1, such that under preselected conditions, e.g., in aqueous buffer, e.g., at pH 7, in saline, e.g., at pH 7, or under physiological conditions, at least 75%, 80, 90, 95, 98, 99 99.5 or 99.9 % of the multifunctional antibody molecule molecules have a HCP 1 complexed, or interfaced with, a HCP2.

[0521] In another aspect, described herein is a method for making, or producing, a multifunctional antibody molecule. The method includes:(i) providing a first heavy chain polypeptide (e.g., a heavy chain polypeptide comprising one, two, three or all of a first heavy chain variable region (first VH), a first CHI, a first heavy chain constant region(e.g. , a first CH2, a first CH3, or both));(ii) providing a second heavy chain polypeptide (e.g., a heavy chain polypeptide comprising one, two, three or all of a second heavy chain variable region (second VH), a second CHI, a second heavy chain constant region (e.g., a second CH2, a second CH3, or both));(iii) providing a lambda chain polypeptide (e.g., a lambda light variable region (VL ). a lambda light constant chain (VL ). or both) that preferentially associates with the first heavy chain polypeptide (e.g., the first VH); and(iv) providing a kappa chain polypeptide (e.g., a lambda light variable region (VL ). a lambda light constant chain (VL ). or both) that preferentially associates with the second heavy chain polypeptide (e.g., the second VH), under conditions where (i)-(iv) associate.

[0522] In some embodiments, the first and second heavy chain polypeptides form an Fc interface that enhances heterodimerization.

[0523] In some embodiments, (i)-(iv) (e.g., nucleic acid encoding (i)-(iv)) are introduced in a single cell, e.g., a single mammalian cell, e.g., a CHO cell. In some embodiments, (i)-(iv) are expressed in the cell. In some embodiments, (i)-(iv) (e.g., nucleic acid encoding (i)-(iv)) are introduced in different cells, e.g., different mammalian cells, e.g. , two or more CHO cell. In some embodiments, (i)-(iv) are expressed in the cells.

[0524] In some embodiments, the method further comprises purifying a cell-expressed antibody molecule, e.g., using a lambda- and / or- kappa-specific purification, e.g., affinity chromatography.

[0525] In some embodiments, the method further comprises evaluating the cell-expressed multifunctional antibody molecule. For example, the purified cell-expressed multifunctional antibody molecule can be analyzed by techniques known in the art, include mass spectrometry. In some embodiments, the purified cell-expressed antibody molecule is cleaved, e.g., digested with papain to yield the Fab moieties and evaluated using mass spectrometry.

[0526] In some embodiments, the method produces correctly paired kappa / lambda multispecific, e.g., bispecific, antibody molecules in a high yield, e.g., at least 75%, 80, 90, 95, 98, 99 99.5 or 99.9 %.

[0527] In other embodiments, the multispecific, e.g., a bispecific, antibody molecule that includes:(i) a first heavy chain polypeptide (HCP1) (e.g., a heavy chain polypeptide comprising one, two, three or all of a first heavy chain variable region (first VH), a first CHI, a first heavy chain constant region (e.g., a first CH2, a first CH3, or both)), e.g., wherein the HCP1 binds to a first epitope;(ii) a second heavy chain polypeptide (HCP2) (e.g., a heavy chain polypeptide comprising one, two, three or all of a second heavy chain variable region (second VH), a second CHI, a second heavy chain constant region (e.g., a second CH2, a second CH3, or both)), e.g, wherein the HCP2 binds to a second epitope;(iii) a lambda light chain polypeptide (LLCP1) (e.g., a lambda light variable region (VLZ). a lambda light constant chain (VLZ). or both) that preferentially associates with the first heavy chain polypeptide (e.g., the first VH), e.g., wherein the LLCP1 binds to a first epitope; and(iv) a kappa light chain polypeptide (KLCP2) (e.g., a kappa light variable region (VLK), a kappa lightconstant chain (VLK), or both) that preferentially associates with the second heavy chain polypeptide (e.g., the second VH), e.g., wherein the KLCP2 binds to a second epitope.

[0528] In some embodiments, the first and second heavy chain polypeptides form an Fc interface that enhances heterodimerization. In some embodiments, the multifunctional antibody molecule has a first binding specificity that includes a hybrid VLZ-CLZ heterodimerized to a first heavy chain variable region connected to the Fc constant, CH2-CH3 domain (having a knob modification) and a second binding specificity that includes a hybrid VLK-CLK heterodimerized to a second heavy chain variable region connected to the Fc constant, CH2-CH3 domain (having a hole modification).Multispecific or multifunctional antibody molecules

[0529] Exemplary structures of multispecific and multifunctional molecules defined herein are described throughout. Exemplary structures are further described in: Weidle U et al. (2013) The Intriguing Options of Multispecific Antibody Formats for Treatment of Cancer. Cancer Genomics & Proteomics 10: 1-18 (2013); and Spiess C et al. (2015) Alternative molecular formats and therapeutic applications for bispecific antibodies. Molecu lar Immunology 67: 95-106; the full contents of each of which is incorporated by reference herein).

[0530] In some embodiments, multispecific antibody molecules can comprise more than one antigenbinding site, where different sites are specific for different antigens. In some embodiments, multispecific antibody molecules can bind more than one (e.g., two or more) epitopes on the same antigen. In some embodiments, multispecific antibody molecules comprise an antigen-binding site specific for a target cell (e.g., cancer cell) and a different antigen-binding site specific for an immune effector cell. In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibody molecules can be classified into five different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG appended with an additional antigen-binding moiety; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates.

[0531] BsIgG is a format that is monovalent for each antigen. Exemplary BsIgG formats include but are not limited to crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pair, Fab-arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, KA-body. orthogonal Fab. See Spiess et al. Mol. Immunol. 67(2015): 95- 106. Exemplary BsIgGs include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm), which contains an anti-CD3 arm and an anti-EpCAM arm; and ertumaxomab (Neovii Biotech, Fresenius Biotech), which targets CD3 and HERZ. In some embodiments, BsIgG comprises heavy chains that are engineered for heterodimerization. For example, heavy chains can be engineered for heterodimerization using a “knobs-into-holes” strategy, a SEED platform, a common heavy chain (e.g., in KX-bodies), and use of heterodimeric Fc regions. See Spiess et al. Mol. Immunol. 67(2015): 95- 106. Strategies that have been used to avoid heavy chain pairing of homodimers in BsIgG include knobs-in-holes, duobody, azymetric, charge pair, HA-TF, SEEDbody, and differential protein A affinity. See Id. BsIgG can be produced by separate expression of the component antibodies in different host cells and subsequent purification / assembly into a BsIgG. BsIgGcan also be produced by expression of the component antibodies in a single host cell. BsIgG can be purified using affinity chromatography, e.g., using protein A and sequential pH elution.

[0532] IgG appended with an additional antigen-binding moiety is another format of bispecific antibody molecules. For example, monospecific IgG can be engineered to have bispecificity by appending an additional antigen-binding unit onto the monospecific IgG, e.g. , at the N- or C- terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single domain antibodies (e.g., variable heavy chain or variable light chain), engineered protein scaffolds, and paired antibody variable domains (e.g., single chain variable fragments or variable fragments). See Id. Examples of appended IgG formats include dual variable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv- (L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG- 2scFv, scFv4-Ig, zybody, and DVI-IgG (four-in-one). See Spiess et al. Mol. Immunol. 67(2015):95-106. An example of an IgG-scFv is MM-141 (Merrimack Pharmaceuticals), which binds IGF-1R and HER3. Examples of DVD-Ig include ABT-981 (AbbVie), which binds IL-la and IL-1[3; and ABT-122 (AbbVie), which binds TNF and IL-17A.

[0533] Bispecific antibody fragments (BsAb) are a format of bispecific antibody molecules that lack some or all of the antibody constant domains. For example, some BsAb lack an Fc region. In some embodiments, bispecific antibody fragments include heavy and light chain regions that are connected by a peptide linker that permits efficient expression of the BsAb in a single host cell. Exemplary bispecific antibody fragments include but are not limited to nanobody, nanobody-HAS, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab’)2, F(ab’)2-scFv2, scFv-KIH, Fab-scFv- Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, tandem scFv-Fc, and intrabody. See Id. For example, the BiTE format comprises tandem scFvs, where the component scFvs bind to CD3 on T cells and a surface antigen on cancer cells.

[0534] Bispecific fusion proteins include antibody fragments linked to other proteins, e.g., to add additional specificity and / or functionality. An example of a bispecific fusion protein is an immTAC, which comprises an anti-CD3 scFv linked to an affinity-matured T-cell receptor that recognizes HLA- presented peptides. In some embodiments, the dock-and-lock (DNL) method can be used to generate bispecific antibody molecules with higher valency. Also, fusions to albumin binding proteins or human serum albumin can be extend the serum half-life of antibody fragments. See Id.

[0535] In some embodiments, chemical conjugation, e.g., chemical conjugation of antibodies and / or antibody fragments, can be used to create BsAb molecules. See Id. An exemplary bispecific antibody conjugate includes the CovX-body format, in which a low molecular weight drug is conjugated site- specifically to a single reactive lysine in each Fab arm or an antibody or fragment thereof. In some embodiments, the conjugation improves the serum half-life of the low molecular weight drug. An exemplary CovX-body is CVX-241 (NCTO 1004822), which comprises an antibody conjugated to two short peptides inhibiting either VEGF or Ang2. See Id.

[0536] The antibody molecules can be produced by recombinant expression, e.g., of at least one or more component, in a host system. Exemplary host systems include eukaryotic cells (e.g., mammalian cells, e.g., CHO cells, or insect cells, e.g., SF9 or S2 cells) and prokaryo...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A composition for treating a disease or condition in a subject in need thereof comprising:(a) a first agent comprising a first domain that binds to a TCR P variable (TCR V) region; and(b) a second agent comprising a histone deacetylase (HDAC) inhibitor.

2. A kit for treating a disease or condition in a subject in need thereof comprising:(a) a first agent comprising a first domain that binds to a TCR variable (TCRpV) region; and(b) a second agent comprising a histone deacetylase (HDAC) inhibitor.

3. The composition of claim 1 or the kit of claim 2, wherein the composition or the kit comprises a therapeutically effective amount of the first agent and the second agent.

4. The composition of claim 1 or 3 or the kit of claim 2 or 3, wherein the subject exhibits an improved therapeutic effect after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

5. The composition of any one of claims 1, 3, and 4, or the kit of any one of claims 2-4, wherein the subject comprises more activated and / or expanded T cells after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

6. The composition of any one of claims 1 and 3-5, or the kit of any one of claims 2-5, wherein the subject comprises less exhausted T cells after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

7. The composition of any one of claims 1 and 3-6, or the kit of any one of claims 2-6, wherein the subject exhibits more anti -turn or efficacy after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

8. The composition of any one of claims 1 and 3-7, or the kit of any one of claims 2-7, wherein the subject comprises T cells that are more cytotoxic against tumor cells or cancer cells after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

9. The composition of any one of claims 1 and 3-8, or the kit of any one of claims 2-8, wherein the subject comprises more T cells that are cytotoxic against tumor cells or cancer cells after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

10. The composition of any one of claims 1 and 3-9, or the kit of any one of claims 2-9, wherein the subject exhibits more cytotoxic activity against tumor cells or cancer cells after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

11. The composition of any one of claims 1 and 3-10, or the kit of any one of claims 2-10, wherein the subject comprises tumors that are more immune infiltrated after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

12. The composition of any one of claims 1 and 3-11, or the kit of any one of claims 2-11, wherein the subject comprises more immune permissive tumor environment after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

13. The composition of any one of claims 5, 6, 8, and 9, wherein the T cells are CD8+ or CD4+.

14. The composition of any one of claims 5, 6, 8, 9, and 13, wherein the T cells are granzyme B+.

15. The composition of any one of claims 5, 6, 8, 9, 13, and 14, wherein the T cells are IFNy+ or TNFa+.

16. The composition of any one of claims 5, 6, 8, 9, and 13-15, wherein the T cells are tumor-infiltrated T cells.

17. The composition of any one of claims 5, 6, 8, 9, and 13-15, wherein the T cells express one or more of TCRPV6-5, TCRp VI 0-1, and TCRp VI 0-2.

18. The composition of any one of claims 7, 10, 11, and 12, wherein the anti -tumor efficacy, the cytotoxic activity, the immune infiltration, or the immune permissive tumor environment is measured by number of CD8+ T cells or CD4+ T cells.

19. The composition of claim 16, wherein the CD8+ T cells are granzyme B+.

20. The composition of any one of claims 7, 10, 11, and 12, wherein the anti -tumor efficacy, the cytotoxic activity, the immune infiltration, or the immune permissive tumor environment is measured by number of Ml macrophages.

21. The composition of claim 20, wherein the Ml macrophages are MHCII+.

22. The composition of any one of claims 7, 10, 11, 12, and 18-21, wherein the anti-tumor efficacy, the cytotoxic activity, the immune infiltration, or the immune permissive tumor environment is measured by IFNy or TNFa level.

23. The composition of claim 1 or 3 or the kit of claim 2 or 3, wherein the subject exhibits an improved therapeutic effect after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

24. The composition of any one of claims 1, 3, and 23, or the kit of any one of claims 2, 3, and 23, wherein the subject comprises more activated and / or expanded T cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

25. The composition of any one of claims 1, 3, 23, and 24, or the kit of any one of claims 2, 3, 23, and24, wherein the subject comprises less exhausted T cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

26. The composition of any one of claims 1, 3, and 23-25, or the kit of any one of claims 2, 3, and 23-25, wherein the subject exhibits more anti -tumor efficacy after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

27. The composition of any one of claims 1, 3, and 23-26, or the kit of any one of claims 2, 3, and 23-26, wherein the subject comprises T cells that are more cytotoxic against tumor cells or cancer cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

28. The composition of any one of claims 1, 3, and 23-27, or the kit of any one of claims 2, 3, and 23-27, wherein the subject comprises more T cells that are cytotoxic against tumor cells or cancer cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

29. The composition of any one of claims 1, 3, and 23-28, or the kit of any one of claims 2, 3, and 23-28, wherein the subject exhibits more cytotoxic activity against tumor cells or cancer cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

30. The composition of any one of claims 1, 3, and 23-29, or the kit of any one of claims 2, 3, and 23-29, wherein the subject comprises tumors that are more immune infiltrated after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

31. The composition of any one of claims 1 , 3, and 23-30, or the kit of any one of claims 2, 3, and 23- 30, wherein the subject comprises more immune permissive tumor environment after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

32. The composition of any one of claims 24, 25, 27, and 28, wherein the T cells are tumor-infiltrated T cells.

33. The composition of any one of claims 24, 25, 27, 28, and 32, wherein the T cells express one or more of TCRPV6-5, TCRp VI 0-1, and TCRp VI 0-2.

34. The composition of any one of claims 24, 25, 27, 28, 32, and 33, wherein the T cells are CD8+ or CD4+.

35. The composition of any one of claims 24, 25, 27, 28 and 32-34, wherein the T cells are granzyme B+.

36. The composition of any one of claims 24, 25, 27, 28, and 32-35, wherein the T cells are IFNy+ or TNFa+.

37. The composition of any one of claims 26, 29, 30, and 31, wherein the anti-tumor efficacy, the cytotoxic activity, the immune infiltration, or the immune permissive tumor environment is measured by number of CD8+ T cells or CD4+ T cells.

38. The composition of claim 37, wherein the CD8+ T cells are granzyme B+.

39. The composition of any one of claims 26, 29, 30, and 31, wherein the anti-tumor efficacy, the cytotoxic activity, the immune infiltration, or the immune permissive tumor environment is measured by number of Ml macrophages.

40. The composition of claim 39, wherein the Ml macrophages are MHCII+.

41. The composition of any one of claims 26, 29, 30, 31, and 37-40, wherein the anti-tumor efficacy, the cytotoxic activity, the immune infiltration, or the immune permissive tumor environment is measured by IFNy or TNFa level.

42. The composition of any one of claims 1 and 3-31, or the kit of any one of claims 2-11, wherein the TCRpV region is a human TCRpV region.

43. The composition or the kit of claim 32, wherein the first domain binds to one or more of a TCRpV subfamily selected from the group consisting of TCRP V6 subfamily and TCRP VI 0 subfamily.

44. The composition or the kit of claim 43, wherein the first domain binds to one or more of a TCRpV subfamily selected from the group consisting of:(a) TCRP V6 subfamily comprising one or more selected from TCRP V6-l*01, TCRP V6-2*01, TCRP V6-3*01, TCRP V6-4*01, TCRP V6-4*02, TCRP V6-5*01, TCRP V6-6*01, TCRP V6-6*02, TCRP V6-6*03, TCRP V6-6*04, TCRP V6-6*05, TCRP V6-7*01, TCRP V6- 8*01, and TCRP V6-9*01; or(b) TCRP V10 subfamily comprising one or more selected from TCRP V10-l*01, TCRP V10- 1*02, TCRP V10-l*03, TCRP V10-2*01, TCRP V10-2*02, TCRP V10-3*01, TCRP V10- 3*02, TCRP V10-3*03, and TCRP V10-3*04.

45. The composition or the kit of any one of claims 42-44, wherein the first domain is a full antibody, an antigen binding domain thereof, or a functional fragment or variant thereof, a bivalent antibody, a bispecific antibody, or a biparatopic antibody that binds to a TCRpV region.

46. The composition or the kit of claim 45, wherein the full antibody, an antigen binding domain thereof, or a functional fragment or variant thereof is a Fab, a Fab', a F(ab’)2, a F(ab)2, a variable fragment (Fv), a single domain antibody, a diabody, a nanobody, a camelid single domain antibody, or a single chain variable fragment (scFv).

47. The composition of any one of claims 1 and 3-46, or the kit of any one of claims 2-46, wherein the first agent comprises one or more heavy chain constant regions selected from the group consisting of IgGl heavy chain constant region or fragment thereof, IgG2 heavy chain constant region or fragment thereof, IgG3 heavy chain constant region or fragment thereof, IgGAl heavy chain constant region or fragment thereof, IgGA2 heavy chain constant region or fragment thereof, IgG4 heavy chain constant region or fragment thereof, IgJ heavy chain constant region or fragment thereof, IgM heavy chain constant region or fragment thereof, IgD heavy chain constant region or fragment thereof, and IgE heavy chain constant region or fragment thereof.

48. The composition of any one of claims 1 and 3-47, or the kit of any one of claims 2-47, wherein the first agent comprises a kappa light chain constant region or fragment thereof, a lambda light chain constant region or fragment thereof, or a combination thereof.

49. The composition of any one of claims 1 and 3-48, or the kit of any one of claims 2-48, wherein the first agent is a multifunctional molecule.

50. The composition of any one of claims 1 and 3-49, or the kit of any one of claims 2-49, wherein the first agent comprises at least two non-contiguous polypeptide chains; wherein a first polypeptide chain of the at least two non-contiguous polypeptide chains comprises a first Fc region, and a second polypeptide chain of the at least two non-contiguous polypeptide chains comprises a second Fc region.

51. The composition or the kit of claim 50, wherein the first Fc region and the second Fc region comprise an Fc interface with a knob-in-a hole.

52. The composition or the kit of claim 50 or 51, wherein the first Fc region, the second Fc region, or a combination thereof comprises an Asn297Ala mutation, a Leu234Ala / Leu235Ala mutation, or a combination thereof.

53. The composition of any one of claims 1 and 3-52, or the kit of any one of claims 2-52, wherein the first agent is a multispecific molecule that further comprises one or more of a tumor-targeting moiety, a cytokine molecule, a stromal modifying moiety, or an immune cell engager.

54. The composition of any one of claims 1 and 3-53, or the kit of any one of claims 2-53, wherein the first agent is a multispecific molecule that further comprises a cytokine molecule.

55. The composition of any one of claims 1 and 3-54, or the kit of any one of claims 2-54, wherein the first agent comprises a dimerization module comprising a first immunoglobulin chain constant region and a second immunoglobulin chain constant region.

56. The composition or the kit of claim 55, wherein the first immunoglobulin chain constant region comprises the first fragment crystallizable region (Fc region) and the second first immunoglobulin chain constant region comprises the second fragment crystallizable region (Fc region).

57. The composition or the kit of claim 56, wherein dimerization of the first Fc region and the second Fc region is enhanced by providing an Fc interface of the first Fc region and the second Fc region with one or more of a paired cavity-protuberance, an electrostatic interaction, or a strand-exchange, such that a greater ratio of heteromultimer to homomultimer forms relative to a non-engineered interface.

58. The composition or the kit of any one of claims 53-57, wherein the immune cell engager is selected from the group consisting of a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, a macrophage cell engager, and any combination thereof.

59. The composition or the kit of any one of claims 53-58, wherein the cytokine molecule is selected from the group consisting of inter-leukin-2 (IL-2) or functional variant thereof, interleukin-7 (IL-7) or functional variant thereof, in-terleukin- 12 (IL-12) or functional variant thereof, interleukin- 15 (IL- 15) or functional variant thereof, interleukin- 18 (IL- 18) or functional variant thereof, interleukin-21 (IL-21) or functional variant thereof, interferon gamma or functional variant thereof, and any combination thereof.

60. The composition or the kit of any one of claims 53-59, wherein the cytokine molecule comprises interleukin-2 (IL-2) or functional variant thereof.

61. The composition or the kit of claim 60, wherein the interleukin-2 (IL-2) or functional variant thereof comprises the sequence of SEQ ID NO: 2270 or SEQ ID NO: 2191.

62. The composition of any one of claims 1 and 3-61, or the kit of any one of claims 2-61, wherein the second agent is a small molecule inhibitor.

63. The composition of any one of claims 1 and 3-62, or the kit of any one of claims 2-62, wherein the second agent is a Class I HDAC inhibitor.

64. The composition of any one of claims 1 and 3-63, or the kit of any one of claims 2-63, wherein the second agent is a HDAC1 inhibitor.

65. The composition of any one of claims 1 and 3-64, or the kit of any one of claims 2-64, wherein the second agent is a HDAC3 inhibitor.

66. The composition of any one of claims 1 and 3-65, or the kit of any one of claims 2-65, wherein the second agent is a HDAC1 and HDAC3 inhibitor.

67. The composition of any one of claims 1 and 3-66, or the kit of any one of claims 2-66, wherein the second agent is a benzamide HDAC inhibitor.

68. The composition of any one of claims 1 and 3-62, or the kit of any one of claims 2-62, wherein the second agent is selected from the group consisting of: entinostat, tacedinaline, mocetinostat, vorinostat, belinostat, LAQ824, and romidepsin panobinostat.

69. The composition of any one of claims 1 and 3-68, or the kit of any one of claims 2-68, wherein the subject comprises higher level of protein lysine acetylation after administration of the first agent and the second agent, relative to a subject administered the first agent without the second agent.

70. The composition of any one of claims 1 and 3-69, or the kit of any one of claims 2-69, wherein the first agent further comprises a tumor-targeting moiety.

71. The composition or the kit of claim 70, wherein the tumor-targeting moiety comprises a full antibody, an antigen binding domain thereof, or a functional fragment or variant that binds to a cancer antigen.

72. The composition or the kit of claim 71, wherein the cancer antigen is selected from the group consisting of BCMA, CD19, CD20, CD22, FcRH5, PDL1, CD47, gangloside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PMSA), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), Ron Kinase, c-Met, Immature laminin receptor, TAG-72, BING-4, Calcium-activated chloride channel 2, Cyclin-Bl, 9D7, Ep-CAM, EphA3, Her2 / neu, Telomerase, SAP-1, Survivin, NY-ESO-l / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gpl00 / pmell7, Tyrosinase, TRP-1 / -2, MC1R, P-catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, p53, Ras, TGF-B receptor, AFP, ETA, MAGE, MUC-1, CA-125, BAGE, GAGE, NY-ESO-1, p-catenin, CDK4, CDC27, a actinin-4, TRPl / gp75, TRP2, gplOO, Melan-A / MARTl, gangliosides, WT1, EphA3, Epidermal growth factor receptor (EGFR), MART-2, MART-1, MUC1, MUC2, MUM1, MUM2, MUM3, MSLN, NA88-1, NPM, OA1, OGT, RCC, RUI1, RUE, SAGE, TRG, TSTA, Folate receptor alpha, LI -CAM, CAIX, gpA33, GD3, GM2, VEGFR, Intergrins, carbohydrates, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, TGF-beta, hyaluronic acid, collagen, tenascin C, and tenascin W.

73. The composition of any one of claims 1 and 3-72, or the kit of any one of claims 2-72, wherein the condition or disease is cancer.

74. The composition or the kit of claim 73, wherein the cancer is a solid tumor, a hematological cancer, a metastatic cancer, a soft tissue tumor, or a combination thereof.

75. The composition or the kit of claim 73 or 74, wherein the cancer is non-responsive to an immune checkpoint inhibitor therapy.

76. The composition or the kit of any one of claims 73-75, wherein the cancer is non-responsive to an anti-PDl therapy or an anti-PDLl therapy.

77. The composition or the kit of any one of claims 74-76, wherein the cancer is the solid tumor, and wherein the solid tumor is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, and prostate cancer, and a combination thereof.

78. The composition or the kit of claim 77, wherein the solid tumor is the colorectal cancer and wherein the tumor comprises a KRAS mutation.

79. The composition or the kit of claim 78, wherein the KRAS mutation is a G12D mutation.

80. The composition or the kit of claim 77, wherein the solid tumor is the breast cancer and wherein the breast cancer is Triple Negative Breast Cancer (TNBC).

81. The composition or the kit of any one of claims 74-76, wherein the cancer is the hematological cancer, and wherein the hematological cancer is selected from the group consisting of Hodgkin’s lymphoma, Non-Hodgkin’s lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia, myelodysplastic syndrome, multiple myeloma, T-cell lymphoma, acute lymphocytic leukemia, and a combination thereof.

82. The composition or the kit of claim 81, wherein the Non-Hodgkin’s lymphoma is selected from the group consisting of B cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (B-CLL), mantle cell lymphoma, marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, and a combination thereof.

83. The composition or the kit of claim 81, wherein the T-cell lymphoma is peripheral T-cell lymphoma.

84. The composition or the kit of any one of claims 73-83, wherein the cancer is characterized by a cancer antigen present on the cancer.

85. The composition or the kit of claim 84, wherein the cancer antigen is a tumor antigen, a stromal antigen, or a hematological antigen.

86. The composition or the kit of claim 84 or 85, wherein the cancer antigen is selected from the group consisting of BCMA, CD19, CD20, CD22, FcRH5, PDL1, CD47, ganglioside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PMSA), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), Ron Kinase, c-Met, Immature laminin receptor, TAG-72, BING-4, Calcium-activated chloride channel 2, Cyclin-Bl, 9D7, Ep-CAM, EphA3, Her2 / neu, Telomerase, SAP-1, Survivin, NY-ESO-l / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gpl00 / pmell7, Tyrosinase, MC1R, -catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, p53, Ras, TGF-B receptor, AFP, ETA, MAGE, CA-125, BAGE, GAGE, CDC27, a actinin-4, TRPl / gp75, TRP2, gangliosides, WT1, Epi-dermal growth factor receptor (EGFR), MART-2, MUC1, MUC2, MUM1, MUM2, MUM3, MSLN, NA88-1, NPM, OA1, OGT, RCC, RU11, RU12, SAGE, TRG, TSTA, Folate receptor alpha, LI -CAM, CAIX, gpA33, GD3, GM2, VEGFR, Integrins, Carbohydrates, IGF1R,TRAILR1, TRAILR2, RANKL, FAP, TGF-beta, hyaluronic acid, collagen, tenascin C, and tenascin W.

87. The composition of any one of claims 1 and 3-86, wherein the composition is pharmaceutical composition.

88. The composition of any one of claims 1 and 3-87, or the kit of any one of claims 2-86 , wherein the composition or kit further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

89. A method of treating a disease or condition in a subject in need thereof comprising:(a) administering a first agent comprising a first domain that binds to a TCR P variable (TCR V) region; and(b) administering a second agent comprising a histone deacetylase (HDAC) inhibitor; wherein a therapeutically effective amount of the first agent and the second agent is administered to the subject, thereby treating the disease or condition in the subject.

90. A method of treating a disease or condition in a subject in need thereof comprising administering to the subject a first agent comprising a first domain that binds to a TCR variable (TCRpV) region, wherein the subject has been previously treated with a second agent comprising a histone deacetylase (HDAC) inhibitor, thereby treating the disease or condition in the subject.

91. A method of improving a therapeutic efficacy of a second agent comprising a histone deacetylase (HDAC) inhibitor in a subject in need thereof comprising: administering a first agent comprising a first domain that binds to a TCR P variable (TCRpV) region, thereby improving the therapeutic efficacy of the second agent in the subject.

92. A method of treating a disease or condition in a subject in need thereof comprising:(a) administering a first agent comprising a first domain that binds to a TCR P variable (TCRpV) region; and(b) administering a second agent comprising a histone deacetylase (HDAC) inhibitor; wherein the disease is non-responsive to an immune checkpoint inhibitor therapy, and wherein a therapeutically effective amount of the first agent and the second agent is administered to the subject, thereby treating the disease or condition in the subject.

93. A method of treating a disease or condition in a subject in need thereof comprising:(a) administering a first agent comprising a first domain that binds to a TCR P variable (TCRpV) region; and(b) administering a second agent comprising a histone deacetylase (HDAC) inhibitor; wherein a therapeutically effective amount of the first agent and the second agent is administered to the subject to promote immune response or immune infiltration, thereby treating the disease or condition in the subject.

94. A method of expanding and / or activating T-cells in a subject in need thereof comprising:(a) administering a first agent comprising a first domain that binds to a TCR P variable (TCRpV) region; and(b) administering a second agent comprising a histone deacetylase (HDAC) inhibitor;wherein a therapeutically effective amount of the first agent and the second agent is administered to the subject, thereby expanding and / or activating T-cells in the subject.

95. A combination therapy for treating a disease or condition in a subject in need thereof a first agent comprising a first domain that binds to a TCR variable (TCR V) region, and a second agent comprising a histone deacetylase (HDAC) inhibitor.

96. The method of any one of claims 89-93 or the combination therapy of claim 95, wherein administering the first agent and the second agent induces an improved therapeutic effect in the subject, relative to a subject administered the second agent without the first agent.

97. The method of any one of claims 89-93 and 96 or the combination therapy of claim 95 or 96, wherein administering the first agent and the second agent induces more activated and / or expanded T cells in the subject, relative to a subject administered the second agent without the first agent.

98. The method of any one of claims 89-93, 96 and 97 or the combination therapy of any one of claims 95-97, wherein administering the first agent and the second agent induces less exhausted T cells in the subject, relative to a subject administered the second agent without the first agent.

99. The method of any one of claims 89-93 and 96-98 or the combination therapy of any one of claims 95-98, wherein administering the first agent and the second agent induces more anti-tumor efficacy in the subject, relative to a subject administered the second agent without the first agent.

100. The method of any one of claims 89-93 and 96-99 or the combination therapy of any one of claims 95-99, wherein administering the first agent and the second agent induces more T cells that are more cytotoxic against tumor cells or cancer cells in the subject, relative to a subject administered the second agent without the first agent.

101. The method of any one of claims 89-93 and 96-99 or the combination therapy of any one of claims 95-99, wherein administering the first agent and the second agent induces more cytotoxicity against tumor cells or cancer cells in the subject, relative to a subject administered the second agent without the first agent.

102. The method of any one of claims 89-93 and 96-100 or the combination therapy of any one of claims 95-100, wherein administering the first agent and the second agent expands or activates more T cells that are cytotoxic against tumor cells or cancer cells in the subject, relative to a subject administered the second agent without the first agent.

103. The method of any one of claims 89-93 and 96-102 or the combination therapy of any one of claims 95-102, wherein administering the first agent and the second agent induces more cytotoxic activity against tumor cells or cancer cells in the subject, relative to a subject administered the second agent without the first agent.

104. The method of any one of claims 89-93 and 96-103 or the combination therapy of any one of claims 95 -103, wherein the subject comprises more immune permissive tumor environment after administration of the first agent and the second agent, relative to a subject administered the second agent without the first agent.

105. The composition of any one of claims 97, 98, 100, and 102, wherein the T cells are tumor- infiltrated T cells.

106. The composition of any one of claims 97, 98, 100, 102, and 105, wherein the T cells express one or more of TCRPV6-5, TCRp VI 0-1, and TCRp VI 0-2.

107. The composition of any one of claims 97, 98, 100, 102, 105, and 106, wherein the T cells are CD8+ or CD4+.

108. The composition of any one of claims 97, 98, 100, 102, and 105-107, wherein the T cells are granzyme B+.

109. The composition of any one of claims 97, 98, 100, 102, and 105-108, wherein the T cells are IFNy+ or TNFa+.

110. The composition of any one of claims 99, 101, 103, and 104, wherein the anti -tumor efficacy, the cytotoxic activity, the immune infiltration, or the immune permissive tumor environment is measured by number of CD8+ T cells or CD4+ T cells.

111. The composition of claim 110, wherein the CD8+ cells are granzyme B+.

112. The composition of any one of claims 99, 101, 103, and 104, wherein the anti -tumor efficacy, the cytotoxic activity, the immune infiltration, or the immune permissive tumor environment is measured by number of Ml macrophages.

113. The composition of claim 112, wherein the Ml macrophages are MHCII+.

114. The composition of any one of claims 99, 101, 103, 104, and 110-113, wherein the anti -tumor efficacy, the cytotoxic activity, the immune infiltration, or the immune permissive tumor environment is measured by IFNy or TNFa level.

115. The method of any one of claims 89-93 and 96-104 or the combination therapy of any one of claims 95-102, wherein the subject exhibits an improved therapeutic effect after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

116. The method of any one of claims 89-93, 96-102, and 115 or the combination therapy of any one of claims 95-102 and 105, wherein the subject comprises more activated and / or expanded T cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

117. The method of any one of claims 89-93, 96-102, 115, and 116, or the combination therapy of any one of claims 95-102, 105, and 116, wherein the subject comprises less exhausted T cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

118. The method of any one of claims 89-93, 96-102, and 115-117, or the combination therapy of any one of claims 95-102 and 105-117, wherein the subject exhibits more anti -tumor efficacy after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

119. The method of any one of claims 89-93, 96-102, and 115-118, or the combination therapy of any one of claims 95-102 and 105-118, wherein the subject comprises T cells that are more cytotoxic against tumor cells or cancer cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

120. The method of any one of claims 89-93, 96-102, and 115-119, or the combination therapy of any one of claims 95-102 and 105-119, wherein the subject comprises more T cells that are cytotoxic against tumor cells or cancer cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

121. The method of any one of claims 89-93, 96-102, and 115-120, or the combination therapy of any one of claims 95-102 and 105-120, wherein the subject exhibits more cytotoxic activity against tumor cells or cancer cells after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

122. The method of any one of claims 89-93, 96-102, and 115-121, or the combination therapy of any one of claims 95-102 and 105-121, wherein the subject comprises tumors that are more immune infiltrated after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

123. The method of any one of claims 89-93, 96-102, and 115-122, or the combination therapy of any one of claims 95-102 and 105-122, wherein the subject comprises more immune permissive tumor environment after administration of the first agent and the second agent, relative to a subject not administered the first agent or the second agent.

124. The composition of any one of claims 116, 117, 119, and 120, wherein the T cells are tumor- infiltrated T cells.

125. The composition of any one of claims 116, 117, 119, 120, and 124, wherein the T cells express one or more of TCRPV6-5, TCRp V10-1, and TCRp V10-2.

126. The composition of any one of claims 116, 117, 119, 120, 124, and 125, wherein the T cells are CD8+ or CD4+.

127. The composition of any one of claims 116, 117, 119, 120, and 124-126, wherein the T cells are granzyme B+.

128. The composition of any one of claims 116, 117, 119, 120, and 124-127 , wherein the T cells are IFNy+ or TNFa+.

129. The composition of any one of claims 99, 101, 103, and 104, wherein the anti -tumor efficacy, the cytotoxic activity, the immune infiltration, or the immune permissive tumor environment is measured by number of CD8+ T cells or CD4+ T cells.

130. The composition of claim 110, wherein the CD8+ cells are granzyme B+.

131. The composition of any one of claims 116, 117, 119, and 120, wherein the anti -tumor efficacy, the cytotoxic activity, the immune infiltration, or the immune permissive tumor environment is measured by number of Ml macrophages.

132. The composition of claim 131, wherein the Ml macrophages are MHCII+.

133. The composition of any one of claims 116, 117, 119, and 120, and 129-132, wherein the antitumor efficacy, the cytotoxic activity, the immune infiltration, or the immune permissive tumor environment is measured by IFNy or TNFa level.

134. The method of any one of claims 89-93 and 96-133 or the combination therapy of any one of claims 95-133, wherein the first agent and the second agent are not concomitantly administered to the subject.

135. The method of any one of claims 89-93 and 96-133 or the combination therapy of any one of claims 95-133, wherein the first agent and the second agent are concomitantly administered to the subject.

136. The method of any one of claims 89-93 and 96-135 or the combination therapy of any one of claims 95-135, wherein the method comprises administering the first agent to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, at least 1, 2, 3, 4, 5, 6, or 7 days, at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years after the subject has been treated with a dose of the second agent.

137. The method of any one of claims 89-93 and 96-135 or the combination therapy of any one of claims 95-135, wherein the method comprises administering the first agent to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, at least 1, 2, 3, 4, 5, 6, or 7 days, at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years after the subject has been treated with a first dose of the second agent.

138. The method of any one of claims 89-93 and 96-135 or the combination therapy of any one of claims 95-135, wherein the method comprises administering the first agent to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, at least 1, 2, 3, 4, 5, 6, or 7 days, at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years after the subject has been treated with a last dose of the second agent.

139. The method of any one of claims 89-93 and 96-135 or the combination therapy of any one of claims 95-135, wherein the method comprises administering the first agent to the subject at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, at most 1, 2, 3, 4, 5, 6, or 7 days, at most 1, 2, 3, or 4 weeks, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at most 1, 2, or 3 years after being treated with the second agent.

140. The method of any one of claims 89-93 and 96-135 or the combination therapy of any one of claims 95-135, wherein the method comprises administering the first agent to the subject at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, at most 1, 2, 3, 4, 5, 6, or 7 days, at most 1, 2, 3, or 4 weeks, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at most 1, 2, or 3 years after being treated with a dose of the second agent.

141. The method of any one of claims 89-93 and 96-135 or the combination therapy of any one of claims 95-135, wherein the method comprises administering the first agent to the subject at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, at most 1, 2, 3, 4, 5, 6, or 7 days, at most 1, 2, 3, or 4 weeks,at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at most 1, 2, or 3 years after being treated with a first dose of the second agent.

142. The method of any one of claims 89-93 and 96-135 or the combination therapy of any one of claims 95-135, wherein the method comprises administering the first agent to the subject at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, at most 1, 2, 3, 4, 5, 6, or 7 days, at most 1, 2, 3, or 4 weeks, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at most 1, 2, or 3 years after being treated with a last dose of the second agent.

143. The method of any one of claims 89-93 and 96-142 or the combination therapy of any one of claims 95-142, wherein the method comprises administering the first agent to the subject from 1 hour to 24 hours after being treated with a dose of the second agent.

144. The method of any one of claims 89-93 and 96-142 or the combination therapy of any one of claims 95-142, wherein the method comprises administering the first agent to the subject from 1 day to 7 days after being treated with a dose of the second agent.

145. The method of any one of claims 89-93 and 96-142 or the combination therapy of any one of claims 95-142, wherein the method comprises administering the first agent to the subject from 1 week to 4 weeks after being treated with a dose of the second agent.

146. The method of any one of claims 89-93 and 96-142 or the combination therapy of any one of claims 95-142, wherein the method comprises administering the first agent to the subject from 1 month to 12 months after being treated with a dose of the second agent.

147. The method of any one of claims 89-93 and 96-142 or the combination therapy of any one of claims 95-142, wherein the method comprises administering the first agent to the subject from 1 year to 3 years after being treated with a dose of the second agent.

148. The method of any one of claims 89-93 and 96-142 or the combination therapy of any one of claims 95-142, wherein the method comprises administering the first agent to the subject from 1 hour to 24 hours after being treated with a first dose of the second agent.

149. The method of any one of claims 89-93 and 96-142 or the combination therapy of any one of claims 95-142, wherein the method comprises administering the first agent to the subject from 1 day to 7 days after being treated with a first dose of the second agent.

150. The method of any one of claims 89-93 and 96-142 or the combination therapy of any one of claims 95-142, wherein the method comprises administering the first agent to the subject from 1 week to 4 weeks after being treated with a first dose of the second agent.

151. The method of any one of claims 89-93 and 96-142 or the combination therapy of any one of claims 95-142, wherein the method comprises administering the first agent to the subject from 1 month to 12 months after being treated with a first dose of the second agent.

152. The method of any one of claims 89-93 and 96-142 or the combination therapy of any one of claims 95-142, wherein the method comprises administering the first agent to the subject from 1 year to 3 years after being treated with a first dose of the second agent.

153. The method of any one of claims 89-93 and 96-142 or the combination therapy of any one of claims 95-142, wherein the method comprises administering the first agent to the subject from 1 hour to 24 hours after being treated with a last dose of the second agent.

154. The method of any one of claims 89-93 and 96-142 or the combination therapy of any one of claims 95-142, wherein the method comprises administering the first agent to the subject from 1 week to 4 weeks after being treated with a last dose of the second agent.

155. The method of any one of claims 89-93 and 96-142 or the combination therapy of any one of claims 95-142, wherein the method comprises administering the first agent to the subject from 1 month to 12 months after being treated with a last dose of the second agent.

156. The method of any one of claims 89-93 and 96-142 or the combination therapy of any one of claims 95-142, wherein the method comprises administering the first agent to the subject from 1 year to 3 years after being treated with a last dose of the second agent.

157. The method of any one of claims 89-93 and 96-142 or the combination therapy of any one of claims 95-142, wherein the method comprises administering the first agent to the subject on the same day the subject had been previously treated with the second agent.

158. The method of any one of claims 89-93 and 96-157 or the combination therapy of any one of claims 95-157, wherein the method is repeated twice or more.

159. The method of any one of claims 89-93 and 96-158 or the combination therapy of any one of claims 95-158, wherein the administration of the first agent comprises one or more doses.

160. The method of any one of claims 89-93 and 96-159 or the combination therapy of any one of claims 95-159, wherein the administration of the first agent comprises two or more doses.

161. The method of any one of claims 89-93 and 96- 160 or the combination therapy of any one of claims 95-160, wherein the administration of the first agent comprises one dose per week.

162. The method of any one of claims 89-93 and 96-160 or the combination therapy of any one of claims 95-160, wherein the administration of the first agent comprises two or more doses per week.

163. The method of any one of claims 89-93 and 96-160 or the combination therapy of any one of claims 95-160, wherein the first agent is administered to the subject once every two weeks.

164. The method of any one of claims 89-93 and 96-160 or the combination therapy of any one of claims 95-160, wherein the first agent is administered to the subject once every three weeks.

165. The method of any one of claims 89-93 and 96-161 or the combination therapy of any one of claims 95-161, wherein the first agent is administered to the subject once every week for at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years.

166. The method of any one of claims 89-93 and 96-160, and 162 or the combination therapy of any one of claims 95-160, and 162, wherein the administration of the first agent comprises two or more doses per week for at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years.

167. The method of any one of claims 89-93 and 96-160, and 163 or the combination therapy of any one of claims 95-160, and 163, wherein the first agent is administered to the subject once every twoweeks for at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years.

168. The method of any one of claims 89-93 and 96-160, and 164 or the combination therapy of any one of claims 95-160, and 164, wherein the first agent is administered to the subject once every three weeks for at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years.

169. The method of any one of claims 89-93 and 96-168 or the combination therapy of any one of claims 95-168, wherein the administration of the second agent comprises one or more doses.

170. The method of any one of claims 89-93 and 96-169 or the combination therapy of any one of claims 95-169, wherein the administration of the second agent comprises two or more doses.

171. The method of any one of claims 89-93 and 96-170 or the combination therapy of any one of claims 95-170, wherein the administration of the second agent comprises one dose per week.

172. The method of any one of claims 89-93 and 96-170 or the combination therapy of any one of claims 95-170, wherein the administration of the second agent comprises two or more doses per week.

173. The method of any one of claims 89-93 and 96-170 or the combination therapy of any one of claims 95-170, wherein the second agent is administered to the subject once every two weeks.

174. The method of any one of claims 89-93 and 96-170 or the combination therapy of any one of claims 95-170, wherein the second agent is administered to the subject once every three weeks.

175. The method of any one of claims 89-93 and 96-171 or the combination therapy of any one of claims 95-171, wherein the second agent is administered to the subject once every week for at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years.

176. The method of any one of claims 89-93, 96-170, and 172 or the combination therapy of any one of claims 95-170, and 172, wherein the administration of the second agent comprises two or more doses per week for at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years.

177. The method of any one of claims 89-93, 96-170, and 173 or the combination therapy of any one of claims 95-170, and 173, wherein the second agent is administered to the subject once every two weeks for at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years.

178. The method of any one of claims 89-93, 96-170, and 174 or the combination therapy of any one of claims 95-170, and 174, wherein the second agent is administered to the subject once every three weeks for at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1,2, or 3 years.

179. The method of any one of claims 89-93 and 96-178 or the combination therapy of any one of claims 95-178, wherein the method comprises administering the first agent to the subject at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, at least 1, 2, 3, 4, 5, 6, or 7 days, at least 1, 2, 3, or 4 weeks,at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 1, 2, or 3 years prior to administering the second agent to the subject.

180. The method of any one of claims 89-93 and 96-179 or the combination therapy of any one of claims 95-179, wherein the method comprises administering the first agent to the subject at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, at most 1, 2, 3, 4, 5, 6, or 7 days, at most 1, 2, 3, or 4 weeks, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at most 1, 2, or 3 years prior to administering the second agent to the subject.

181. The method of any one of claims 89-93 and 96- 180 or the combination therapy of any one of claims 95-180, wherein the method comprises administering the first agent to the subject from 1 hour to 24 hours prior to administering the second agent to the subject.

182. The method of any one of claims 89-93 and 96-180 or the combination therapy of any one of claims 95-180, wherein the method comprises administering the first agent to the subject from 1 day to 7 days prior to administering the second agent to the subject.

183. The method of any one of claims 89-93 and 96-180 or the combination therapy of any one of claims 95-180, wherein the method comprises administering the first agent to the subject from 1 week to 4 weeks prior to administering the second agent to the subject.

184. The method of any one of claims 89-93 and 96-180 or the combination therapy of any one of claims 95-180, wherein the method comprises administering the first agent to the subject from 1 month to 12 months prior to administering the second agent to the subject.

185. The method of any one of claims 89-93 and 96-180 or the combination therapy of any one of claims 95-180, wherein the method comprises administering the first agent to the subject from 1 year to 3 years prior to administering the second agent to the subject.

186. The method of any one of claims 89-93 and 96-185 or the combination therapy of any one of claims 95-185, wherein the administration of the first agent comprises two or more doses, and each dose comprises a same amount of the first agent.

187. The method of any one of claims 89-93 and 96-185 or the combination therapy of any one of claims 95-185, wherein the administration of the first agent comprises two or more doses, and each dose comprises different amounts of the first agent.

188. The method of any one of claims 89-93 and 96-187 or the combination therapy of any one of claims 95-187, wherein the administration of the second agent comprises two or more doses, and each dose comprises a same amount of the second agent.

189. The method of any one of claims 89-93 and 96-187 or the combination therapy of any one of claims 95-187, wherein the administration of the second agent comprises two or more doses, and each dose comprises different amounts of the second agent.

190. The method of any one of claims 89-93 and 96-189 or the combination therapy of any one of claims 95-189, wherein the first agent is administered to the subject at a dose of from about 0.001 mg / kg to about 20 mg / kg.

191. The method of any one of claims 89-93 and 96- 190 or the combination therapy of any one of claims 95-190, wherein the second agent is administered to the subject at a dose of from about 0.001 mg / kg to about 20 mg / kg.

192. The method of any one of claims 89-93 and 96-191 or the combination therapy of any one of claims 95-191, wherein the method comprises administering the first agent and the second agent to the subject simultaneously or on the same day.

193. The method of any one of claims 89-93 and 96-192 or the combination therapy of any one of claims 95-192, wherein the disease or condition in the subject is treated to a higher extent relative to a corresponding method in which the first agent is administered and the second agent is not administered.

194. The method of any one of claims 89-93 and 96-193 or the combination therapy of any one of claims 95-193, wherein the method is more effective to treat the disease or condition in the subject relative to a method in which the first agent is administered to the subject but not the second agent.

195. The method of any one of claims 89-93 and 96-194 or the combination therapy of any one of claims 95-194, wherein the method is more effective to treat the disease or condition in the subject relative to a method in which the second agent is administered to the subject but not the first agent.

196. The method of any one of claims 89-93 and 96-195 or the combination therapy of any one of claims 95-195, wherein the TCRpV region is a human TCRpV region.

197. The method or combination therapy of claim 196, wherein the first domain binds to one or more of a TCRpV subfamily selected from the group consisting of TCRP V6 subfamily or TCRP V10 subfamily.

198. The method or combination therapy of claim 196 or 197, wherein the first domain binds to one or more of a TCRpV subfamily selected from the group consisting of:(a) TCRP V6 subfamily comprising one or more selected from TCRP V6-l*01, TCRP V6-2*01, TCRP V6-3*01, TCRP V6-4*01, TCRP V6-4*02, TCRP V6-5*01, TCRP V6-6*01, TCRP V6-6*02, TCRP V6-6*03, TCRP V6-6*04, TCRP V6-6*05, TCRP V6-7*01, TCRP V6- 8*01, and TCRP V6-9*01; or(b) TCRP V10 subfamily comprising one or more selected from TCRP V10-l*01, TCRP V10- 1*02, TCRP V10-l*03, TCRP V10-2*01, TCRP V10-2*02, TCRP V10-3*01, TCRP V10- 3*02, TCRP V10-3*03, and TCRP V10-3*04.

199. The method or combination therapy of any one of claims 196-198, wherein the first domain is a full antibody, an antigen binding domain thereof, or a functional fragment or variant thereof, a bivalent antibody, a bispecific antibody, or a biparatopic antibody that binds to a TCRpV region.

200. The method or combination therapy of claim 199, wherein the full antibody, an antigen binding domain thereof, or a functional fragment or variant thereof is a Fab, a Fab', a F(ab’)2, a F(ab)2, a variable fragment (Fv), a single domain antibody, a diabody, a nanobody, a camelid single domain antibody, or a single chain variable fragment (scFv).

201. The method of any one of claims 89-93 and 96-200 or the combination therapy of any one of claims 95-200, wherein the first agent comprises one or more heavy chain constant regions selected from the group consisting of IgGl heavy chain constant region or fragment thereof, IgG2 heavy chain constant region or fragment thereof, IgG3 heavy chain constant region or fragment thereof, IgGAl heavy chain constant region or fragment thereof, IgGA2 heavy chain constant region or fragment thereof, IgG4 heavy chain constant region or fragment thereof, IgJ heavy chain constant region or fragment thereof, IgM heavy chain constant region or fragment thereof, IgD heavy chain constant region or fragment thereof, and IgE heavy chain constant region or fragment thereof.

202. The method of any one of claims 89-93 and 96-201 or the combination therapy of any one of claims 95-201, wherein the first agent comprises a kappa light chain constant region or fragment thereof, a lambda light chain constant region or fragment thereof, or a combination thereof.

203. The method of any one of claims 89-93 and 96-202 or the combination therapy of any one of claims 95-202, wherein the first agent is a multifunctional molecule.

204. The method of any one of claims 89-93 and 96-203 or the combination therapy of any one of claims 95-203, wherein the first agent comprises at least two non-contiguous polypeptide chains; wherein a first polypeptide chain of the at least two non-contiguous polypeptide chains comprises a first Fc region, and a second polypeptide chain of the at least two non-contiguous polypeptide chains comprises a second Fc region.

205. The method or the combination therapy of claim 204, wherein the first Fc region and the second Fc region comprise an Fc interface with a knob-in-a hole.

206. The method or the combination therapy of claim 204 or 205, wherein the first Fc region, the second Fc region, or a combination thereof comprises an Asn297Ala mutation, a Leu234Ala / Leu235Ala mutation, or a combination thereof.

207. The method of any one of claims 89-93 and 96-206 or the combination therapy of any one of claims 95-206, wherein the first agent is a multispecific molecule that further comprises one or more of a tumor-targeting moiety, a cytokine molecule, a stromal modifying moiety, or an immune cell engager.

208. The method of any one of claims 89-93 and 96-207 or the combination therapy of any one of claims 95-207, wherein the first agent is a multispecific molecule that further comprises a cytokine molecule.

209. The method of any one of claims 89-93 and 96-208 or the combination therapy of any one of claims 95-208, wherein the first agent comprises a dimerization module, comprising a first immunoglobulin chain constant region and a second immunoglobulin chain constant region.

210. The method or the combination therapy of claim 209, wherein the first immunoglobulin chain constant region comprises the first fragment crystallizable region (Fc region) and the second first immunoglobulin chain constant region comprises the second fragment crystallizable region (Fc region).

211. The method or the combination therapy of claim 210, wherein dimerization of the first Fc region and the second Fc region is enhanced by providing an Fc interface of the first Fc region and the second Fc region with one or more of a paired cavity-protuberance, an electrostatic interaction, or a strand -exchange, such that a greater ratio of heteromultimer to homomultimer forms relative to a non-engineered interface.

212. The method or the combination therapy of any one of claims 207-211, wherein the immune cell engager is selected from the group consisting of a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, a macrophage cell engager, and any combination thereof.

213. The method or the combination therapy of any one of claims 207-212, wherein the cytokine molecule is selected from the group consisting of inter-leukin-2 (IL-2) or functional variant thereof, interleukin-7 (IL-7) or functional variant thereof, in-terleukin- 12 (IL- 12) or functional variant thereof, interleukin- 15 (IL-15) or functional variant thereof, interleukin- 18 (IL-18) or functional variant thereof, interleukin-21 (IL-21) or functional variant thereof, interferon gamma or functional variant thereof, and any combination thereof.

214. The method or the combination therapy of any one of claims 207-213, wherein the cytokine molecule comprises interleukin-2 (IL-2) or functional variant thereof.

215. The method or the combination therapy of claim 214, wherein the interleukin-2 (IL-2) or functional variant thereof comprises the sequence of SEQ ID NO: 2270 or SEQ ID NO: 2191.

216. The method of any one of claims 89-93 and 96-215 or the combination therapy of any one of claims 95-215, wherein the second agent is a small molecule inhibitor.

217. The method of any one of claims 89-93 and 96-216 or the combination therapy of any one of claims 95-216, wherein the second agent is a Class I HDAC inhibitor.

218. The method of any one of claims 89-93 and 96-217 or the combination therapy of any one of claims 95-217, wherein the second agent is a HDAC1 inhibitor.

219. The method of any one of claims 89-93 and 96-217 or the combination therapy of any one of claims 95-217, wherein the second agent is a HDAC3 inhibitor.

220. The method of any one of claims 89-93 and 96-219 or the combination therapy of any one of claims 95-219, wherein the second agent is a HDAC1 and HDAC3 inhibitor.

221. The method of any one of claims 89-93 and 96-220 or the combination therapy of any one of claims 95-220, or the kit of any one of claims 2-66, wherein the second agent is a benzamide HDAC inhibitor.

222. The method of any one of claims 89-93 and 96-216 or the combination therapy of any one of claims 95-216, wherein the second agent is selected from the group consisting of: entinostat, tacedinaline, mocetinostat, vorinostat, belinostat, LAQ824, and romidepsin panobinostat.

223. The method of any one of claims 89-93 and 96-222 or the combination therapy of any one of claims 95-222, wherein the subject comprises higher level of protein lysine acetylation after administration of the first agent and the second agent, relative to a subject administered the first agent without the second agent.

224. The method of any one of claims 89-93 and 96-223 or the combination therapy of any one of claims 95-223, wherein the first agent further comprises a tumor-targeting moiety.

225. The method or the combination therapy of claim 224, wherein the tumor-targeting moiety comprises a full antibody, an antigen binding domain thereof, or a functional fragment or variant that binds to a cancer antigen.

226. The method or the combination therapy of claim 225, wherein the cancer antigen is selected from the group consisting of BCMA, CD 19, CD20, CD22, FcRH5, PDL1, CD47, gangloside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PMSA), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), Ron Kinase, c-Met, Immature laminin receptor, TAG-72, BING-4, Calcium-activated chloride channel 2, Cyclin-Bl, 9D7, Ep-CAM, EphA3, Her2 / neu, Telomerase, SAP-1, Survivin, NY-ES0-1 / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gpl00 / pmell7, Tyrosinase, TRP-1 / -2, MC1R, P-catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, p53, Ras, TGF-B receptor, AFP, ETA, MAGE, MUC-1, CA-125, BAGE, GAGE, NY-ESO-1, p- catenin, CDK4, CDC27, a actinin-4, TRPl / gp75, TRP2, gplOO, Melan-A / MARTl, gangliosides, WT1, EphA3, Epidermal growth factor receptor (EGFR), MART-2, MART-1, MUC1, MUC2, MUM1, MUM2, MUM3, MSLN, NA88-1, NPM, OA1, OGT, RCC, RUI1, RUE, SAGE, TRG, TSTA, Folate receptor alpha, Ll-CAM, CAIX, gpA33, GD3, GM2, VEGFR, Intergrins, carbohydrates, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, TGF-beta, hyaluronic acid, collagen, tenascin C, and tenascin W.

227. The method of any one of claims 89-93 and 96-226 or the combination therapy of any one of claims 95-226, wherein the condition or disease is cancer.

228. The method or the combination therapy of claim 227, wherein the cancer is a solid tumor, a hematological cancer, a metastatic cancer, a soft tissue tumor, or a combination thereof.

229. The method or the combination therapy of claim 227 or 228, wherein the cancer is non- responsive to an immune checkpoint inhibitor therapy.

230. The method or the combination therapy of any one of claims 227-229, wherein the cancer is non- responsive to an anti-PDl therapy or an anti-PDLl therapy.

231. The method or the combination therapy of any one of claims 228-230, wherein the cancer is the solid tumor, and wherein the solid tumor is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, prostate cancer, and a combination thereof.

232. The method or the combination therapy of claim 231, wherein the solid tumor is the colorectal cancer and wherein the tumor comprises a KRAS mutation.

233. The method or the combination therapy of claim 232, wherein the KRAS mutation is a G12D mutation.

234. The method or the combination therapy of any one of claims 228-230, wherein the solid tumor is the breast cancer and wherein the breast cancer is Triple Negative Breast Cancer (TNBC).

235. The method or the combination therapy of any one of claims 228-230, wherein the cancer is the hematological cancer, and wherein the hematological cancer is selected from the group consisting ofHodgkin’s lymphoma, Non-Hodgkin’s lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia, myelodysplastic syndrome, multiple myeloma, T-cell lymphoma, acute lymphocytic leukemia, and a combination thereof.

236. The method or the combination therapy of claim 235, wherein the Non-Hodgkin’s lymphoma is selected from the group consisting of B cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (B-CLL), mantle cell lymphoma, marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, and a combination thereof.

237. The method or the combination therapy of claim 235, wherein the T-cell lymphoma is peripheral T-cell lymphoma.

238. The method or the combination therapy of any one of claims 227-237, wherein the cancer is characterized by a cancer antigen present on the cancer.

239. The method or the combination therapy of claim 238, wherein the cancer antigen is a tumor antigen, a stromal antigen, or a hematological antigen.

240. The method or the combination therapy of claim 238 or 239, wherein the cancer antigen is selected from the group consisting of BCMA, CD19, CD20, CD22, FcRH5, PDL1, CD47, ganglioside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PMSA), prostate -specific antigen (PSA), carcinoembryonic antigen (CEA), Ron Kinase, c-Met, Immature laminin receptor, TAG-72, BING-4, Calcium -activated chloride channel 2, Cyclin-Bl, 9D7, Ep-CAM, EphA3, Her2 / neu, Telomerase, SAP-1, Survivin, NY-ESO-l / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gpl00 / pmell7, Tyrosinase, MC1R, p-catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, p53, Ras, TGF-B receptor, AFP, ETA, MAGE, CA-125, BAGE, GAGE, CDC27, a actinin-4, TRPl / gp75, TRP2, gangliosides, WT1, Epi-dermal growth factor receptor (EGFR), MART-2, MUC1, MUC2, MUM1, MUM2, MUM3, MSLN, NA88-1, NPM, OA1, OGT, RCC, RU11, RU12, SAGE, TRG, TSTA, Folate receptor alpha, LI -CAM, CAIX, gpA33, GD3, GM2, VEGFR, Integrins, Carbohydrates, IGF1R, TRAILR1, TRAILR2, RANKL, FAP, TGF-beta, hyaluronic acid, collagen, tenascin C, and tenascin W.

241. The method of any one of claims 89-93 and 96-240 or the combination therapy of any one of claims 95-240, wherein the first agent is administered to the subject prior to, concurrently, or after administration of the second agent to the subject.

242. A kit comprising the composition of any one of claims 1, 3-87, and 88, or the kit of any one of claims 2-86 and 88, and instructions for administering the first agent and the second agent to a human subject in need thereof to treat a disease or condition in the human subject.

243. The composition of any one of claims 1, 3-87, and 88, the kit of any one of claims 2-86 and -88, the method of any one of claims 89-93 and 96-241 or the combination therapy of any one of claims 95-241, wherein the first agent comprises a multispecific molecule comprising a first polypeptide, a second polypeptide, and a third polypeptide; wherein the first polypeptide, the second polypeptide and the third polypeptide are non-contiguous, wherein(i) the first polypeptide comprises a first portion of a dimerization module linked to a first portion of the TCRpV6-binding moiety comprising a VH of the TCRpV6-binding moiety;(ii) the second polypeptide comprises a second portion of the dimerization module, wherein the IL-2 or functional fragment or functional variant thereof is covalently linked to the second polypeptide; and(iii) the third polypeptide comprises a second portion of the TCRpV6-binding moiety comprising a VL of the TCRpV6-binding moiety.

244. The composition, kit, method, or combination therapy of claim 243, wherein the first polypeptide comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 3517, 4000, 4004, 4006, 4008, 4010, 4011, 4014, 4016 and 4018, the second polypeptide comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 3521, 4002, 4007, 4003, 4013 and 4015 and the third polypeptide comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 3518, 4005, 4009, 4012 and 4017.

245. The composition of any one of claims 1, 3-87, and 88, the kit of any one of claims 2-86 and 88, the method of any one of claims 89-93 and 96-241 or the combination therapy of any one of claims 95-241, wherein the first agent comprises a multispecific molecule comprising a first polypeptide and a second polypeptide; wherein the first polypeptide and the second polypeptide are non-contiguous, wherein the TCRpV6-binding moiety comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), or a single domain antibody, wherein(i) the first polypeptide comprises a first portion of a dimerization module linked to the TCRPV6- binding moiety; and(ii) the second polypeptide comprises a second portion of the dimerization module, wherein the IL-2 or functional fragment or functional variant thereof is covalently linked to the second polypeptide.

246. The composition, kit, method, or combination therapy of claim 245, wherein the first polypeptide comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 4019, 4021, 4023, 4025 and 4027, and the second polypeptide comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 4020, 4022, 4024, 4026 and 4028.

247. A method of activating and / or expanding T cells, wherein the method comprises:(a) administering a first agent comprising a first domain that binds to a TCR P variable (TCRpV) region on the T cells; and(b) administering a second agent comprising a histone deacetylase (HDAC) inhibitor, wherein the T cells are activated or expanded.

248. A method of preventing or reducing T cell exhaustion, wherein the method comprises:(a) Administering a first agent comprising a first domain that binds to a TCR P variable (TCRpV) region on a T cell; and(b) administering a second agent comprising a histone deacetylase (HDAC) inhibitor, wherein the administrations prevent or reduced the T cells exhaustion.

249. A method of increasing T cell cytotoxicity, wherein the method comprises:(a) administering a first agent comprising a first domain that binds to a TCR P variable (TCR V) region; and(b) administering a second agent comprising a histone deacetylase (HDAC) inhibitor, wherein the administrations increase the T cell cytotoxicity.

250. A method of increasing tumor or cancer immune infiltration, wherein the method comprises:(a) administering a first agent comprising a first domain that binds to a TCR variable (TCRpV) region; and(b) administering a second agent comprising a histone deacetylase (HDAC) inhibitor, wherein the administrations increase the tumor or cancer immune infiltration.

251. A method of promoting immune permissive tumor environment, wherein the method comprises:(a) administering a first agent comprising a first domain that binds to a TCR P variable (TCRpV) region; and(b) administering a second agent comprising a histone deacetylase (HDAC) inhibitor, wherein the administration promotes the immune permissive tumor environment.

252. The method of any one of claim 247-249, wherein the T cell is a tumor-infiltrated T cell.

253. The method of any one of claims 247-249 and 252, wherein the T cell expresses one or more ofTCRPV6-5, TCRp VI 0-1, and TCRp VI 0-2.

254. The method of any one of claim 247-249, 252, and 253, wherein the T cell is CD8+ or CD4+.

255. The method of any one of claims 247-249, and 252-254, wherein the T cell is granzyme B+.

256. The method of any one of claims 247-249, and 252- 255, wherein the T cell is IFNy+ or TNFa+.

257. The method of claim 250 or 251, wherein the immune infiltration or the immune permissive tumor environment is measured by number of CD8+ T cells or CD4+ T cells.

258. The method of claim 257, wherein the CD8+ T cells are granzyme B+.

259. The method of claim 250 or 251, wherein the immune infiltration or the immune permissive tumor environment is measured by number Ml macrophages.

260. The method of claim 259, wherein the Ml macrophages are MHCII+.

261. The method of any one of claims 250, 251, and 257-260, wherein the immune infiltration or the immune permissive tumor environment is measured by IFNy or TNFa level.

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