Multifunctional molecules and methods of treatment

Abstract

Described are multifunctional antigen-binding molecules, for example, a trispecific or bispecific antigen-binding molecule, wherein the molecule comprises i. a first antigen- binding domain that specifically binds to a tumor-associated antigen (TAA); and ii. a second antigen-binding domain that specifically binds to a yδ TCR, wherein the second antigen- binding domain binds to more than one yδ T cell subtype. Also described are pharmaceutical compositions comprising the multifunctional molecule as well as methods of treating a disease or condition such as a cancer, a B-cell mediated autoimmune disease, and a B-cell mediated inflammatory condition.

Description

[0001] MULTIFUNCTIONAL MOLECULES AND METHODS OF TREATMENT

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 665,609 filed June 28, 2024, U.S. Provisional Application No. 63 / 765,174 filed February 28, 2025, U.S. Provisional Application No. 63 / 777,097 filed March 25, 2025. U.S. Provisional Application No. 63 / 790,150 filed April 17, 2025, and U.S. Provisional Application No. 63 / 804,107 filed May 12, 2025. The entire contents of the above applications are encompassed by reference herein.

[0004] BACKGROUND OF THE INVENTION

[0005] Gamma-delta (y3) T-cells are an important and unique subset of T lymphocytes as they can recognize a broad range of antigens without antigen priming and are not major histocompatibility7complex (MHC) restricted, meaning they can go from donor to recipient without modification or MHC match. They can target and kill cells directly through their cytotoxic activity7or indirectly through the activation of other immune cell types. y5 T-cell functional responses are induced by several factors including the recognition of stress antigens, which promotes cytokine production and regulates pathogen clearance, inflammation, and tissue homeostasis in response to stress (e.g., a chemotherapeutic agent environment). The cytotoxicity of yo T-cells to tumors can be induced through the expression of cell surface receptors, including natural killer group 2D ligand (NKG2DL), on tumor cells. Thus, yo T cells are a unique subset of T lymphocytes that can directly kill malignant cells through a variety of mechanisms including the recognition of tumor and / or stress antigens that are not generally expressed on normal healthy tissue.

[0006] It would be desirable to enhance the targeting of y8 T cells to cancers, and also to enhance the efficacy of y5 T cell immunotherapy.

[0007] SUMMARY OF THE INVENTION

[0008] The present invention is based, at least partially, on the discovery that a multifunctional pan-y8 TCR engager efficiently expands multiple y5 T cell subtypes including both V51 and Vd2 subpopulations. Such expansion of y5 T cells has not been previously reported for any other T-cell engager, let alone at the magnitude described herein. The invention is also based, at least partially , on the discovery7that administration of anti- CD33 and anti-CD19 multifunctional y5 T-cell engager molecules, such as bi-specific and tri- specific y3 T-cell engager molecules, enhanced cytotoxicity of y8 T cells against target cancer cells even at low effectortarget ratios. For example, the examples show that administration of y8 T cells in combination with a multifunctional y8 T-cell engager molecule with affinity for both CD33 and y8 T-cell receptor (y5 TCR) enhanced the cytotoxicity of the y8 T cells up to 200-fold against acute myeloid leukemia (AML) cells even at low effector to target (E:T) ratios. The invention is also based, at least partially, on the discovery of a novel method for the production of an engager molecule, wherein the method comprises expressing (e.g., overexpressing) the engager molecule as a membrane-bound protein (that comprises the engager in the extracellular domain) such that the extracellular domain (or ectodomain) of the membrane-bound protein is isolated from the cell membrane, and further comprising recovering the extracellular domain.

[0009] The invention encompasses a multifunctional antigen-binding molecule, for example, a trispecific or bispecific antigen-binding molecule, wherein the molecule comprises i. a first antigen-binding domain that specifically binds to a tumor-associated antigen (TAA); and li. a second antigen-binding domain that specifically binds to a y8 T cell, or to a y8 TCR, wherein the second antigen-binding domain binds to more than one y8 T cell subtype (or to the y8 TCR of more than one y8 T cell subtype). For example, the second antigen-binding domain can bind to an epitope expressed by more than one y8 T cell subtype or to a region of a y8 TCR common to more than one y8 T cell subtype. In certain aspects, the second antigenbinding domain binds to both V81 and V82 subpopulations of y8 T cells, for example, binding to an epitope expressed by both V81 and V82 subpopulations of y8 T cells. In yet additional aspects, the second antigen-binding domain binds to another y8 T cell subtype (in addition to both V81 and V82 subpopulations of y8 T cells), for example, the epitope is also expressed by another y8 T cell subtype, or in other words. anon-V81V82 subpopulation. In yet further aspects, the second antigen-binding domain is a pan y8 TCR antigen-binding moiety. In yet further aspects, the multifunctional antigen-binding molecule comprises a third domain, for example, is a tri-specific antigen-binding molecule. The third domain can, for example, promote expansion of y8 T cells, for example, it can promote expansion of the V81 and V82 subpopulations of y8 T cells, and optionally promote expansion of one or more other y8 T cell subtypes. In certain aspects, the third domain binds or specifically binds to an epitope expressed by y8 T cells wherein the epitope is different from that which the second antigen-binding domain binds, and the binding of the third domain to the different epitope promotes the expansion of the y8 T cells. The epitope can, for example, be an epitope expressed by a y8 TCR, such as an epitope expressed by the y8 TCR of more than one y8 T cell subt pe. In certain aspects, the binding of the third domain to the different epitope promotes the expansion of the V81 and V82 subpopulations of y8 T cells. In further aspects the binding of the third domain to the different epitope promotes the expansion of the V81 and V82 subpopulations of y8 T cells and one or more other y8 T cell subty pes. In certain specific aspects, one or more antigen-binding domain is a single-chain variable fragment (scFv). In further aspects, the antigen-binding domains are attached by a linker; optionally the linker is a peptide linker. Also encompassed is a pharmaceutical composition comprising the multifunctional molecule or bispecific antigen-binding molecule described herein and a pharmaceutically acceptable excipient.

[0010] In certain aspects, the invention is directed to a multifunctional antigen-binding molecule comprising i. a first antigen-binding domain that binds to CD33, ii. a second antigen-binding domain that binds to a y8 TCR, for example, a human y8 TCR. The invention additionally includes a multifunctional antigen-binding molecule comprising i. a first antigen-binding domain that specifically binds to CD33, and ii. a second antigen-binding domain that specifically binds to a y8 TCR (e.g., a human y8 TCR). In certain aspects, the second antigen-binding domain specifically binds to the y8 TCR of more than one y8 T cell subtype or to an epitope expressed by more than one y8 T cell subtype. In certain aspects, the second antigen-binding domain binds to the y8 TCR of both the V81 and V82 subpopulations of y8 T cells, or to an epitope expressed by both V81 and V82 subpopulations of y8 T cells. In yet additional aspects, the epitope is also expressed by another y8 T cell subtype in addition to the V81 and V82 subpopulations, or in other words, the epitope is also expressed by a non- V81V82 subpopulation. In yet further aspects, the second antigen-binding domain is a pan y8 TCR antigen-binding moiety7. In yet further aspects, the multifunctional antigen-binding molecule comprises a third domain, for example, is a tri-specific antigen-binding molecule. The third domain can, for example, promote expansion of y8 T cells, for example, it can promote expansion of the V81 and V82 subpopulations of y8 T cells, and optionally promote expansion of one or more other y8 T cell subtypes. In certain aspects, the third domain binds or specifically binds to an epitope expressed by y8 T cells, wherein the epitope is different from that which the second antigen-binding domain binds, and the binding of the third domain to the different epitope promotes the expansion of the y8 T cells. The epitope can, for example, be an epitope expressed by a yb TCR, such as an epitope expressed by the yb TCR of more than one yb T cell subtype. In certain aspects, the binding of the third domain to the different epitope promotes the expansion of the V51 and Vb2 subpopulations of yb T cells. In further aspects the binding of the third domain to the different epitope promotes the expansion of the Vbl and Vb2 subpopulations of yb T cells and one or more other yb T cell subtypes. In certain specific aspects, one or more antigen-binding domains is a single-chain variable fragment (scFv). In certain specific aspects, the first and second antigen-binding domains are scFvs and the VH of the first scFv is attached to the VH of the second scFv. In additional aspects, the antigen-binding domains are attached by a linker; optionally the linker is a peptide linker. Also encompassed is a pharmaceutical composition comprising the multifunctional molecule described herein and a pharmaceutically acceptable excipient. In yet further aspects, the multifunctional antigen-binding molecule comprises a third antigenbinding domain.

[0011] The invention further includes a bispecific antigen-binding molecule comprising i. a first antigen-binding domain that binds to CD33, and ii. a second antigen-binding domain that binds to a yb TCR (e g., a human yb TCR). The invention encompasses a bispecific antigenbinding molecule comprising i. a first and gen-binding domain that specifically binds to CD33, and ii. a second antigen-binding domain that specifically binds to a yb TCR, for example, a human yb TCR. In certain aspects, the second antigen-binding domain specifically binds to the yb TCR of more than one yb T cell subtype or to an epitope expressed by more than one yb T cell subtype. In certain aspects, the second antigen-binding domain binds to the yb TCR of both the Vbl and Vb2 subpopulations of yb T cells, or to an epitope expressed by both Vbl and Vb2 subpopulations of yb T cells. In yet additional aspects, the epitope is also expressed by another yb T cell subtype, or in other words, a non- V lVb2 subpopulation. In yet further aspects, the second antigen-binding domain is a pan yb TCR antigen-binding moiety'. In certain specific aspects, each antigen-binding domain is a single-chain variable fragment (scFv). In certain specific aspects, the first and second antigen-binding domains are scFvs and the VH of the first scFv is attached to the VH of the second scFv. In additional aspects, the two antigen-binding domains are attached by a linker; optionally the linker is a peptide linker. Additionally encompassed is a pharmaceutical composition comprising the bispecific molecule described herein and a pharmaceutically acceptable excipient. The invention additionally encompasses a multifunctional antigen-binding molecule comprising i. a first antigen-binding domain that binds to CD 19, ii. a second antigen-binding domain that binds to ay8 TCR, for example, a human y8 TCR. The invention further encompasses a multifunctional antigen-binding molecule comprising i. a first antigen-binding domain that specifically binds to CD 19, and ii. a second antigen-binding domain that specifically binds to a y8 TCR (e.g.. a human y8 TCR). In certain aspects, the second antigenbinding domain specifically to the y8 TCR of more than one y8 T cell subtype, or to an epitope expressed by more than one y8 T cell subtype. In certain aspects, the second antigenbinding domain binds to the y8 TCR of both the V81 and V82 subpopulations of y8 T cells, or to an epitope expressed by both V81 and V82 subpopulations of y8 T cells. In yet additional aspects, the epitope is expressed by another y8 T cell subtype, or in other words, a non- V81V82 subpopulation. In yet further aspects, the second antigen-binding domain is a pan y8 TCR antigen-binding moiety. In yet further aspects, the multifunctional antigen-binding molecule comprises a third domain, for example, is a tri-specific antigen-binding molecule. The third domain can, for example, promote expansion of y8 T cells, for example, it can promote expansion of the V81 and V82 subpopulations of y8 T cells, and optionally promote expansion of one or more other y8 T cell subtypes. In certain aspects, the third domain binds or specifically binds to an epitope expressed by y8 T cells, wherein the epitope is different from that which the second antigen-binding domain binds, and the binding of the third domain to the different epitope promotes the expansion of the y8 T cells. The epitope can, for example, be an epitope expressed by a y8 TCR, such as an epitope expressed by the y8 TCR of more than one y8 T cell subtype. In certain aspects, the binding of the third domain to the different epitope promotes the expansion of the V81 and V82 subpopulations of y8 T cells. In further aspects the binding of the third domain to the different epitope promotes the expansion of the V81 and V82 subpopulations of y8 T cells and one or more other y8 T cell subtypes. In certain specific aspects, one or more antigen-binding domain is a single-chain variable fragment (scFv). In certain specific aspects, the first and second antigen-binding domains are scFvs and the VH of the first scFv is attached to the VH of the second scFv. In additional aspects, the antigen-binding domains are attached by a linker; optionally the linker is a peptide linker. Also encompassed is a pharmaceutical composition comprising the multifunctional molecule described herein and a pharmaceutically acceptable excipient. The invention further includes a bispecific antigen-binding molecule comprising i. a first antigen-binding domain that binds to CD 19, and ii. a second antigen-binding domain that binds to a y8 TCR (e.g., a human y8 TCR). The invention encompasses a bispecific antigenbinding molecule comprising i. a first antigen-binding domain that specifically binds to CD19, and ii. a second antigen-binding domain that specifically binds to a y8 TCR (e.g., a human y8 TCR). In certain aspects, the second antigen-binding domain specifically to the y8 TCR of more than one y8 T cell subtype, or to an epitope expressed by more than one y8 T cell subtype. In certain additional aspects, the second antigen-binding domain binds to the y5 TCR of both the V51 and V82 subpopulations of y8 T cells, or to an epitope expressed by both V81 and V82 subpopulations of y8 T cells. In yet additional aspects, the epitope is expressed by another y8 T cell subtype, or in other words. anon-V81V82 subpopulation. In yet further aspects, the second antigen-binding domain is a pan y8 TCR antigen-binding moiety. In certain specific aspects, each antigen-binding domain is a single-chain variable fragment (scFv). In certain specific aspects, the first and second antigen-binding domains are scFvs and the VH of the first scFv is attached to the VH of the second scFv. In additional aspects, the antigen-binding domains are attached by a linker; optionally the linker is a peptide linker. Also encompassed is a pharmaceutical composition comprising the bispecific antigen-binding molecule described herein and a pharmaceutically acceptable excipient.

[0012] The invention also includes a method for treating cancer a subject in need thereof, the method comprising administering to said subject an effective amount of a multifunctional antigen-binding molecule, e.g., trispecific or bispecific antigen-binding molecule, described herein. In certain aspects, the second antigen-binding domain specifically binds to the y8 TCR of more than one y8 T cell subtype or to an epitope expressed by more than one y8 T cell subtype. In certain aspects, the second antigen-binding domain binds to the y8 TCR of both the V81 and V82 subpopulations of y8 T cells, or to an epitope expressed by both V81 and V82 subpopulations of y8 T cells. In yet additional aspects, the epitope is expressed by another y8 T cell subtype, or in other words, anon-V81V82 subpopulation. In yet further aspects, the second antigen-binding domain is a pan y8 TCR antigen-binding moiety. In yet further aspects, the multifunctional antigen-binding molecule comprises a third domain, for example, is a tri-specific antigen-binding molecule. The third domain can, for example, promote expansion of y8 T cells, for example, it can promote expansion of the V81 and V82 subpopulations of y8 T cells, and optionally promote expansion of one or more other y8 T cell subtypes. In certain aspects, the third domain binds or specifically binds to an epitope expressed by y8 T cells, wherein the epitope is different from that which the second antigenbinding domain binds, and the binding of the third domain to the different epitope promotes the expansion of the y8 T cells. The epitope can, for example, be an epitope expressed by a yo TCR. such as an epitope expressed by the y8 TCR of more than one yd T cell subtype. In certain aspects, the binding of the third domain to the different epitope promotes the expansion of the V81 and V82 subpopulations of y8 T cells. In further aspects the binding of the third domain to the different epitope promotes the expansion of the V81 and V82 subpopulations of y8 T cells and one or more other y8 T cell subtypes. In certain embodiments, a population of the patient’s y8 T cells is expanded, and optionally activated, after administration of the antigen-binding molecule; for example, the expansion of a population of the patient’s y8 T cells includes V81 and V82 subpopulations of y8 T cells; for example, without transplanting or administering a population of y8 T cells (e.g., autologous or allogeneic). In certain additional aspects, a transplanted or administered population of y8 T cells (e.g., allogeneic y8 T cells) is expanded in vivo after administration of the antigenbinding molecule to the patient. In certain additional aspects, the antigen-binding molecule mediated expansion persists for hours, days, weeks or months. In further aspects, the antigenbinding molecule mediates an increase in the in vivo persistence of y8 T cells, including expanded y8 T cells. The first antigen-binding domain can, for example, specifically bind a TAA is selected from the group consisting of CD19, CD33. CD123, CD20. CD22, EGFRvIII, IL13ra2, folate receptor 1 (FOLR1), CLL-1, MMP-2, and mesothelin. In certain specific aspects, the first antigen-binding domain specifically binds CD33. In certain other aspects, the first antigen-binding domain specifically binds CD19. In certain additional aspects, the cancer is a hematologic cancer, such as leukemia. In yet additional aspects, the cancer is a solid tumor, including, but not limited to, glioma and glioblastoma.

[0013] Also encompassed herein is a method of enhancing the cytotoxicity of a population of y8 T cells, the method comprising administering to a subject suffering from cancer an effective amount of a multifunctional or bispecific antigen-binding molecule described herein. Cytotoxicity of population of y8 T cell can, for example, be enhanced by increasing cellular activation and / or increasing the level of y8 T cells. In some embodiments, the cytotoxicity of a population of the patient’s y8 T cells is enhanced. In further embodiments, the cytotoxicity of a transplanted or administered population of y8 T cells (e.g., allogeneic y8 T cells) is enhanced. In certain aspects, the second antigen-binding domain specifically binds to the yd TCR of more than one y8 T cell subtype or to an epitope expressed by more than one y8 T cell subtype. In certain additional aspects, the second antigen-binding domain binds to both V81 and V82 subpopulations of y8 T cells or to an epitope expressed by both V81 and V82 subpopulations of y8 T cells. In yet additional aspects, the epitope is expressed by another y8 T cell subtype, or in other words, a non-V81V82 subpopulation. In yet further aspects, the second antigen-binding domain is a pan y8 TCR antigen-binding moiety. In yet further aspects, the multifunctional antigen-binding molecule comprises a third domain, for example, is a tri-specific antigen-binding molecule. The third domain can, for example, promote expansion of y8 T cells, for example, it can promote expansion of the V81 and V82 subpopulations of y8 T cells, and optionally promote expansion of one or more other y8 T cell subtypes. In certain aspects, the third domain binds, for example, specifically binds, to an epitope expressed by y8 T cells, wherein the epitope is different from that which the second antigen-binding domain binds, and the binding of the third domain to the different epitope promotes the expansion of the y8 T cells. The epitope can, for example, be an epitope expressed by a y8 TCR, such as an epitope expressed by the y8 TCR of more than one y8 T cell subtype. In certain aspects, the binding of the third domain to the different epitope promotes the expansion of the V81 and V82 subpopulations of y8 T cells. In further aspects, the binding of the third domain to the different epitope promotes the expansion of the V81 and V82 subpopulations of y8 T cells and one or more other y8 T cell subtypes. In certain additional embodiments, a population of the patient’s y8 T cells is expanded, and optionally activated, after administration of the antigen-binding molecule; for example, the population of the patient’s y8 T cells includes V81 and V82 subpopulations of y8 T cells. In certain additional aspects, a transplanted or administered population of y8 T cells (e.g., allogeneic y8 T cells) is expanded in vivo after administration of the antigen-binding molecule. In further aspects, the antigen-binding molecule mediates an increase in the in vivo persistence of y8 T cells, including expanded y8 T cells. The first antigen-binding domain can, for example, specifically bind a TAA is selected from the group consisting of CD19, CD33, CD123, CD20, CD22, EGFRvIII, IL13ra2, folate receptor 1 (FOLR1), CLL-1, MMP-2, and mesothelin. In certain specific aspects, the first antigen-binding domain specifically binds CD33. In certain additional aspects, the first antigen-binding domain specifically binds CD19. In certain additional aspects, the cancer is a hematologic cancer, such as leukemia. In yet additional aspects, the cancer is a solid tumor, including, but not limited to, glioma and glioblastoma.

[0014] Also encompassed herein is a method of treating a B-cell mediated inflammatory disease or B-cell mediated autoimmune disease in a patient in need thereof comprising administering to said patient an effective amount of a multifunctional antigen-binding molecule, a trispecific antigen-binding molecule, or a bispecific antigen-binding molecule comprising i. a first antigen-binding domain that specifically binds to CD 19, and ii. a second antigen-binding domain that specifically binds to a y8 TCR (e.g., a human y8 TCR). The invention also encompasses a method for reducing the number of B cells in a patient in need thereof (for example, an autoimmune disease patient) comprising administering to said patient an effective amount of the multifunctional antigen-binding molecule, trispecific antigen-binding molecule, or the bispecific antigen-binding molecule. In certain aspects, the second antigen-binding domain specifically binds to an epitope expressed by more than one yd T cell subtype. In certain aspects, the second antigen-binding domain binds to an epitope expressed by both V81 and V82 subpopulations of y8 T cells. In yet additional aspects, the epitope is also expressed by another y8 T cell subtype, or in other words, a non-V81V82 subpopulation. In yet further aspects, the second antigen-binding domain is a pan y8 TCR antigen-binding moiety. In yet further aspects, the multifunctional antigen-binding molecule comprises a third domain, for example, is a tri-specific antigen-binding molecule. The third domain can, for example, promote expansion of y8 T cells, for example, it can promote expansion of the V81 and V82 subpopulations of y8 T cells, and optionally promote expansion of one or more other y8 T cell subtypes. In certain aspects, the third domain binds or specifically binds to an epitope expressed by y8 T cells, wherein the epitope is different from that which the second antigen-binding domain binds. The epitope can, for example, be an epitope expressed by a y8 TCR, such as an epitope expressed by the y8 TCR of more than one y8 T cell subtype. In further aspects, the binding of the third domain to the different epitope promotes the expansion of the y8 T cells, and the binding of the third domain to the different epitope promotes the expansion of the y8 T cells. In further aspects the binding of the third domain to the different epitope promotes the expansion of the V81 and V82 subpopulations of y8 T cells and one or more other y8 T cell subtypes. In certain specific aspects, one or more antigen-binding domain is a single-chain variable fragment (scFv). Nonlimiting examples of a B-cell mediated autoimmune disease include systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, antiphospholipid syndrome, pemphigus, cicatricial pemphigoid, myasthenia gravis, neuromyelitis optica, and immune thrombocytopenia. Non-limiting examples of a B-cell mediated inflammatory disease are type 2 diabetes, periodontal disease, and graft-versus-host disease.

[0015] The invention also encompasses a method of treating cancer or tumor in a subject in need thereof, the method comprising administering to said subject an effective amount of a population of y8 T cells and a multifunctional antigen-binding molecule, wherein the multifunctional molecule comprises i) an antigen-binding domain that binds to a tumor- associated antigen (TAA) and ii) an antigen-binding domain that binds to a y8 TCR (e.g., a human y8 TCR). In certain aspects, the multifunctional antigen-binding molecule is a bispecific antigen-binding molecule or a tri-specific antigen-binding molecule; optionally each antigen-binding domain is a single-chain variable fragment (scFv). In yet additional aspects, the multifunctional antigen-binding molecule, for example, a bispecific antigen-binding molecule, comprises i. a first antigen-binding domain that specifically binds to a tumor- associated antigen (TAA); and ii. a second antigen-binding domain that specifically binds to a y8 TCR, wherein the second antigen-binding domain binds to the y8 TCR of more than one y8 T cell subtype. For example, the second antigen-binding domain can bind to an epitope expressed by more than one y8 T cell subtype or a region of a y8 TCR common to the y8 TCR of more than one y8 T cell subtype. In certain aspects, the second antigen-binding domain binds to both V81 and V82 subpopulations of y8 T cells, for example, binding to an epitope expressed by both V81 and V82 subpopulations of y8 T cells. In yet additional aspects, the second antigen-binding domain binds to another y8 T cell subtype (in addition to both V81 and V82 subpopulations of y8 T cells), for example, the epitope is also expressed by another y8 T cell subtype, or in other words, a non-V81V82 subpopulation. In yet further aspects, the second antigen-binding domain is a pan y8 TCR antigen-binding moiety'. In yet further aspects, the multifunctional antigen-binding molecule comprises a third domain, for example, is a tri-specific antigen-binding molecule. The third domain can, for example, promote expansion of y8 T cells, for example, it can promote expansion of the V81 and V82 subpopulations of y8 T cells, and optionally promote expansion of one or more other y8 T cell subtypes. In certain aspects, the third domain binds or specifically binds to an epitope expressed by y8 T cells, wherein the epitope is different from that which the second antigenbinding domain binds, and the binding of the third domain to the different epitope promotes the expansion of the y8 T cells. The epitope can, for example, be an epitope expressed by a y8 TCR. such as an epitope expressed by the yd TCR of more than one yd T cell subtype. In certain aspects, the binding of the third domain to the different epitope promotes the expansion of the Vdl and Vd2 subpopulations of yd T cells. In further aspects the binding of the third domain to the different epitope promotes the expansion of the Vdl and Vd2 subpopulations of yd T cells and one or more other yd T cell subtypes. In certain specific aspects, one or more antigen-binding domains is a single-chain variable fragment (scFv). In yet further aspects, the TAA is selected from the group consisting of CD19, CD33, CD123, CD20, CD22, EGFRvIII, IL13ra2, folate receptor 1 (FOLR1), CLL-1, MMP-2, and mesothelin. In certain additional embodiments, the TAA is CD33. In further aspects, the TAA is CD 19. In yet further aspects, the population of yd T cells is an ex vivo expanded population of yd T cells. In certain additional aspects, the cancer is a hematologic cancer, such as leukemia. In yet additional aspects, the cancer is a solid tumor, including, but not limited to, glioma and glioblastoma.

[0016] The invention additionally encompasses a method of treating cancer or tumor in a subject in need thereof, the method comprising administering to said subject an effective amount of a population of yd T cells and a bispecific antigen-binding molecule, wherein the bispecific antigen-binding molecule comprises i) an antigen-binding domain that binds to a tumor-associated antigen (TAA) and ii) an antigen-binding domain that binds to a yd TCR (e.g., a human yd TCR). In certain aspects, each antigen-binding domain is a single-chain variable fragment (scFv). In yet additional aspects, the bispecific antigen-binding molecule comprises i. a first antigen-binding domain that specifically binds to a tumor-associated antigen (TAA); and ii. a second antigen-binding domain that specifically binds to a yd TCR, wherein the second antigen-binding domain binds to the yd TCR of more than one yd T cell subtype. For example, the second antigen-binding domain can bind to an epitope expressed by more than one yd T cell subtype or a region of a yd TCR common to the yd TCR of more than one yd T cell subtype. In certain aspects, the second antigen-binding domain binds to both Vdl and Vd2 subpopulations of yd T cells, for example, binding to an epitope expressed by both V l and Vd2 subpopulations of yd T cells. In yet additional aspects, the second antigen-binding domain binds to another yd T cell subty pe (in addition to both Vdl and Vd2 subpopulations of yd T cells), for example, the epitope is also expressed by another yd T cell subtype, or in other words, a non-Vdl Vd2 subpopulation. In yet further aspects, the second antigen-binding domain is a pan yd TCR antigen-binding moiety7. In certain specific aspects, each antigen-binding domain is a single-chain variable fragment (scFv). In yet further aspects, the TAA is selected from the group consisting of CD19, CD33, CD123, CD20, CD22, EGFRvIII, IL13ra2, folate receptor 1 (FOLR1), CLL-1, MMP-2, and mesothelin. In certain additional embodiments, the TAA is CD33. In further aspects, the TAA is CD19. In yet further aspects, the population of 78 T cells is an ex vivo expanded population of 78 T cells. The population of 78 T cells can, for example, be allogeneic or autologous. In certain additional aspects, the cancer is a hematologic cancer, such as leukemia. In yet additional aspects, the cancer is a solid tumor, including, but not limited to, glioma and glioblastoma.

[0017] The invention also includes a method of increasing the cytotoxicity of a population of 76 T cells to cancer cells, the method comprising administering to a subject suffering from cancer an effective amount of the population of y8 T cells, and further comprising administering to said subject a multifunctional antigen-binding molecule, wherein the multifunctional molecule comprises i) an antigen-binding domain that binds to a tumor- associated antigen (TAA) and ii) a antigen-binding domain that binds to a 78 TCR (e.g., a human 78 TCR). In certain aspects, the multifunctional antigen-binding molecule is a bispecific antigen-binding molecule; optionally one or more antigen-binding domains is a single-chain variable fragment (scFv). In additional aspects, the multifunctional antigenbinding molecule, for example, a bispecific antigen-binding molecule, comprises i. a first antigen-binding domain that specifically binds to a tumor-associated antigen (TAA); and ii. a second antigen-binding domain that specifically binds to a 78 TCR, wherein the second antigen-binding domain binds to more than one 78 T cell subtype. For example, the second antigen-binding domain can bind to an epitope expressed by more than one 78 T cell subtype or a region of a 78 TCR common to the 78 TCR of more than one 78 T cell subtype. In certain aspects, the second antigen-binding domain binds to both V81 and V82 subpopulations of 78 T cells, for example, binding to an epitope expressed by both V81 and V82 subpopulations of 78 T cells. In yet additional aspects, the second antigen-binding domain binds to another 78 T cell subtype (in addition to both V81 and V82 subpopulations of 78 T cells), for example, the epitope is also expressed by another 78 T cell subtype, or in other words, a non-V81V82 subpopulation. In yet further aspects, the second antigen-binding domain is a pan 78 TCR antigen-binding moiety. In yet further aspects, the multifunctional antigen-binding molecule comprises a third domain, for example, is a tri-specific antigen-binding molecule. The third domain can, for example, promote expansion of 78 T cells, for example, it can promote expansion of the V81 and V82 subpopulations of y8 T cells, and optionally promote expansion of one or more other y8 T cell subtypes. In certain aspects, the third domain binds or specifically binds to an epitope expressed by y8 T cells, wherein the epitope is different from that which the second antigen-binding domain binds, and the binding of the third domain to the different epitope promotes the expansion of the y5 T cells. The epitope can, for example, be an epitope expressed by a y8 TCR, such as an epitope expressed by the y8 TCR of more than one y8 T cell subtype. In certain aspects, the binding of the third domain to the different epitope promotes the expansion of the V51 and V82 subpopulations of y8 T cells. In further aspects the binding of the third domain to the different epitope promotes the expansion of the V31 and V82 subpopulations of y8 T cells and one or more other y8 T cell subtypes. In certain specific aspects, one or more antigen-binding domains is a single-chain variable fragment (scFv). In yet further aspects, the TAA is selected from the group consisting of CD19, CD33, CD123, CD20, CD22, EGFRvIII, IL13ra2, folate receptor 1 (FOLR1), CLL-1, MMP-2, and mesothelin. In certain embodiments, the TAA is CD33. In further aspects, the TAA is CD19. In yet further aspects, the population of y8 T cells is an ex vivo expanded population ofy8 T cells. The population of y8 T cells can, for example, be allogeneic or autologous. In certain additional aspects, the cancer is a hematologic cancer, such as leukemia. In yet additional aspects, the cancer is a solid tumor, including, but not limited to, glioma and glioblastoma.

[0018] In yet further aspects, the invention includes a method of increasing the cytotoxicity of a population of y8 T cells, the method comprising administering to a subject suffering from cancer an effective amount of the population of y8 T cells and further comprising administering to said subject a bispecific antigen-binding molecule, wherein the bispecific molecule comprises i) an antigen-binding domain that binds to a tumor-associated antigen (TAA) and ii) an antigen-binding domain that binds to a y8 TCR (e.g., a human y8 TCR). In certain aspects, each antigen-binding domain is a single-chain variable fragment (scFv). In yet additional aspects, the bispecific antigen-binding molecule comprises i. a first antigenbinding domain that specifically binds to a tumor-associated antigen (TAA); and ii. a second antigen-binding domain that specifically binds to a y8 TCR, wherein the second antigenbinding domain binds to more than one y8 T cell subty pe. For example, the second antigenbinding domain can bind to an epitope expressed by more than one y8 T cell subtype or a region of a y8 TCR common to the y8 TCR of more than one y8 T cell subtype. In certain aspects, the second antigen-binding domain binds to both V81 and V82 subpopulations of y8 T cells, for example, binding to an epitope expressed by both V81 and V82 subpopulations of y8 T cells. In yet additional aspects, the second antigen-binding domain binds to another y8 T cell subtype (in addition to both V81 and V82 subpopulations of 76 T cells), for example, the epitope is also expressed by another y8 T cell subtype, or in other words, a non-V81V82 subpopulation. In yet further aspects, the second antigen-binding domain is a pan y8 TCR antigen-binding moiety. In certain specific aspects, each antigen-binding domain is a singlechain variable fragment (scFv). In yet further aspects, the TAA is selected from the group consisting of CD19, CD33, CD123, CD20, CD22, EGFRvIII, IL13ra2, folate receptor 1 (FOLR1), CLL-1, MMP-2, and mesothelin. In certain embodiments, the TAA is CD33. In further aspects, the TAA is CD19. In yet further aspect, the population of y8 T cells is an ex vivo expanded population of y8 T cells. In certain additional aspects, the cancer is a hematologic cancer, such as leukemia. In yet additional aspects, the cancer is a solid tumor, including but not limited to, glioma and glioblastoma.

[0019] The invention also encompasses a nucleic acid or vector encoding a multifunctional, trispecific, or bispecific antigen-binding molecule as described herein. In certain aspects, the nucleic acid molecule further comprises a transmembrane domain and optionally, an extracellular hinge domain and / or a co-stimulatory domain. Also encompassed herein is a nucleic acid or vector encoding the bispecific antigen-binding molecule. Additionally encompassed herein is a nucleic acid or vector encoding the trispecific antigen-binding molecule.

[0020] In certain aspects, the nucleic acid or vector encodes a multifunctional, trispecific or bispecific antigen-binding molecule comprising: i. a first antigen-binding domain that binds to CD33; ii. a second antigen-binding domain that binds to y8 TCR; iii. a linker that attaches the first antigen-binding domain the second antigenbinding domain.

[0021] In additional aspects, the multifunctional or bispecific antigen-binding molecule further comprises a transmembrane domain and optionally, an extracellular hinge domain and / or a co-stimulatory domain. In yet additional aspects, the multifunctional antigen-binding molecule comprises a third domain for example, a third domain that promotes expansion of y8 T cells as described herein. In certain aspects, the nucleic acid or vector encodes a multifunctional, trispecific or bispecific antigen-binding molecule comprising: i. a first antigen-binding domain that binds to CD 19; ii. a second antigen-binding domain that binds to y8 TCR; iii. a linker that attaches the first antigen-binding domain to the second antigenbinding domain.

[0022] In certain aspects, the antigen-binding molecule further comprises a transmembrane domain and optionally, an extracellular hinge domain and / or a co-stimulatory domain. In yet additional aspects, the multifunctional antigen-binding molecule comprises a third domain for example, a third domain that promotes expansion of y8 T cells as described herein.

[0023] The invention also includes a nucleic acid encoding a membrane-bound multifunctional antigen-binding and immune cell engaging molecule, wherein the membranebound molecule comprises an extracellular domain and a transmembrane / intracellular region, wherein the nucleic acid encodes a molecule comprising: i. a first antigen-binding domain that binds to a tumor-associated antigen (TAA); ii. a second antigen-binding domain that binds to an immune cell antigen (for example, a T cell antigen or a y8 T cell antigen); iii. a linker that attaches the first antigen-binding domain to the second antigenbinding domain; and iv. a transmembrane domain; and wherein the extracellular domain comprises the antigen-binding domain that binds to the TAA, the antigen-binding domain that binds to the immune cell antigen, and the linker; and further wherein the transmembrane / intracellular region comprises the transmembrane domain. In certain specific aspects, the immune cell antigen is a y8 T cell antigen. In some examples, the second antigen-binding domain binds to the y6 TCR of more than one y5 T cell subtype, for example, an epitope expressed by more than one y8 T cell subtype. In certain examples, the second antigen-binding domain binds to an epitope expressed by both V51 and V82 subpopulations of y8 T cells. In some examples, the second antigen-binding domain is a pan y8 TCR antigen-binding moiety7. In yet additional aspects, the multifunctional antigenbinding molecule comprises a third domain, for example, a third domain that promotes expansion of y8 T cells as described herein. Also described herein is a vector comprising the nucleic acid encoding the membrane-bound multifunctional antigen-binding and immune cell engaging molecule and a host cell comprising the vector. In yet other aspects, the invention is a process for the production of a multifunctional antigen-binding molecule or bispecific antigen-binding molecule, the process comprising culturing the host cell under conditions suitable for expression and harvesting of the extracellular domain, and purifying the extracellular domain from the culture.

[0024] Also provided herein are the inventions described in the Enumerated Embodiments section below.

[0025] Each specific delineated embodiment stated above and herein can be taken in combination with one, any or all other specific delineated embodiments.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 is a schematic showing the interaction between a y8 T cell engager (TCE), e.g., a pan-y5 T cell engager, and a target cell, e.g., a hematologic tumor cell. Pan- 6 TCEs as described herein selectively activate and significantly expand both V81 and V62 y5 T cell subsets, enhancing their tumor-targeting potential. As shown in the schematic, the T-cell engager activates and directs the y8 T cells to the target cell expressing the tumor associated antigen (TAA) and results in robust activation and expansion of pan-y3 T cell subsets.

[0028] FIG. 2A is a schematic summarizing the preparation of an exemplary CD33 / y8 TCR multifunctional y8 T cell engager (33xGD engager) and use of the CD33 / y8 TCR engager in combination with expanded y8 T cells for targeted lysis of CD33-positive AML cells. In summary, an exemplary construct comprising a CD33 binding domain and y8 TCR binding domain is cloned into a lentiviral vector and the vector is used to transfect HEK-293T cells. After integration and cloning, HEK293T cells expressing the 33xGD y8 T cell engager were isolated and expanded. The CD33 / y8 TCR engager was purified and concentrated. The addition of the engager activates and directs the y8 T cells to CD33 (activated and CD33- redirected y8 T cells) on the surface of tumor cells. The activated and CD33-redirected y8 T cells are contacted with AML cells and their interaction results in release of granzyme and perforin, and targeted cell lysis. The construct in the schematic has the following elements (wherein the left of the construct corresponds to the N-terminal side): signal peptide — target / TAA binding domain— G4S linker— y8 T cell binding domain— detection tag— y8 T cell expansion domain (GDED)— P2A— selection marker; - can represent direct or indirect linkage. FIG. 2B is an exemplary map of lentiviral transfer vector for 33xGD. The construct equipped with a human EFla promoter encoding a transcript contains a signal peptide, a CD33-binding domain, a vST-cell targeting domain, a CD28 hinge domain and transmembrane domain, and a selection marker.

[0029] FIGs. 3A-3C are graphs showing cytotoxicity (%) of y8 T cells (donor 1) in combination with crude comprising the 33xGD engager C'33xGD293) or medium control cocultured with leukemia cell lines HL-60 (AML), KG- la (AML) and K562 cells (CML) at different effector to target ratios (E:T). 100 pl of 33xGD crude was added to 400 pl total of y8 T cell and target cell co-culture. The figure shows that the addition of the engager significantly enhanced the cytotoxicity of y8 T cells against AML cells HL-60 and KG- la, and had less effect on K562 CML cells likely because of low CD33 expression on K562.

[0030] FIG. 4A is a bar graph showing cytotoxicity (%) of y8 T cells in combination with different clones of stable 33xGD expression HEK293T cells (C 1 -C6) or a control (null x y8 TCR-binding domain, GD293) after 24 hours of co-culture with HL-60, KG-la, MOLM-13 and K562 cells at an E:T of 1. 100 pl of crude was added to 400 pl total of y8 T cell and target cell co-culture. The clones were established for optimal production of the 33xGD engager. The figure shows that crude from all of the different clones enhanced cytotoxicity of y8 T cells against the CD33-positive HL-60, KG-la and MOLM-13 cells, and that the cytotoxic effect of the engager was retained by different clones generated using the process described herein.

[0031] FIG. 4B is a schematic showing a multifunctional antigen-binding molecule (a y8 TCE) with a CD33-binding domain, a y8 T cell binding domain, and a third domain (‘ GDED”). “GDED” is a y8 T cell expansion domain, a domain that promotes expansion of the y8 T cells. Also shown, is a control (null y8TCE control) that lacks the CD33-binding domain (bottom).

[0032] FIG. 4C is a schematic showing a first and second antigen-binding domain, wherein the binding domains are scFv domains. An exemplary 33xGD293 engager is shown at the top, and a GD293 control which has only the y8 TCR domain is shown on the bottom.

[0033] FIG. 5 is a graph showing cytotoxicity (%) of y8 T cells treated with the 33xGD engager ('‘33xGD”) at the indicated volumes of the engager after 24 hours co-culture with HL-60, KG-la, MOLM-13 and K-562 cells; the indicated volume of the engager was added to 400 pl total of y8 T cell and target cell co-culture with an E:T of 1. The figure shows that cytotoxicity against the AML cell lines increased in a dose-dependent manner. FIG. 6 shows flow cytometric analysis of y8 T cells co-cultured with the AML cell lines HL-60, KG- la and MOLM-13 at E:T of 1 for 24 hours with or without the 33xGD engager and stained with anti-CD107A. CD107A is a marker of degranulation and release of cytotoxic granules (CGs). The light gray represents HL-60, KG-la or MOLM-13 (as indicated) and y8 T cells without engager, and the dark gray is HL-60, KG-la or MOLM-13 (as indicated) and y8 T cells with the engager. The figure shows that treatment of y8 T cells with the 33xGD engager enhances y8 T cells degranulation.

[0034] FIG. 7 shows cytokine release by 33xGD engager expanded and activated y8 T cells co-cultured with M0LM13 cells. The figure shows bar graphs of concentration of cytokine released (pg / ml) after y8 T cell treated with indicated volumes of 33xGD crude were co- cultured with M0LM13 at E:T of 1 for 24 hours using a commercial assay. The bars (going left to right) are M0LM13 only (no treatment), 0 pl 33xGD, 6 pl 33xGD, 12.5 pl 33xGD, 25 pl 33xGD, 50 pl 33xGD, 100 pl 33xGD and 200 pl 33xGD. The graph shows the concentration of cytokine released. Treatment with the engager enhanced cytokine release for many of the cytokines measured. The concentration of cytokine released was highest for IFN- y, Granzyme A, Granzyme B, Perforin and Granzyme, which are involved in y8 T cells killing ability, showing the cells have enhanced potency after treatment with the 33xGD engager, which are expressed in a dose-dependent manner.

[0035] FIG. 8 shows flow cytometric analysis of MOLM-13 cells without (left) and with (right) the purified 33xGD engager, and stained with anti-FLAG antibody and anti-CD33 antibody. The 33xGD engager was purified from a serum-free suspension culture. The figure shows that purified 33xGD engager binds to the CD33+ AML cells.

[0036] FIG. 9 shows flow cytometric analysis of y8 T cells (top) and non-y8 T cells (bottom) in a population of cells enriched for y8 T cells (and containing other immune cell types) without (left) and with (right) the 33xGD engager, and stained with anti-FLAG antibody and the anti-y8 TCR. The figure shows that the purified 33xGD engager specifically binds y8 T cells.

[0037] FIG. 10 is a graph of percent cytotoxicity over increasing concentration of CD33xGD engager (pM) for MOLM-13 (CD33+) cells after co-culture with y8 T cells. The figure shows that the CD33xGD engager induces dose-dependent cytotoxicity of y8 T cells against MOLM-13 cells after co-culture. Experiments were performed with multiple y8 T cell donors (N=3), and donor variability is reflected in the differences in EC50 (82.7 pM, 169.43 pM and 66.36 pM). EC50 values define cytotoxic efficacy. This figure shows that the CD33xGD engager enhances y8 T cell cytotoxicity against MOLM-13 (CD33+) AML cells in a dosedependent manner.

[0038] FIG. 11A is a graph of percent cytotoxicity over increasing concentration of CD33xGD engager (pM) (E:T=1; 24 h) for MOLM-13 (CD33+) cells and Raji (CD33-) cells co-cultured with yd T cells. This figure shows that the engager induces dose-dependent, target-specific killing activity.

[0039] FIG. 1 IB are graphs showing activation / degranulation markers present in cultures of MOLM-13 (CD33+) cells and Raji (CD33-) cells co-cultured with gd T cells in the presence and absence of CD33xGD engager (pM) (E:T=1; 24 h).

[0040] FIG. 12 shows flow cytometric analysis of healthy donor PBMCs alone (left panel), healthy PBMCs co-cultured with MOLM-13 cells at E:T=10 for 4 days alone (middle panel), healthy PBMCs co-cultured with MOLM-13 cells (E:T=10 for 4 days) with the CD33xGD engager (right panel). This figure demonstrates elimination of AML cells (MOLM-13 cells) that express the target CD33 by PBMCs with the addition of CD33xGD engager. Control: PBMC only at day 4 (left panel).

[0041] FIG. 13 is a schematic showing the interaction between a CD19 / y8 T cell engager (“CD19xGD engager" or CD 19xGD TCE "; both terms used interchangeably herein), a y8 T cell and a B-ALL or Lupus B cell. The CD19xGD TCE activates and directs the y8 T cells to CD19 on the surface of the B-ALL / Lupus B cell. The interaction between the activated and CD19-redirected y8 T cells with the B-ALL / Lupus B cell cells results in release of granzyme and perforin, and targeted cell lysis.

[0042] FIG. 14 is a diagram for an exemplary construct of the CD19xGD engager. The construct includes a CD8a signal peptide, a CD 19 targeting domain, a G4S linker, a y8T cell binding domain, a Flag-tag, a gd T cell expansion domain, . a P2A ribosomal-skipping peptide and a selection marker.

[0043] FIG. 15 is an exemplary vector map for the CD19xGD engager. The construct equipped with a promoter encoding a transcript contains a signal peptide, a CD19-binding domain, a y8T-cell targeting domain, a CD28 hinge domain and transmembrane domain, and a selection marker

[0044] FIGs. 16A and 16B are line graphs showing % live target cells co-cultured for 24 hours with y8T cells from two different donors as a function of the amount of CD19xGD engager (pL at E:T of 1 : 1). The target cells are CD19- MOLM-13 cells (circles) and CD19+ NALM-6 cells (squares). The graphs shows specific killing of the CD19+ target cells and specifically, that the engager enhances y8 T-cell cytotoxicity to CD19+ cells in a dosedependent manner and at a very low E:T. CD19- (circles) and CD19+ (squares) in 24 h coculture with 78 T cells at a very low E:T=1 and increasing concentration of 19xGD crude shows specific dose-dependent killing of CD19+ cells.

[0045] FIGs. 17A and 17B are microscopic images of Nalm6 cells co-cultured with 76 T cells in the absence of the CD19xGD engager (FIG. 17A) and in the presence of the CD19xGD engager. FIG. 17B shows a marked increase in cell death in the presence of the CD19xGD engager which can be seen by the reduction in cell number which is shown by an increase in negative space in 17B.

[0046] FIGs. 18A and 18B shows flow cytometric analysis of purified CD19xGD incubated with 5x105Nalm-6, MOLM-13 and expanded y8 TCR+cells and expanded ySTCR" cells. Cells were stained anti-FLAG mAb before flow analysis. 19xGD engager showed strong and selective binding to Nalm-6 (CD19+) and y8TCR+ cells, and did not bind to MOLM-13 (CD 19) and ySTCR negative cells. These figures show' that purified 19xGD TCE binds to CD19+ and y3TCR+ cells but not CD19- or non-y3T cells.

[0047] FIGs. 19A and 19B are bar graphs showing y3 T cells expanded from PBMCs obtained from two separate donors and treated with increasing concentration of purified 19xGD engager. “No Txt’" are control PBMCs not treated with the 19xGD engager or . "Zoledronate’’ are PBMCs treated with Zoledronate, which is known to expand the V32 subpopulations of y8 T cells. The figures show results on day 10, y3 T cell absolute numbers out of total cell count and (FIG. 19 A) and y3 T cell frequency within the CD45+ population (FIG. 19B). The figure show's that CD19xGD engager expands y3 T cells from PBMCs during and after the cytotoxic lysis of normal B cells, V31 and V32 subpopulations of y8 T cells as well as non-V31, V32 subpopulations had expanded. Zoledronate (positive control) expanded primarily V82+ cells from PBMCs, as expected. y8 T cells from PBMCs without the CD19xGD engager (“No Txt”; negative control) did not expand. No other T-cell engager has been shown to drive significant expansion and proliferation of y3 T cells.

[0048] FIG. 20A is a graph of percent cytotoxicity over increasing concentrations of CD19xGD engager (pM) for Nalm6 (CD19+) cells. The figure shows that the CD19xGD engager induces dose-dependent, CD19-specific cytotoxicity of y3 T cells against NALM-6 cells, and that the enhanced cytotoxicity is induced at very low concentrations of the engager. Experiments w ere performed with PBMCs from multiple donors (N=3), and donor variability is reflected in the differences in EC50 (26.18 pM, 29.47 pM and 51.74 pM). EC50 values define cytotoxic efficacy.

[0049] FIG. 20B is a graph of percent cytotoxicity (normalized) over increasing concentration of CD19xGD engager (pM) for Nalm6 (CD19+) cells and MOLM-13 (CD19-) cells. The figure shows that the engager did not induce cytotoxicity of 78 T cells against MOLM-13 cells, which do not express CD19. This confirms that the engager induces CD19- specific cytotoxicity of the target cells by 78 T cells. These figures show that the engager induces potent and target-specific 78 T cytotoxicity.

[0050] FIG. 21 are graphs showing the upregulation of CD 107a. CD69, and PD-1 for 78 T cells alone, no treatment (No Txt), and increasing concentration of the engager. The figure shows that the CD19xGD engager dose-dependently upregulates the activation markers CD69 and CD107a, and also upregulates PD-1 to a lesser degree, after treatment (N=3).

[0051] FIG. 22 is a heatmap showing the levels of selected secreted cytokines and cytotoxic molecules in the supernatants of cocultures of Nalm6 (B-ALL) cells and 78 T cells from 3 different donors exposed to various concentrations of the 19xGD engager (“TCE”) and controls as indicated. The fold-change of cytokine release was highest for TNF-a and IFN-y, and the engager enhanced cytokine release for these enzymes in a dose-dependent manner.

[0052] FIG. 23 shows flow cytometric analysis of 78 T cells and B cells in peripheral blood samples obtained from systemic lupus erythematosus (SLE) donors with active disease. As shown in the figure, there were low levels of 78 T cells and high levels of B cells, representative of chronic autoimmune disease.

[0053] FIGs. 24A, 24B and 24C are graphs showing the relative percentages of live B cells and 78 T cells over time (days) for cultures prepared from three SLE donor PBMC samples treated with the 19xGD engager. Each donor sample showed 78 T cell expansion (squares) and depletion of B cells (circles). The depletion accelerates as the 78 T cells expand suggesting that the expansion is important for the ultimate near-complete depletion of the B cells. These figures show that the engager drives B cell depletion and 78 T cell expansion in SLE donor sample culture.

[0054] FIG. 25 is a graph showing the change (in folds) over no treatment for the 78 T cell cytotoxic activation marker CD107A over time (days) in 19xGD engager treated cultures prepared from three SLE donor peripheral blood samples. The figure show s the degranulation of 78 T cells upon treatment with the engager as measured by surface expression of CD107A on both V81T cells (green / gray squares) and V82T cells (blue / black squares activation markers increase during B cell depletion. V81+ (green / gray squares) and V82+ cells (blue / black squares), subtypes undergo degranulation / activation and this further demonstrates that the increases follow the trajectories of B cell depletion in all three donors.

[0055] FIGs. 26A and 26B are graphs showing expression (logio ng / ml; culture supernatant at Day 1 was compared to Day 8) of IgG and IgM antibodies in cultures prepared from three SLE donor peripheral blood samples treated with 19xGD engager (5 nM) and control cultures (no treatment: 0 nM). The depletion of B cells in all three donors by 19xGD engager treatment was further confirmed by this data showing the loss of IgG (left) and IgM (right) antibodies in an ELISA test performed with culture supernatants from Day 8.

[0056] FIGs. 27A and 27B are representative bar graphs showing the absolute number of 78 T cells expanded from PBMCs obtained from a healthy donor (left) and a SLE donor (right) treated with increasing concentration of purified 19xGD engager on Day 10. No engager (No Txt) and Zoledronate serve as controls. The figures shows that the engager induces pan -78 T cell expansion in both healthy and SLE donors.

[0057] FIG. 28 are graphs showing percent B cells for two donors treated with serial dilutions (7 total) of the CD19xGD engager, zoledronate, or no treatment. Serial dilutions of the CD19xGD engager showed dose-dependent B cell killing of PBMCs from two separate healthy donors using only the unmanipulated 78 T cells present in the PBMCs as compared to PBMC without the CD19xGD engager and PBMC with y8 T cell stimulation (Zoledronate) demonstrating complete ablation of the B cells only when in combination with the engager at day 8.

[0058] FIG. 29 are bar graphs showing expansion and activation (CD95+) of y8 T cell subsets from PBMCs treated with 20 nM CD19xGD engager, 5 nM CD19xGD engager and no engager. Circles are total expanded V81 and V82 out of CD45 and rectangles are total CD95+ V81 and V82 out of CD45. This figure shows that the engager expands and activates both the V81+ and V82+ y8 T cell subsets from PBMCs. The CD19xGD engager activates both Vdl+ and Vd2+ T cells as shown by the expression of CD95 (FAS) from PBMCs. No significant expansion or activation is seen without the CD19xGD engager ('‘No Txt”) over 10-day culture of PBMCs.

[0059] FIG. 30 are bar graphs showing expansion and activation (CD95+) of V81+ and V82+ y6 T cell subsets from PBMCs. This figure shows that the CD19xGD engager expands V82+ cells within physiologic ranges. The CD19xGD engager expands V82+ cells within the same range as Zoledronate, which is known to expand V82+ cells and is approved for some medical uses in patients as a therapeutic and is known to be safe. Suggesting that the level of expansion from Zoledronate does not result in patient toxicities. The CD19xGD engager. The CD19xGD engager induces V82+ cell expansion without off-target effects that can occur from broad cytokine stimulation, and provides a potentially safer and targeted way to expand and activate y8 T cells.

[0060] FIG. 31 are graphs showing that the CD19xGD engager drives B cell depletion and y8 expansion in PBMCs from SLE donor with active disease. Each SLE donor demonstrates y8 T cell expansion (top) and depletion of B cells (bottom). The depletion accelerates as yS T cells expand suggesting that the expansion may be important for the efficient and ultimate near-complete depletion of the B cells.

[0061] DETAILED DESCRIPTION OF THE INVENTION

[0062] Various bi-specific T-cell engagers (TCEs) have been described in the literature for redirecting T cells’ cytotoxic activity to target tumors. Many such TCEs are engagers that target the CD3 receptor (including, for example, an anti-CD3 scFv) and are therefore pan T- cell engagers. CD3-TCEs activate all T cells indiscriminately, which can lead to several challenges, including cytokine release syndrome (CRS), T cell exhaustion, and activation of regulatory T cells (Tregs). TCEs that specifically target y8 T cells have also been described, for example, targeting the V82 subpopulation. Limitations associated with these therapies include cancer-associated depletion of y8 T cells as well as T-cell exhaustion, which reduces the effectiveness of current yS T cell-based therapies due to the extremely low levels of yS T cells in cancer patients or patients with some chronic illnesses. In these instances, the low levels of y8 T cells have been insufficient and, in the absence of yS T cell targets to engage, the therapies are unable to effectively target and eradicate the intended cancer or TAA of interest. Notably, y8 T cell levels decline in patients as cancer progresses and these low levels combined with the lack of expansion of y8 T cells poses a significant impediment to the effectiveness of previous y8 TCEs in the clinic. In contrast, the multifunctional, trispecific and bispecific pan-y8 TCR engagers described herein drive expansion and proliferation of multiple y8 T cells subtypes, including V81 and V82 subpopulations of y8 T cells, as well as non-V81V82 y8 T cell subpopulations. Such expansion, let alone at this magnitude, has not been previously reported for any other T-cell engager (TCE), therefore the antigen-binding molecules and methods described herein provide the potential for deeper and more durable responses. The experimental work described herein shows that the multifunctional antigen- binding molecule expands yd T cells present in PBMCs and that the yd T cells expanded from the PBMCs can exert cytotoxic effects against target cells. Therefore the methods described herein encompass administration of the multifunctional antigen-binding molecule, wherein the antigen-binding molecule expands the patient's own yd T cells in vivo, without administering or transplanting a cell product comprising yd T cells, e.g., autologous or allogeneic yd T cells. The multifunctional, trispecific, and bispecific yd T cell engagers described herein can leverage both the antigen presenting properties of Vd2+ T cells and the longer-term resistance against exhaustion and tissue residence properties of Vdl+ T cells. The multifunctional engagers described herein can potentially be used in the treatment of patients with low levels of yd T and / or treatment of patients without co-administration of IL-2 and / or without co-administration of yd T cells. The multifunctional engagers described herein can, for example, be used to recruit, activate and expand yd T cells in vivo at the site of the target cells (that expresses the antigen), e.g., tumor cells. For example, the CD19-directed multifunctional, trispecific, and bispecific engagers described herein can be used in the treatment of cancer or autoimmune disease, potentially providing a treatment that can overcome disease-mediated yd T cell exhaustion.

[0063] Unless defined otherwise, 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 invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. As used in the specification and the appended claims, the singular forms “a,” “an,’' and “the"’ include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell" includes a plurality of cells. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

[0064] It should be noted that ratios, concentrations, doses, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of “about 0. 1% to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt. % to about 5 wt. %, but also include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range. The term “about” can include ±1%. ±2%. ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%, or more of the numerical value(s) being modified. In addition, the phrase “about ‘x’ to ‘y’” includes “about ‘x’ to about ‘y’”. Numbers, ratios, concentrations, amounts, ranges and other numerical data should be construed as modified by the term “about” unless inconsistent with the context.

[0065] By “administration” is meant introducing a compound, biological materials including a cell population, or a combination thereof, or a composition comprising any of the aforementioned compounds, biological materials (e.g., a cell population), or a combination thereof, of the present invention into a human or animal subject. One preferred route of administration of the compounds is intravenous. Another preferred route is parenteral. "Parenteral" refers to a route of administration that is associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrastemal, intrathecal, intracranial, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Other exemplary routes of administration of the compounds may be intraperitoneal or intrapleural, or via a catheter to the brain. However, any route of administration, such as oral, topical, subcutaneous, peritoneal, intra-arterial, inhalation, vaginal, rectal, nasal, introduction into the cerebrospinal fluid, intracranial, or instillation into body compartments can be used. Direct injection into a target tissue site such as a solid tumor is also contemplated. For example, intracranial administration of the y8 T-cells for the treatment of a glioma or other intracranial tumor is encompassed.

[0066] The term “y5 T-cells.” “y3 T cells,” “gd T-cells,” “gd T cells,” and “gamma delta T- cells” as used herein refers to a subset of T-cells that express a distinct T-cell receptor (TCR) on their surface. The majority of T-cells have a TCR composed of two glycoprotein chains called a- and P-TCR chains (“a.p T cells”). In contrast, in y8 T-cells, the TCR is made up of one y-chain and one 5-chain. This group of T-cells is much less common than aP T-cells. The y5 T-cell is classified as an innate immune cell but sits at the nexus of the immune system with properties of both adaptive T-cells and innate immune cells. y3 T-cells are unique amongst T-cell types in that they do not require antigen processing and MHC presentation of peptide epitopes in order to identify and kill unhealthy cells. y5 T-cells are also not MHC-restricted, meaning they can go from a donor to a recipient without inducing graft versus host disease, or GvHD, from MHC mismatch. Furthermore, y5 T-cells are believed to have a prominent role in recognition of lipid antigens, cell-surface antigens and to respond to stress-related antigens such as MIC-A and MIC-B and other ligands of the NKG2D receptor. Upon recognition of an unhealthy cell, they can kill directly through perforin and granzyme, in a manner similar to an NK cell. y8 T cells are classified in different types based on how the TCR chains are grouped together, as there are several different 5 chains: V51, V52, V83, and V85; and seven different y chains Vy2, 3, 4, 5, 8. 9, and 11 in humans. Subsets of human y5 T cells include, for example, V51, V82, V83 and V55 y8 T cells. Specific examples of V81 y8 T cells include, but are not limited to, Vy2V81, Vy3V81, Vy4V61, Vy5V61, Vy8V81, and Vy9V81 y8 T cells. V82 y8 T cells include Vy9V82 y8 T cells and Vy2V82 y8 T cells. Specific examples of V83 y8 T cells are Vy2V83 and Vy3V82 y8 T cells. Specific examples of V85 y8 T cells are Vy4V85 y8 T cells. In humans, V81 and V82 y8 T-cells are the two main subpopulations of y8 T-cells as based on their TCR expression. V82 y8 T-cells are ty pical ly circulating lymphocytes and constitute the majority of peripheral blood y8 T-cells and perform immune surveillance. However, upon sensing cellular stress or damage, they can infiltrate into tissues to seek out and kill the damaged or cancerous cells. Meanwhile, V81 y8 T-cells are generally tissue-resident lymphocytes, abundant in mucosal surfaces and epithelia of the digestive, respiratory and urogenital tracts (Caron et al. (2021), Front Immunol, https: / / doi.org / 10.3389 / fimmu.2021.666983). y8 T cells include Vy9V82 T-cells and V81 T-cells. In certain aspects, the y8 T cells in the population of y8 T cells comprise or consist of V81 y8 T-cells. In other aspects, the y8 T cells in the population of y8 T cells comprise or consist of V82 y8 T-cells. In further aspects, the y8 T cell population comprises V81 T-cells and V82 y8 T-cells. In certain examples, the y8 T cell population comprises a greater proportion of V81 y8 T-cells than V82 y8 T-cells. In yet other aspects, y8 T cell population comprises a greater proportion of V82 y8 T-cells than V81 y8 T- cells. A non-V81V82 subpopulation can include a V83 y8 T or V85 yS T cells, or a combination thereof.

[0067] Human y8 T-cells can also exhibit an antigen-presenting capacity. Similar to dendritic cells (DCs), blood Vy9V82 T-cells are able to respond to signals from microbes and tumors and prime CD4+and CD8+T-cells. y8 T-APCs are believed to cross-present antigens directly to CD8+T-cells. The intracellular protein degradation and endosomal acidification are significantly delayed in y8 T-cells in comparison to monocyte-derived DCs. The antigens are transported across IRAP (Insulin-Regulated Amino Peptidase)-positive early and late endosomes, and their processing consists of an export to the cytosol for degradation by the proteasome before being imported into an MHC-I-loading compartment. Activated y5 T-cells are able to phagocytose tumor antigens and apoptotic or live cancer cells possibly through the scavenger receptor CD36 in a C / EBPa (CCAAT / enhancer-binding protein a)-dependent mechanism and mount a tumor antigen-specific CD8+T-cell response. y3 T-cells can also induce DC maturation through TNF-a production. Overall, y3 T-cells can process a wide range of antigens for presentation and stimulate other immune cells. Therefore. y8 T-cells' role in response to infections or cancer may be leveraged to design new strategies in order to improve clinical response of human y8 T-cell-based immunotherapy. Increased tumor immunogenicity (e.g., increased upregulation of ligands for the NKG2D receptor), e.g., resulting from a chemotherapeutic agent or DDR inhibition (as described, for example, in W02020097306 and U.S. Pat. Nos. 10,322,145 and 12.005,078) is uniquely conducive to yo T-cell-mediated tumor immunosurveillance, and ultimately tumor cell killing by yo T-cells.

[0068] A composition, y8 T cell product, cell composition or population of y8 T cells can be enriched for the y5 T-cells, for example. The term “enriched,"’ as used herein, refers to increasing the total percentage of one or more cytotoxic immune cell types present (e.g.. y6 T-cells and / or NK cells) in a sample, relative to the total percentage of the same one or more cell types prior to enrichment, as disclosed herein. For example, a sample that is “enriched” for one or more types of cytotoxic immune cell may comprise between about 10% to 100% of the one or more cytotoxic immune cell types in the sample, whereas the total percentage of one or more of the cytotoxic immune cell types in a sample prior to enrichment was, for example, between 0% and 10%. Preferably, an enriched sample comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, of one or more types of cytotoxic immune cell. Samples may be enriched for one or more cell types using standard techniques, for example, flow cytometry techniques. The term “highly enriched”, as used herein, refers to increasing the total percentage of one or more cytotoxic immune cell types in a sample such that the one or more cytotoxic immune cell types may comprise between at least about 70% to about 100% of the cytotoxic immune cell type in the sample, whereas the total percentage of that same type of cytotoxic immune cell prior to enrichment was, for example, between 0% and 10%. Preferably, a highly enriched sample comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of one or more types of cytotoxic immune cell. Samples may be highly enriched for one or more cell types using standard techniques, for example, flow cytometry techniques. As used herein, the “y8 T cell product'’ is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and expanded and optionally activated 78 T cells (e.g., allogeneic or autologous), wherein the 78 T cell product is depleted in ap T cells. The 78 T cell product or the expanded population of y3 T cells can thus be enriched in y8 T cells and depleted in o. T cells. The y8 T cell product or population of yS T cells (for example, to be administered) can be prepared, for example, by collecting peripheral blood sample or an apheresis product from a donor, and expanding and activating the population of y8 T cells ex vivo therefrom. In certain aspects, the y8 T cell product is allogeneic. The y8 T cell product can also be expanded from patient’s PBMC, for example, for autologous therapy. Methods of preparing a y8 T cell product and for expanding and activating y8 T-cells have been described, for example, in WO 2017035375 Al and U.S. Pat. No. 7,078,034, incorporated herein by reference in their entireties.

[0069] The y8 T cell product can be prepared by expanding and optionally activating a population of y8 T cells ex vivo. The terms ‘"expanded” and “expanding” and the like as used herein mean expansion of one or more cytotoxic immune cells in a sample means to increase in the number of one or more cytotoxic immune cells in a sample by, for example about at least 2-fold, preferably by about 5-fold, preferably by at least 10-fold, preferably about at least 50-fold or more. Expansion of a cytotoxic immune cell population can be accomplished by any number of methods as are known in the art. For example, T-cells can be rapidly expanded using non-specific T-cells receptor stimulation (optionally, in the presence of feeder lymphocytes) and either interleukin-2 (IL-2) or interleukin- 15 (IL-15), with IL-2 and / or zoledronate being preferred. The non-specific T-cell receptor stimulus can, for example, include, a mouse monoclonal anti-CD3 antibody (available from ORTHOMCNEIL®, Raritan, N.J.). Alternatively T-cells can be rapidly expanded by stimulation of an apheresis sample or peripheral blood mononuclear cells (PBMC) in vitro or ex vivo with one or more antigens (including antigenic portions thereof, such as epitope(s), or a cell) of the cancer, which can be optionally expressed from a vector, such as an human leukocyte antigen A2 (HLA-A2) binding peptide, e g., 0.3 pM MART-1 (26-35,27 L) or gplOO (209- 217,210M), in the presence of a T-cell grow th factor, such as IL-2 or IL-15 (e.g., at about 300 lU / ml). with IL-2 being preferred. Methods of expanding y8 T-cells, for example, from an apheresis sample, have been described, for example, in WO2017035375 and WO2011053750; the contents of which are expressly incorporated by reference herein. As described herein, the antigen-binding molecules described herein can enhance or induce in vivo expansion of a population of y3 T cells. “Expansion / ’ “expanding.” “expand,” and the like, in the in vivo context refers to the growth or proliferation of the cells in vivo, for example of administered y8 T cells or a patient’s own y8 T cells, after administration of the multifunctional antigen-binding molecule to the recipient. The expansion can be of administered y8 T cells or the cellular therapy product (e g. the y8 T cell product) and / or the patient's own y8 T cells as compared to that at baseline or as compared to that in the absence of the antigen-binding molecule: wherein baseline is before administration of the antigenbinding molecule described herein or prior to initiating the treatment comprising the antigenbinding molecule described herein. For example, as described herein, a population of the patient's y8 T cells (or in other words, y8 T cells in the patient’s circulation) can be expanded in vivo using an antigen-binding molecule described herein. A population of the patient’s y6 T cells is expanded, for example, when there is an increase in the number of y8 T cells per milliliter of peripheral blood as compared to that over baseline. A population of transplanted or administered y6 T cells (e.g., allogeneic or autologous) is expanded, for example, when there is an increase in the number of y8 T cells per milliliter of peripheral blood after administration. In certain embodiments, the population of the patient's y8 T cells or transplanted or administered y8 T cells is expanded such that the circulating y6 T cells as measured in a peripheral blood sample obtained from the patient is at least about 10%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or greater than that at baseline. In yet further aspects, in vivo expansion is promoted, induced or increased such that circulating y5 T cells as measured in a peripheral blood sample obtained from the patient is at least about 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, or 50 times more than that at baseline.

[0070] In certain aspects, the in vivo persistence of y5 T cells, for example, of a population of the patient’s y8 T cells or a transplanted or administered population of y8 T cells, is enhanced or increased using an antigen-binding molecule described herein. “Persistence” and in vivo persistence in the context of the present invention refers to the survival or maintenance of the cells in the patient after administration of the cellular therapy product (e.g., the y8 T cell product) to the recipient patient and / or the patient’s own circulating y8 T cells as compared to that at baseline. In certain aspects, the antigen-binding molecules described herein increase in vivo persistence and expansion of a population of 78 T cells. Increasing “the in vivo persistence and expansion of 78 T cells” or increasing “persistence and expansion” in the in vivo context refers to increasing the persistence and / or expansion of the 78 T cells after infusion or administration of the engager or practicing the methods described herein. In certain aspects, the increase in in vivo persistence is such that there are at least 10,000 76 T cells / ml in a peripheral blood sample obtained from the patient days, weeks, or months after initiating the treatment. In yet other aspects, there is an increase in in vivo persistence when the expanded 78 T cells persist for at least about 30 days, about 60 days, about 100 days, about 180 days, about 365 days, about 18 months, or about 2 years.

[0071] The 78 T-cells of the y8 T cell product or composition can also be derived from human induced pluripotent stem cells (hiPSCs). The pluripotent stem cells can. for example, be isolated from the patient having the cancer. In other aspects, the pluripotent stem cells may be isolated from a source other than the patient with cancer. The optionally enriched and / or optionally expanded compositions comprising 78 T-cells can also comprise natural killer (NK) cells and optionally further comprise other immunocompetent cells including, but not limited to monocytes, macrophages and dendritic cells. Methods for generating y8 T- cells from induced pluripotent stem cells have been described, for example, in W02023200761, Watanabe et al. 2017, Stem Cells Transl Med 7(1): 34-44, Zeng et al. (2019), PLoS One 14(5): e0216815; the contents of each of which are expressly incorporated by reference herein.

[0072] The terms “isolated’ and “isolated population” of cells as used herein refers to a cell or a plurality of cells removed from the tissue or state in which they are found in a subject. The terms may further include cells that have been separated according to such parameters as, but not limited to, cell surface markers, a reporter marker such as a dye or label.

[0073] The y8 T-cells are present in the y8 T cell product or in the transplanted or administered population of y8 T cells (e.g., the transplanted population of y8 T cells) at greater than or equal to about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the total cell population, for example, as determined by flow cytometry. In yet further aspects, the y8 T-cells are present in the 78 T cell product or in the population of 78 T cells at greater than or equal to about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the total viable cell population, for example, as determined by flow cytometry. In certain embodiments, the composition comprises y8 T-cells and NK cells, wherein the y8 T-cells are present at greater than or equal to about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the total cell population or the total viable cell population and the NK cells are present at less than or equal to about 35%, 30%, 25%, or 20% (for example, as determined by flow cytometry). In additional aspects, the composition comprises the aP T-cells at less than or equal to 10% of the total cell population or the total viable cell population, for example, as determined by flow cytometry. In additional aspects, the composition comprises the a.p T-cells at less than or equal to 5% of the total cell population or the total viable cell population, for example, as determined by flow cytometry. In certain embodiments, the composition comprises the yd T-cells and o.p T-cells, wherein the yd T-cells are present at greater than or equal to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the total cell population or the total viable cell population, for example, as determined by flow cytometry and wherein the composition comprises aP T- cells at less than or equal to 10% of the total cell population or the total viable cell population, for example, as determined by flow cytometry. In yet additional aspects, the composition comprises the aP T-cells at less than or equal to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the total cell population or the total viable cell population, for example, as determined by flow cytometry. In certain embodiments, the composition comprises the yd T- cells, aP T-cells and NK cells, wherein the yd T-cells are present at greater than or equal to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the total cell population or the total viable cell population, for example as determined by flow cytometry, the aP T- cells are present at less than or equal to 10%, 9%, 8%, 7%, 6% or 5% of the total cell population or the total viable cell population, and the NK cells are present at less than or equal to about 35%, 30%, 25%, or 20% of the total cell population or the total viable cell population, as determined by flow cytometry. In yet additional aspects, the yd T cell product or the population of yS T cells comprises the y5 T-cells, aP T-cells and NK cells, wherein the yd T-cells are present at greater than or equal to 60%, the aP T-cells are present at less than or equal to 5%, and the NK cells are present at less than or equal to about 30% of the total cell population or the total viable cell population, as determined by flow cytometry. In yet additional aspects, the y8 T cell product or the population of yd T cells comprises the yd T- cells, aP T-cells and NK cells, wherein the yd T-cells are present at greater than or equal to 60%, the aP T-cells are present at less than or equal to 5%, and the NK cells are present at less than or equal to about 25% of the total cell population or the total viable cell population, as determined by flow cytometry. In certain additional aspects, the yd T cell product or the population of yd T cells comprises the yd T-cells, aP T-cells and NK cells, wherein the yd T- cells are present at greater than or equal to 70%. the aP T-cells are present at less than or equal to 3%, and NK cells are present at less than or equal to 30% of the total cell population or the total viable cell population, as determined by flow cytometry. In certain additional aspects, the y8 T cell product or the population of y8 T cells comprises the yS T-cells, a T- cells and NK cells, wherein the y8 T-cells are present at greater than or equal to 90%, the aP T-cells are present at less than or equal to 3%. and NK cells are present at less than or equal to 7% of the total cell population or the total viable cell population, as determined by flow cytometry. In a further aspect, the y8 T cell product or the population of y8 T cells comprises the y8 T-cells, aP T-cells and NK cells, wherein the y8 T-cells are present at greater than or equal to 95%, the aP T-cells and are present at less than or equal to 2%, and NK cells are present at less than or equal to 3% of the total cell population or the total viable cell population, as determined by flow cytometry'. In yet additional embodiment, the y8 T cell product or the population of y8 T cells comprises the y8 T-cells, aP T-cells and NK cells, wherein the y5 T-cells are present at greater than or equal to 99%, and the o.p T-cells and the NK cells together are present at less than or equal to 1% of the total cell population or the total viable cell population, as determined by flow cytometry.

[0074] As used herein any form of administration of a combination of agents, compounds, or compositions, ‘'combined therapy’’ and / or '‘combined treatment regimen,” or '‘coadministration” or '‘co-administering,” or the like, refers to administration of at least two therapeutically active drugs or compositions (e.g., administration or transplantation of the y8 T-cells and a bispecific antigen-binding molecule, or pharmaceutical compositions thereof), simultaneously or substantially simultaneously in either separate or combined formulations, or sequentially at different times separated by minutes, hours, days, weeks, or months, but in some way act together to provide the desired therapeutic response, for example, as part of the same treatment regimen.

[0075] The terms “enhance.” “enhancing.” “enhanced,” or the like, as used herein, refers to an increase in the response or outcome referred to. For example, “enhancing cytotoxicity” refers to increasing cytotoxicity. An enhanced response can comprise an increase in responsiveness (e.g., cytotoxicity) of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%. 45%. 50%. 55%. 60%. 65%. 70%. 75%. 80%. 85%. 90%. 95% or 98% or more.

[0076] As used herein, the phrase '‘enhancing cytotoxicity of a population of y8 T cells, ” “increasing cytotoxicity of a population of y8 T cells,” “enhancing cytotoxicity of a of y8 T cell product,” “increasing cytotoxicity of a y8 T cell product,” and the like, with respect to activity of the engager (e.g., the multifunctional antigen-binding molecule, the trispecific antigen-binding molecule, or the bispecific antigen-binding molecule as described) is intended to mean enhancing or increasing the targeting and killing efficacy of the yS T cells and / or increasing the potency of the y5 T cells. “Killing efficacy’' is the ability of the y8 T cell to identify and kill target or tumor cells.

[0077] The terms “reduce,” “reducing,” “reduced,” or the like, as used herein, refers to a decrease in the response or outcome referred to. For example, “reduced cytotoxicity” to a normal, non-cancerous cell refers to a decreased cytotoxicity to that normal, non-cancerous cell, for example, as compared with the cytotoxicity to a cancer cell. A reduced cytotoxicity can comprise a decrease in cytotoxicity of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more.

[0078] The term “cancer,” as used herein, shall be given its ordinary meaning, as a general term for diseases in which abnormal cells divide without control. Cancer cells can invade nearby tissues and can spread through the bloodstream and lymphatic system to other parts of the body. When normal cells lose their ability to behave as a specified, controlled and coordinated unit, a tumor is formed. Generally, a solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas (some brain tumors do have cysts and central necrotic areas filled with liquid). A single tumor may even have different populations of cells within it. with differing processes that have gone awry. Solid tumors may be benign (not cancerous), or malignant (cancerous). Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors. Carcinoma is cancer that begins in the skin or in tissues that line or cover internal organs. Glioma is a tumor that arises from the supportive (“gluey“) tissue of the brain, called glia, which helps to keep the neurons in place and functioning well. Sarcoma is cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the bloodstream. Lymphoma is cancer that begins in the cells of the immune system.

[0079] Representative cancers include, but are not limited to, Acute Lymphoblastic Leukemia (ALL), Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia (AML), Adult; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS-Related Lymphoma; AIDS-Related Malignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma / Malignant Fibrous Histiocytoma; Glioblastoma, Childhood; Glioblastoma, Adult; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Cerebellar Astrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma / Malignant Glioma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor, Medulloblastoma, Childhood; Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors. Childhood; Brain Tumor, Visual Pathway and Hypothalamic Glioma, Childhood; Brain Tumor, Childhood (Other); Breast Cancer; Breast Cancer and Pregnancy; Breast Cancer, Childhood; Breast Cancer, Male; Bronchial Adenomas / Carcinoids, Childhood: Carcinoid Tumor, Childhood; Carcinoid Tumor, Gastrointestinal; Carcinoma, Adrenocortical; Carcinoma, Isletcell; Carcinoma of Unknown Primary; Central Nervous System Lymphoma, Primary; Cerebellar Astrocytoma, Childhood; Cerebral Astrocytoma / Malignant Glioma, Childhood; Cervical Cancer; Childhood Cancers; Chrome Lymphocy tic Leukemia; Chronic Myelogenous Leukemia; Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of Tendon Sheaths; Colon Cancer; Colorectal Cancer, Childhood; Cutaneous T-Cell Lymphoma; Endometrial Cancer; Ependymoma, Childhood; Epithelial Cancer, Ovarian; Esophageal Cancer; Esophageal Cancer, Childhood; Ewing's Family of Tumors; Extracranial Germ. Cell Tumor. Childhood; Extragonadal Germ. Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer. Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma. Childhood Brain Stem; Glioma. Childhood Visual Pathway and Hypothalamic: Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin's Lymphoma, Adult;

[0080] Hodgkin's Lymphoma, Childhood; Hodgkin's Lymphoma During Pregnancy; Hypopharyngeal Cancer; Hypothalamic and Visual Pathway Glioma, Childhood; Intraocular Melanoma; Islet-cell Carcinoma (Endocrine Pancreas); Kaposi's Sarcoma; Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia, Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood; Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood; Leukemia, Chrome Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer. Adult (Primary); Liver Cancer, Childhood (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphoblastic Leukemia, Adult Acute; Lymphoblastic Leukemia, Childhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma. AIDS -Related; Lymphoma. Central Nervous System (Primary); Lymphoma, Cutaneous T-Cell; Lymphoma, Hodgkin's, Adult; Lymphoma, Hodgkin's; Childhood; Lymphoma, Hodgkin's During Pregnancy; Lymphoma, Non-Hodgkin's, Adult; Lymphoma, Non-Hodgkin's, Childhood; Lymphoma, Non-Hodgkin's During Pregnancy; Lymphoma, Primary Central Nervous System; Macroglobulinemia, Waldenstrom's; Male Breast Cancer; Malignant Mesothelioma. Adult; Malignant Mesothelioma, Childhood; Malignant Thymoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular; Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous Neck Cancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma / Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplasia Syndromes; Myelogenous Leukemia, Chrome; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Nasophary ngeal Cancer, Childhood; Neuroblastoma; Neurofibroma; Non-Hodgkin's Lymphoma, Adult; Non-Hodgkin's Lymphoma, Childhood; Non-Hodgkin's Lymphoma During Pregnancy; Non-Small Cell Lung Cancer; Oral Cancer, Childhood; Oral Cavity and Lip Cancer; Oropharyngeal Cancer; Osteosarcoma / Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; Pancreatic Cancer. Childhood', Pancreatic Cancer, Islet-cell; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm / Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma; Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult; Primary Liver Cancer, Childhood; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Cell Cancer, Childhood; Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma;

[0081] Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Salivary’ Gland' Cancer, Childhood; Sarcoma. Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma Osteosarcoma) / Malignant Fibrous Histiocytoma of Bone; Sarcoma, Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue, Adult; Sarcoma, Soft Tissue, Childhood; Sezary’ Syndrome; Skin Cancer; Skin Cancer, Childhood; Skin Cancer (Melanoma); Skin Carcinoma. Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma. Adult; Soft Tissue Sarcoma. Childhood; Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer, Childhood; Supratentorial Primitive Neuroectodermal Tumors, Childhood; T-Cell Lymphoma. Cutaneous: Testicular Cancer; Thymoma, Childhood; Thymoma, Malignant; Thyroid Cancer; Thyroid Cancer, Childhood; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Unknown Primary Site, Cancer of, Childhood; Unusual Cancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma. Childhood; Vulvar Cancer; Waldenstrom's Macroglobulinemia; and Wilms' Tumor, among others.

[0082] The term “cancer” encompasses tumors. A tumor can be classified as malignant or benign. In both cases, there is an abnormal aggregation and proliferation of cells. In the case of a malignant tumor, these cells behave more aggressively, acquiring properties of increased invasiveness. Ultimately, the tumor cells may even gain the ability to break away from the microscopic environment in which they originated, spread to another area of the body (with a different environment, not normally conducive to their grow th), and continue their rapid growth and division in this new location. This is called metastasis. Once malignant cells have metastasized, achieving a cure or treatment is more difficult. Benign tumors have less of a tendency to invade and are less likely to metastasize.

[0083] The phrase “therapeutically effective amount” or an “effective amount” in the context of the administration of an agent or composition to a subject, refers to an amount capable of having any detectable, positive effect on any symptom, aspect, or characteristic of a disease, disorder or condition, when administered to the subject; the agent or composition can be administered either alone or as part of a pharmaceutical composition and either in a single dose or as part of a series of doses. The therapeutically effective amount or effective amount can be ascertained by measuring relevant physiological effects, and it can be adjusted in connection with the dosing regimen and diagnostic analysis of the subject's condition, and the like. In reference to cancer or pathologies related to unregulated cell division, a therapeutically effective amount or an effective amount can refer to that amount which has the effect of (1) reducing the size of a tumor (i.e. tumor regression), (2) inhibiting (that is, slowing to some extent, preferably stopping) aberrant-cell division, for example cancer cell division, (3) preventing or reducing the metastasis of cancer cells, (4) relieving to some extent (or, preferably eliminating) one or more symptoms associated with a pathology related to or caused in part by unregulated or aberrant-cellular division, including for example, cancer. (5) increasing the survival or life expectancy of the subject, and / or (6) decreasing the risk of relapse. An “effective amount” can also be an amount that results in desirable PD and PK profiles and / or desirable immune cell profiling or reconstitution upon administration of the therapeutically active compositions of the invention. An “effective amount’" can also be an amount that achieves a recited effect or result; for example, an effective amount of a chemotherapeutic agent that, alone or when in combination with another agent, can be an amount that reduces the size of a tumor and / or increases stress antigen expression on the tumor cells, and / or has a cytotoxic effect.

[0084] The terms “treating” or “treatment” of a disease (or a condition or a disorder) as used herein refer to inhibiting the disease (slowing or arresting its development), providing relief from the symptoms or side-effects of the disease (including palliative treatment), preventing or delaying recurrence, and causing regression of the disease. With regard to cancer, these terms also mean that the life expectancy of an individual affected with a cancer may be increased or that one or more of the symptoms of the disease will be reduced. With regard to cancer, “treating” also includes enhancing or prolonging an anti-tumor or anti-cancer response in a subject. In certain aspects, the methods described are methods of reducing a tumor in a patient in need thereof. The term “reducing a tumor” as used herein refers to a reduction in the size or volume of a tumor mass, a decrease in the number of metastasized tumors in a subject, a decrease in the proliferative status (the degree to which the cancer cells are multiplying) of the cancer cells, and the like.

[0085] The terms “subject” and “patient” as used herein include humans, mammals (e.g., cats, dogs, horses, etc.), living cells, and other living organisms. A living organism can be a mammal. Typical patients are mammals, primates, especially humans. In preferred aspects, the subject or patient is a human subject or patient.

[0086] In certain aspects, a population of yb T cells (e.g., a yb T cell product) is transplanted or co-administered with the antigen-binding molecule described herein. The population of yb T cells that are administered is an isolated population of yb T cells. The terms “isolated’ and “isolated population” of cells as used herein refers to a cell or a plurality of cells removed from the tissue or state in which they are found in a subject. The terms may further include cells that have been separated according to such parameters as, but not limited to, cell surface markers, a reporter marker such as a dye or label.

[0087] The term “expressed” or “expression” and the like as used herein has its ordinary meaning in the art and refers to the transcription from a gene to give an RNA nucleic acid molecule at least complementary in part to a region of one of the two nucleic acid strands of the gene. The term “expressed” or “expression” as used herein also refers to the translation from said RNA nucleic acid molecule to give a protein, a polypeptide, or a portion or fragment thereof.

[0088] The term "vector" as used herein refers to a polynucleotide comprised of single strand, double strand, circular, or supercoiled DNA or RNA. A typical vector may be comprised of the following elements operatively linked at appropriate distances for allowing functional gene expression; replication origin, promoter, enhancer, 5' mRNA leader sequence, ribosomal binding site, nucleic acid cassette, termination and polyadenylation sites, and selectable marker sequences. One or more of these elements may be omitted in specific applications. The vector may also contain a nucleic acid cassette, which can include a restriction site for insertion of the nucleic acid sequence to be expressed. In a functional vector the nucleic acid cassette contains the nucleic acid sequence to be expressed including translation initiation and termination sites. A vector is constructed so that the particular coding sequence (for example, a coding sequence for a bispecific antigen-binding molecule of the present disclosure) is located in the vector with the appropriate control sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is operably linked and / or is transcribed "under the control" of the control sequences. Modification of the sequences encoding the particular protein of interest may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it may be operably linked to the control sequences with the appropriate orientation or to maintain the reading frame. The control sequences and / or other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site that is in reading frame with and under regulatory control of the control sequences. The invention includes a vector comprising a nucleic acid sequence encoding the multifunctional or bispecific antigen-binding molecule as described herein. A non-limiting example of a vector is an AAV, a lentiviral vector, or a retroviral vector. In certain aspects, the invention is a vector that comprises a nucleic acid encoding a bispecific antigen-binding molecule as described herein. A specific example of a vector is the Baboon envelope pseudotyped lentiviral vector (BaEv).

[0089] The term "promoter" as used herein refers to the DNA sequence that determines the site of transcription initiation from an RNA polymerase. A "promoter-proximal element" may be a regulatory sequence within about 200 base pairs of the transcription start site. The term “recombinant cell’" refers to a cell that has a new combination of nucleic acid segments that are not covalently linked to each other in nature. A new combination of nucleic acid segments can be introduced into an organism using a wide array of nucleic acid manipulation techniques available to those skilled in the art. A recombinant cell can be a single eukaryotic cell, or a single prokary otic cell, or a mammalian cell. The recombinant-cell may harbor a vector that is extragenomic. An extragenomic nucleic acid vector does not insert into the cell's genome. A recombinant cell may further harbor a vector or a portion thereof that is intragenomic. The term “intragenomic” defines a nucleic acid construct incorporated within the recombinant-cell's genome.

[0090] The terms “recombinant nucleic acid” and “recombinant DNA” as used herein refer to combinations of at least two nucleic acid sequences that are not naturally found in a eukaryotic or prokaryotic cell. The nucleic acid sequences include, but are not limited to, nucleic acid vectors, gene expression regulatory7elements, origins of replication, suitable gene sequences that when expressed confer antibiotic resistance, protein-encoding sequences, and the like. The term “recombinant” with respect to a protein or peptide is meant to include a polypeptide or protein produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location, purity or structure. Generally, such a recombinant polypeptide or protein will be present in a cell in an amount different from that normally observed in nature.

[0091] The terms "operably" or "operatively linked" as used herein refer to the configuration of the coding and control sequences so as to perform the desired function. Thus, control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence. A coding sequence is operably linked to or under the control of transcriptional regulatory7regions in a cell when DNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA that can be translated into the encoded protein. The control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0092] The terms "heterologous" and "exogenous" as they relate to nucleic acid sequences such as coding sequences and control sequences denote sequences that are not normally- associated with a region of a recombinant construct or with a particular chromosomal locus, and / or are not normally associated with a particular cell. Thus, a "heterologous" region of a nucleic acid construct is an identifiable segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a construct could include a coding sequence flanked by sequences not found in association with the coding sequence in nature. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). Similarly, a cell transformed with a construct, which is not normally present in the host cell, would be considered heterologous for purposes of this invention.

[0093] The promoter can be modified by the addition or deletion of sequences, or replaced with alternative sequences, including natural and synthetic sequences as well as sequences that may be a combination of synthetic and natural sequences. Many eukaryotic promoters contain two types of recognition sequences: the TATA box and the upstream promoter elements. The former, located upstream of the transcription initiation site, is involved in directing RNA polymerase to initiate transcription at the correct site, while the latter appears to determine the rate of transcription and is upstream of the TATA box. Enhancer elements can also stimulate transcription from linked promoters, but many function exclusively in a particular cell type. Many enhancer / promoter elements derived from viruses, e.g., the SV40, the Rous sarcoma virus (RSV), and CMV promoters are active in a wide array of cell types and are termed "'constitutive" or ' 'ubiquitous. ’ The nucleic acid sequence inserted in the cloning site may have any open reading frame encoding a polypeptide of interest, with the proviso that where the coding sequence encodes a polypeptide of interest, it should lack cryptic splice sites that can block production of appropriate mRNA molecules and / or produce aberrantly spliced or abnormal mRNA molecules.

[0094] The termination region that is employed primarily will be one of convenience, since termination regions appear to be relatively interchangeable. The termination region may be native to the intended nucleic acid sequence of interest, or may be derived from another source.

[0095] The terms “transformation.” “transduction” and the like denote the introduction of a polynucleotide into a recipient-cell or cells.

[0096] The terms “antigen recognition domain,” “antigen recognition moiety,” “antigen binding domain,” “antigen binding moiety,” and the like, are used interchangeably herein. Similarly, the terms, “transmembrane domain,” “transmembrane moiety ,” “transmembrane region,” and the like are used interchangeably; the terms “hinge domain,” “hinge moiety,” and “hinge region,” and the like are used interchangeably. The multifunctional molecule, such as a trispecific or bispecific antigen-binding molecule, described herein is a fusion protein. The term "fusion protein" or a “fusion polypeptide,’’ as used herein, refers to a chimeric molecule, which comprise, for example, at least to antigen recognition domains, which are not naturally linked in nature. The amino acid sequences may normally exist in separate proteins that are brought together in the fusion polypeptide or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. Fusion proteins may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.

[0097] As discussed above, the invention encompasses multifunctional antigen-binding molecules, such as trispecific and bispecific antigen-binding molecules, comprising i) a first antigen-binding domain that binds to a tumor-associated antigen (TAA) and ii) a second antigen-binding domain that binds to a 78 TCR; as w ell as compositions comprising the multifunctional antigen-binding molecule. In certain aspects, the multifunctional antigenbinding domain comprises a third antigen-binding domain. Also encompassed is a bispecific antigen-binding molecule, wherein the molecule comprises i. a first antigen-binding domain that specifically binds to a tumor-associated antigen; and ii. a second antigen-binding domain that specifically binds to more than one 78 T cell subtype, for example, binds to the 78 TCR of more than one y8 T cell subtype or an epitope expressed by more than one y8 T cell subtype. In certain aspects, the second antigen-binding domain binds an epitope expressed by both V81 and V82 subpopulations of y8 T cells or the second antigen-binding domain is a pan y8 TCR antigen-binding moiety. The antigen-binding molecules comprising a second antigen-binding domain that specifically binds to more than one y8 T cell subty pe (for example, by binding to the y8 TCR of more than one y8 T cell subtype, or to an epitope expressed by more than one y8 T cell subtype, or an epitope expressed by both V81 and V82 subpopulations), can induce expansion of a population of y8 T cells comprising more than one y8 T cell subtype. For example, an antigen-binding molecule as described herein can induce expansion of V81 and V82 subpopulations of y8 T cells. The multifunctional and bispecific antigen binding molecules described herein can selectively induce expansion or enhance the expansion of y8 T cells of more than one subtype by specifically targeting more than one y8 T cell subtype, for example, by binding to a domain or an epitope expressed by more than one y8 T cell subty pe. In additional aspects, the multifunctional antigen-binding molecule comprises a third domain, for example, is a tri-specific antigen-binding molecule. The third domain can, for example, promote expansion of y8 T cells, for example, it can promote expansion of the V81 and V52 subpopulations of y8 T cells, and optionally promote expansion of one or more other y8 T cell subtypes. In certain aspects, the third domain binds, for example, specifically binds, to an epitope expressed by y5 T cells, wherein the epitope is different from that which the second antigen-binding domain binds, and the binding of the third domain to the different epitope promotes the expansion of the y8 T cells. In certain aspects, the binding of the third domain to the different epitope promotes the expansion of the V61 and V52 subpopulations of y8 T cells. In further aspects the binding of the third domain to the different epitope promotes the expansion of the V81 and V82 subpopulations of y8 T cells and one or more other y8 T cell subtypes. Additionally encompassed is a method of treating cancer comprising administering the multifunctional, trispecific or bispecific antigenbinding molecule described herein. In certain aspects, a population of the patient’s y8 T cells are expanded, wherein the method does not comprise transplantation or administration of yS T cells (e.g., autologous or allogeneic y8 T cells). Also described herein are methods of treating cancer comprising administration of an effective amount of a population of y8 T cells and the multifunctional antigen-binding molecule, such as the trispecific or bispecific antigen-binding molecule, described herein. Also included herein are methods of increasing the cytotoxicity of a population of y8 T cells against cancer cells (e.g., a population of the patient's own y3 T cells), the method comprising administration of an antigen-binding molecule, such as the trispecific or bispecific antigen-binding molecule, described herein. Also encompassed are methods of increasing the cytotoxicity of a population of y8 T cells against cancer cells, the method comprising administration of an effective amount of a population of y8 T cells and the multifunctional antigen-binding molecule, such as the bispecific antigen-binding molecule, described herein.

[0098] An “antigen” relates to any substance, such as a peptide or protein, which reacts specifically with antibodies or fragments thereof, or T-lymphocytes, or other immune cell. The term “antigen” can refer to any molecule which comprises at least one epitope.

[0099] An “epitope” refers to the portion of the antigen or target which is specifically bound by an antibody or antigen-binding fragment thereof, or an antigen-binding domain. Epitopes found on protein targets may be defined as “linear epitopes” or “conformational epitopes.” Linear epitopes are formed by a continuous sequence of amino acids in a protein antigen. Conformational epitopes are formed of amino acids that are discontinuous in the protein sequence, but which are brought together upon folding of the protein into its three- dimensional structure.

[0100] The multifunctional, trispecific, and bispecific antigen-binding molecules described herein can include one or more antigen-binding domains that specifically binds to a region or domain of the y8 TCR common to more than one yd T cell subty pe (for example, on either the y chain or the 5 chain or both). For example, the multifunctional and bispecific antigenbinding molecules described herein can include one or more antigen-binding domains that specifically binds to an epitope expressed by more than one yd T cell subtype (e.g., binding to an epitope expressed by both V51 and V62 subpopulations). An example of an epitope can, for example, be an activating epitope. An activating epitope can be one wherein binding to the epitope results in activation of the yd T cell, for example, modulation of a TCR-associated function, such as TCR downregulation, degranulation of the cell, cytotoxicity’, proliferation, increased survival or resistance to exhaustion, intracellular signaling, cytokine or growth factor secretion, phenoty pic change, or a change in gene expression. For example, the binding to the activating epitope may stimulate expansion (i.e., proliferation) of the y5 T cell population.

[0101] The terms “multifunctional,” “multi-functional,” “multi-specific” and “multispecific” are used interchangeably’ herein. Similarly, the terms “bifunctional,” ‘bi -functional,” “bispecific” and “bispecific” are used interchangeably herein. Also, the terms “trifunctional,” “tri-functional,” “tri-specific” and “trispecific” are used interchangeably herein. The term “antigen-binding molecule” can also be used interchangeably with the term “engager.” As used herein, the term “multifunctional antigen-binding molecule” or a “multifunctional engager” is a molecule or fusion protein that has at least two linked antigenbinding domains (e.g., two scFv domains), wherein at least one antigen-binding domain targets a cell-surface molecule on y5 T cells and at least one antigen-binding domain that targets a tumor antigen or TAA. A multifunctional antigen-binding molecule can, for example, be bi-specific (e.g., having a first antigen-binding domain and a second antigenbinding domain as described herein) and a tri-specific antigen binding domain (e g., having a first antigen-binding domain, a second antigen-binding domain, and a third domain as described herein). Two antigen-binding domains can be linked together by a linker, for example, a short flexible linker. A non-limiting example of a linker is a GlySer linker (a linker comprises glycine and serine residues), such as the twenty7residue peptide linker GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 14), Ser-Gly-Gly-Gly (SEQ ID NO: 8). Ser- Gly-Gly-Gly-Gly (SEQ ID NO: 9), (GGGGS)2(SEQ ID NO: 16). or (GGGGS)3(SEQ ID NO: 15), or (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 10; wherein n is an integer from 1 to 10). The term “bispecific antigen binding molecule” is a molecule or fusion protein that has two linked antigen-binding domains (e.g., scFv domains), wherein one antigen-binding domain targets a cell-surface molecule on yb T cells and one antigen-binding domain targets a tumor antigen or TAA. The term “bispecific antigen-binding molecule” can be used interchangeably with the terms “bispecific engager,” “bispecific yb T cell engager,” “yb T cell engager,” “engager,” and the like. A “bispecific antigen-binding molecule has two antigen binding domains, and therefore can recognize and bind two antigens. The bispecific antigen-binding molecule can, for example, be a bispecific antibody. In certain aspects, the bispecific antigenbinding molecule is a fusion protein of two antigen-binding domains (e.g., single-chain variable fragments (scFvs) of different antibodies), or amino acid sequences from four different genes, on a single peptide chain (one antigen-binding domain binds the TAA and the other antigen-binding domain binds the yb TCR). As discussed above, another example of a multifunctional antigen-binding molecule is a trispecific antigen-binding molecule. A “trispecific antigen-binding molecule” or a “trispecific engager” has three antigen binding domains, and therefore can recognize and bind three antigens. The trispecific antigen-binding molecule can, for example, be a trispecific antibody. In certain aspects, the trispecific antigen-binding molecule is a fusion protein of three antigen-binding domains (e g, singlechain variable fragments (scFvs) of different antibodies), or amino acid sequences from six different genes, on a single peptide chain one antigen-binding domain binds the TAA, a second antigen-binding domain binds the yb TCR, and a third antigen-binding domain binds a different antigen).

[0102] Without wishing to be bound by theory', it is believed that by binding to a tumor antigen and to a yb T cell simultaneously, the multifunctional antigen-binding molecule (e.g., the bispecific antigen-binding molecule or the trispecific antigen-binding molecule) described herein direct the yb T cells’ cytotoxic activity against the target cell (e.g., cancer cell that expresses the TAA). In some examples, the antigen-binding molecule can expand yb T cells and also kill target cells which, in turn, can enhance activation and cytokine release which promotes further expansion. When the multifunctional antigen-binding molecule binds to yb T cells (e.g., the yb TCR) of more than one subtype (e.g., including Vbl and Vb2 subpopulations of yb T cells), the antigen-binding molecule directs the cytotoxic activity of multiple subtypes of yb T cells against the target cell (e.g., the tumor cell) and can enhance the expansion of multiple subtypes of yb T cells (for example, the third domain can promote expansion of the y8 T cells). For example, the multifunctional antigen-binding molecule, trispecific antigen-binding molecule, or bispecific antigen-binding molecule described herein can bring the y8 T cell and a cancer cell in proximity, and thereby enhance the cytotoxicity of the y8 T cells against the cancer cells. The formation of a complex or synapse comprising the y8 T cell, the multifunctional / bispecific antigen-binding molecule, and the cancer cell can induce y8 T cell signaling including, for example, the release of cytotoxic mediators. See, for example, FIG. 1 which shows the formation of the lytic immune synapse (comprising a complex of the y8 T cell, the multifunctional engager, and target cell, such as an AML cell) which results in release of mediators such as granzyme and perforin, which in turn, results in targeted lysis of the AML cell. When the engager comprises an antigen-binding domain that binds CD33, the bispecific engager brings a CD33-expressing cell (for example, an AML cell) into proximity' with the y8 T cell so that that y8 T cell can exert its effect on the CD33- expressing target cell. The pre-requisite of co-localization via binding of the CD33 multifunctional antigen-binding molecule leads to selective killing of CD33-positive cells. In other words, the multifunctional, trispecific, or bispecific molecule of the present invention is able to activate T cells following binding of the CD33 antigen-binding domain to CD33 expressed on the surface of target cells. See, also, FIG. 13 w hich shows the formation of the lytic immune synapse (comprising a complex of the y8 T cell, the multifunctional engager, and a B-ALL cell or lupus B-cell) w hich results in release of mediators such as granzyme and perforin, which in turn, results in targeted lysis of the B-ALL cell or lupus B-cell.

[0103] In some examples, the multifunctional antigen-binding molecule is a bispecific antigen-binding molecule comprising i) an antigen-binding domain that binds to a tumor- associated antigen (TAA), and ii) an antigen-binding domain that binds to a y8 TCR. In other examples, the multifunctional antigen-binding molecule is a bispecific antigen-binding molecule comprising i) a first antigen-binding domain that specifically binds to a tumor- associated antigen (TAA). and ii) a second antigen-binding domain that specifically binds to a y8 TCR, wherein the second antigen-binding domain binds to an epitope expressed by more than one y8 T cell subtype. For example, the TAA can be any antigen expressed by a tumor cell including, for example, CD33 and CD 19, and the y8 TCR is a human y8 TCR. In some examples, the bispecific antigen-binding molecule comprises, in the N-terminal to C-terminal direction, a signaling peptide that, for example, directs the engager to proper folding, modification and / or transportation; a first antigen-binding domain that binds to a first tumor- associated antigen (TAA), a linker, and a second antigen-binding domain that binds to a y8 TCR, for example, a human y8 TCR. In additional examples, the bispecific antigen-binding molecule comprises, in the N-terminal to C-terminal direction, a signaling peptide that, for example, directs the engager to properly fold, modification and transportation; an antigenbinding domain that binds to a y8 TCR, for example, a human yd TCR (the “second antigenbinding domain”); a linker; and an antigen-binding domain that binds to a tumor-associated antigen (TAA) (the “first antigen-binding domain”). In certain aspects, the second antigenbinding domain specifically binds to an epitope expressed by more than one y8 T cell subtype.

[0104] In further aspects, the multifunctional antigen-binding molecule is a tri-specific antigen-binding molecule, comprising i) a first antigen-binding domain that binds to a first tumor-associated antigen (TAA), ii) a second antigen-binding domain that binds to a 78 TCR, for example, specifically binds to a 78 TCR, and iii) a third domain, wherein the third domain promotes expansion of 78 T cells, for example, it can promote expansion of the V81 and V82 subpopulations of 78 T cells, and optionally promote expansion of one or more other 78 T cell subtypes. In certain aspects, the third domain specifically binds to an epitope expressed by y8 T cells or the y8 TCR, wherein the epitope is different from that which the second antigenbinding domain binds, and the binding of the third domain to the different epitope promotes the expansion of the y8 T cells. In certain aspects, the binding of the third domain to the different epitope promotes the expansion of the V81 and V82 subpopulations of y8 T cells. In further aspects the binding of the third domain to the different epitope promotes the expansion of the V81 and V82 subpopulations of y8 T cells and one or more other y8 T cell subtypes. In additional aspects, the second antigen-binding domain binds to an epitope expressed by more than one y8 T cell subtype. In certain aspects, the second antigen-binding domain specifically binds to an epitope expressed by more than one y8 T cell subtype. In some examples, the multifunctional antigen-binding molecule comprises, in the N-terminal to C-terminal direction, a signaling peptide that, for example, directs the engager to proper folding, modification and / or transportation; the first antigen-binding domain that binds to a first tumor-associated antigen (TAA), a linker, the second antigen-binding domain that binds to a y8 TCR, for example, a human y8 TCR, and a third domain. In additional examples, the multifunctional antigen-binding molecule comprises, in the N-terminal to C-terminal direction, a signaling peptide that, for example, directs the engager to property fold, modification and transportation; a third domain, a second antigen-binding domain that binds to a y6 TCR, for example, a human 78 TCR (the “second antigen-binding domain”); and antigen-binding domain that binds to a tumor-associated antigen (TAA) (the “first antigenbinding domain”), wherein the domains can be directly linked or linked by a linker.

[0105] In additional examples, the multifunctional antigen-binding molecule is a tri-specific antigen-binding molecule, comprising i) a first antigen-binding domain that binds to a first tumor-associated antigen (TAA), ii) a second antigen-binding domain that binds to ay8 TCR, for example, specifically binds to a y8 TCR, and iii) a third antigen-binding domain that binds to a different tumor-associated antigen than the first antigen-binding domain. In additional examples, the multifunctional antigen-binding molecule is a tri-specific antigen-binding molecule, comprising i) a first antigen-binding domain that binds to a first tumor-associated antigen (TAA), ii) a second antigen-binding domain that binds to a y8 TCR, for example, specifically binds to a y8 TCR, and iii) a third antigen-binding domain that binds to an immune cell antigen. An “immune cell antigen” can, for example, be a T-cell antigen or an NK cell antigen. An example of a T-cell antigen is CD3. In another example, the T-cell antigen is a y8 T cell antigen such as a y8 TCR. Examples of an NK antigen include, for example, CD 16a, NKG2D, NKG2C, Nkp30, Nkp46, Nkp80 and CD 160. In additional aspects, the second antigen-binding domain binds to an epitope expressed by more than one y8 T cell subtype. In certain aspects, the second antigen-binding domain specifically binds to an epitope expressed by more than one y6 T cell subtype.

[0106] The terms “specific binding” and “specifically binds” in the context of an antibody or an antigen-binding fragment thereof or an antigen-binding domain or other molecule, are well understood in the art. For example, a molecule is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration and / or with greater affinity with a particular target antigen or epitope, than it does with alternative targets. An antibody, antigenbinding fragment thereof, or antigen-binding domain can “specifically bind” to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. An antibody, or fragment thereof, or antigenbinding domain may also be considered to specifically bind to a target if the binding is statistically significant compared to a non-relevant binder. For example, an antigen-binding domain that binds specifically to a y8 T cell, to a y8 TCR or to an epitope of any of thereof, when it binds with greater affinity, avidity, and / or greater duration to ay8 T cell, to a y8 TCR or to an epitope of any of thereof, as compared to an ap T cell, an a TCR, or an epitope thereof. Thus, an antigen-binding domain that binds to CD3 is not an antigen-binding domain that specifically binds to a y5 T cell or a y5 TCR, or that specifically binds to an epitope of a y8 T cell or y8 TCR, because it binds both a.p and yo T cells.

[0107] As described above, the multifunctional molecule, trispecific or bispecific antigenbinding molecules comprises an antigen binding domain that binds a tumor antigen. The terms “tumor antigen." “tumor associated antigen” and “TAA” are used interchangeably herein. Non-limiting examples of tumor-associated antigens include CD 19; CD 123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A 24); c-type lectin-like molecule-1 (CLL-1 or CLECL 1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD 2); ganglioside GD3 (aNeu 5Ac (2-8) aNeu5 Ac (2-3) bDGalp (1 -4) bDGlcp (1 -1 ) Cer); TNF receptor family member B Cell Maturation (BCMA); tn antigen ((TnAg) or (GalNAc. Alpha. -Ser / Thr)); prostate Specific Membrane Antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR 1); fins- like tyrosine kinase 3 (FLT 3); tumor associated glycoprotein 72 (TAG 72); CD38: CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD 276); KIT (CD 117); interleukin- 13 receptor subunit alpha-2 (IL-13 Ra2 or CD213 A2); mesothelin; interleukin 11 receptor alpha (IL-11 Ra); prostate Stem Cell Antigen (PSCA); protease serine 21 (Testisin or PRSS 21); vascular endothelial growth factor receptor 2 (VEGFR 2); a Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage specific embryonic antigen-4 (SSEA-4); CD20; a folate receptor alpha (folate receptor 1: FOLR1); receptor ty rosine protein kinase ERBB2 (Her 2 / neu); mucin 1, cell surface associated material (MUC 1); epidermal Growth Factor Receptor (EGFR); neural Cell Adhesion Molecule (NCAM); a Prostase; prostatic Acid Phosphatase (PAP); mutant elongation factor 2 (ELF 2M); ephrin B2; fibroblast activation protein alpha (FAP); insulinlike growth factor 1 receptor (IGF-I receptor), carbonic Anhydrase IX (CAIX); proteasome (Prosome, macropain) subunit beta type 9 (LMP 2); glycoprotein 100 (gp 100); an oncogene fusion protein (BCR-Abl) consisting of the Breakpoint Cluster Region (BCR) and the homolog 1 (Abl) of the Abelson murine leukemia virus oncogene; a tyrosinase enzyme; ephrin type a receptor 2 (EphA 2); fucose GM1; sialic acid Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu 5Ac (2-3) bDGalp (1-4) bDGlcp (1-1) Cer); transglutaminase 5 (TGS 5); high Molecular Weight Melanoma Associated Antigen (HMWMAA); o-acetyl-GD 2 ganglioside (OAcGD 2); folate receptor ty tumor endothelial marker 1 (TEM 1 / CD 248); tumor endothelial marker 7 related (TEM 7R); claudin 6 (CLDN 6); thyroid Stimulating Hormone Receptor (TSHR); a G protein-coupled receptor class C group 5D member (GPRC 5D); chromosome X open reading frame 61 (CXORF 61); CD97; CD 179a; anaplastic Lymphoma Kinase (ALK); polysialic acid: placenta-specific (PLACENTA-specific) 1 (PL AC 1); the hexasaccharide moiety (globoH) of the globoH glycoceramide (glycoceramide); mammary7gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK 2); hepatitis a virus cell receptor 1 (HAVCR 1); adrenergic receptor beta 3 (ADRB 3); pannexin 3 (PANX 3); g protein-coupled receptor 20 (GPR 20); lymphocyte antigen 6 complex, locus K9 (LY 6K); olfactory receptor 51E2 (OR 51E 2); TCR y alternate reading frame protein (TARP); wilms tumor protein (WT 1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1 a); melanoma-associated antigen 1 (MAGE-A1); ETS translocationvariant gene 6 (ETV 6-AML) located on chromosome 12 p; sperm protein 17 (SPA 17); x antigen family member 1 A (XAGE 1); angiogenin binds to cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD- CT-2); fos-related antigen 1; tumor protein p53 (p 53); a p53 mutant; a prostein; survival (surviving); a telomerase; prostate cancer tumor antigen-1 (PCTA-1 or Galectin 8 (Galectin 8)), melanoma antigen 1 recognized by T cells (Melana or MART 1); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); a sarcoma translocation breakpoint; melanoma apoptosis inhibitors (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS 2) ETS fusion gene); n-acetylglucosaminyltransferase V (NA 17); paired box protein Pax-3 (PAX 3); an androgen receptor; cyclin Bl; v-myc avian myelomatosis virus oncogene neuroblastoma derived homolog (MYCN); ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450B 1 (CYP IB 1); CCCTC-binding factor (zinc finger protein) -like (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen recognized by T cells 3 (SART 3); paired box protein Pax-5 (PAX 5); the preproceptorin (proacrosin) binding protein sp32 (OY-TES 1): lymphocytespecific protein tyrosine kinase (LCK); kinase anchoring protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX 2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (renal ubiquitin 1) (RU 1); renal ubiquitin 2 (RU 2); legumain; human papillomavirus E6 (UPV E6); human papilloma virus E7 (UPV E7); an intestinal carboxylesterase; mutated heat shock protein 70-2 (mut hsp 70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR 1); an Fc fragment of IgA receptor (FCAR or CD 89); leukocyte immunoglobulin-like receptor subfamily a member 2 (LILRA 2); CD300 molecule-like family member f (CD 300 LF); c-type lectin domain family 12 member a (CLEC 12A); bone marrow stromal cell antigen 2 (BST 2); mucin-like hormone receptor-like protein 2 (EMR 2) containing an EGF-like module; lymphocyte antigen 75 (LY 75); glypican-3 (Glypican-3. GPC3); fc receptor-like protein 5 (FCRL 5); and immunoglobulin lambda-like polypeptide 1 (IGLL 1). In certain aspects, the tumor antigen includes EphA2, B cell maturation antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD22, CD19, CD20, R0R1, kappa or light chain, carcinoembryonic antigen, alpha-fetoprotein, CA- 125, Glypican-3, epithelial tumor antigen, melanoma-associated antigen, EGP2, EGP40, EPCAM. ERBB3, ERBB4, ErbB3 / 4, PAP, FAR, FBP, fetal AchR, Folate Receptor a, mutated p53, mutated ras, HER2, ERBB2, HER3, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, 5T4, 8H9, GD2, CD123, CD171, CS-1, CD23, CD24, CD33, CD30. CD38, CD56, c-Met, fap, mesothelin, GD3, HERV-K, IL- 1 IRa, IL-13Ra, IL-13Ra2, CSPG4. Lewis- Y, MCSP, MucL Mucl6. NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, EGFR, Spl7, SURVIVIN, TAG72, TEM1, TEM8, epidermal grow th factor receptor variant III, EGFRvIII, VEGFR2. In yet additional aspects, the tumor antigen is an NKG2D ligand selected from the group consisting of ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, MIC-A. and MIC-B.

[0108] In certain specific aspects, the tumor antigen (TAA) is selected from the group consisting of CD19, CD33, CD123, CLL-1, CD20, CD22, EGFRvIII, IL13ra2, folate receptor 1 (FOLR1), MMP2, and mesothelin. In certain additional aspects, the tumor antigen is selected from the group consisting of CD19, CD33. CD123, CD20. and CD22. In yet further aspects, the tumor antigen is selected from the group consisting of CD19, CD33, and CD123.

[0109] In certain aspects, the tumor antigen is CD33. CD33 is a my eloid differentiation antigen and is highly expressed on myeloid progenitor cells but is expressed at low levels in differentiated myeloid cells (i.e., macrophages and granulocytes). CD33 has been reported to be expressed in about 88% of Acute Myeloid Leukemia (AML) (Ehniger et al (2014), Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia, Blood Cancer Journal 4(6): e 218; the contents of which are expressly incorporated by reference herein).

[0110] In certain additional aspects, the tumor antigen is CD 19. The human CD 19 antigen is a 95 kd transmembrane glycoprotein belonging to the immunoglobulin superfamily. CD19 is expressed early in B-cell differentiation and is only lost at terminal B-cell differentiation into plasma cells. Consequently, CD 19 is expressed on all B-cell malignancies except for multiple myeloma. The antigen binding domain can comprise an antibody, antigen-binding fragment thereof, or an antigen binding molecule with specificity for the tumor antigen; for example, an anti-CD33 antibody or antigen binding fragment thereof or an anti-CD19 antibody or antigen-binding fragment thereof. As described herein, an antigen-binding domain can also comprise an antibody, antigen-binding fragment thereof, or an antigen binding molecule with specificity for a y3 T cell or a y3 TCR. The term "antibody" includes fragments that retain the ability to specifically bind to the antigen. Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multi specific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain- antibody heavy chain pair, intrabodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), camelized antibodies, affibodies. Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), anti -idiotypic (anti-Id) antibodies (including, e.g. , anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above. An "antigen binding molecule," "antigen binding portion," "antibody fragment," or ‘’antigen-binding fragment” refers to any molecule that comprises the antigen binding parts (e.g., CDRs) of the antibody from which the molecule is derived. Examples of antibodies include, but are not limited to, Fab. Fab', F(ab')2, and Fv fragments, dAb, linear antibodies. scFv. and multi specific antibodies formed from antigen binding molecules. scFvs or single chain antibodies are encompassed within the term antibody unless otherwise indicated by context. An antigenbinding molecule or domain can include the antigenic complementarity determining regions (CDRs) of an antibody, for example, an antibody that specifically binds to a TAA. For example, the first antigen-binding domain can include the CDRs of an anti-CD33 antibody. In another example, the first antigen -binding domain can include the CDRs of an anti-CD19 antibody. In certain additional aspects, the antigen-binding domain comprises the VH and VL of an antibody, for example, an antibody that specifically binds to a TAA. For example, the first antigen-binding domain can comprises the VH and the VL of an anti-CD33 antibody. In another example, the first antigen-binding domain can comprise the VH and the VL of an anti-CD19 antibody.

[0111] A scFv refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin, connected with a short linker peptide (generally of about 10 to about 25 amino acids) to produce, for example, a VH-linker-VL structure or a VL- linker-VH structure. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This scFv retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. In certain aspects, one or more of the antigen-binding domains of the multifunctional antigen-binding molecule or bispecific antigen-binding molecule is an scFv. In yet further aspects, the antigen recognition domain is a multivalent scFv. A multivalent scFv comprises two immunoglobulin derived antigen binding domains (such as a VH-linker-VL chain) joined by an additional linker, wherein the antigen binding domain recognize different antigens or different portions of the same antigen. Non-limiting examples of structure as described herein are CD33VL- linker-CD33VH-GDVH-linker-GDVL, CD33VH-linker-CD33VL-GDVL-lmker-GDVH, CD33VL-hnker-CD33VH-GDVL-linker-GDVH, or CD33VH-linker-CD33VL-GDVH- linker-GDVL. The VH of the anti-CD33 scFv can, for example, be attached to the VH or VL of the anti-y8 TCR scFv by a linker or the VL of the anti-CD33 scFv can be attached to the VL or VH of the anti -78 TCR scFv by a linker. Additional examples encompassed herein is CD 19VL-hnker-CD 19VH-GDVH-linker-GDVL, CD 19VH-hnker-CD 19VL-GDVL-linker- GDVH, CD19VL-linker-CD19VH-GDVL-linker-GDVH, or CD19VH-linker-CD19VL- GDVH-linker-GDVL. The VH of the anti-CD19 scFv can, for example, be attached to the VH or VL of the anti-y8 TCR scFv by a linker or the VL of the anti-CD19 scFv can be attached to the VL or VH of the anti-y5 TCR scFv by a linker.

[0112] A single domain antibody (sdAb), also known as a nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. sdAbs have been derived from heavy-chain antibodies found in Camelidae species (such as camel, llama, dromedary, alpaca and guanaco) using molecular biology techniques, which are also known as VHH fragments (herein also termed ’VHH" or ’VHH"). sdAbs have also been generated from a heavy chain / light chain of conventional immunoglobulin G (IgGs) by engineering techniques. In certain aspects, at least one antigen-binding domain is an sdAb, for example, a VHH. An antigen-binding domain can also comprise any molecule (recognition element) that can bind to a tumor antigen, e.g., can be a peptide or sequence that specifically binds to the tumor antigen or a receptor ligand that binds to the tumor antigen. For example, the first antigen-binding domain can comprise any molecule (recognition element) that can bind to a tumor antigen, e.g., can be a peptide or sequence that specifically binds to the tumor antigen or a receptor ligand that binds to the tumor antigen, such as CD33 or CD 19. An antigenbinding domain can also comprise any molecule (recognition element) that can bind to a yb TCR, e.g., peptide that specifically binds to the yb TCR or a receptor ligand that binds to the yb TCR. For example, the second antigen-binding domain can comprise any molecule (recognition element) that can bind to a yb TCR, e.g., peptide that specifically binds to the yb TCR or a receptor ligand that binds to the yb TCR. An antigen-binding domain can also comprise any molecule (recognition element) that can bind to a yb T cell, e.g., peptide that specifically binds to an epitope on the yb T cell. For example, the second antigen-binding domain can comprise any molecule (recognition element) that can bind to an epitope on one or more yb T cell subtypes, e.g., peptide that specifically binds to the yb T cell epitope or a receptor ligand that binds to the yb T cell epitope. Examples of suitable soluble receptor ligands include autocrine and paracrine grow th factors, chemokines, cytokines, hormones, and engineered artificial small molecule ligands that exhibit the required specificity. Natural ligand sequences can also be engineered or optimized to increase their specificity for a particular target cell.

[0113] Described herein are multifunctional, trispecific, or bispecific antigen-binding molecules that comprise an antigen-binding domain that binds to CD33, for example, human CD33. In some examples, the antigen binding domain comprises an anti-CD33 scFv. Anti- CD33 antibodies, antigen-binding fragments, and scFvs can, for example, can be derived from gemtuzumab ozogamicin (GO) (hP67.6), vadastuximab talirine (SGN-CD33A), lintuzumab, BI 836858 (Vasu et al. (2021), Haemotologica 107(3): 770-773), IMGN779 (Immungen, Inc.), AL003 ((Alector. Inc.). M195, Hul95, or MY96 (Mylotarg). MY96 has been described, for example, in WO2016014576 and Kenderian et al. (2015), Leukemia 29: 1637-1647. Additional anti-CD33 antibodies and antigen-binding fragments thereof have been described, for example, in US20240409634, US20240262910, US20240173425, US20240124575, US20230250171, US20230190810. In some examples, the antigen-binding domain that binds CD33 comprises gemtuzumab (hP67.6) or an antigen-binding fragment thereof, such as an scFv (Cowan et al.. Front. Biosci. (Landmark Ed ), 18: 1311-1334 (2013) and U.S. Pat. Nos. 5,773,001 and 5,739,116, each incorporated by reference herein). For example, the antigen-binding domain can be an scFv comprising a VH and VL, wherein the VH of the scFv comprises the VH CDRs of gemtuzumab and wherein the VL of the scFv comprises the VL CDRs of gemtuzumab. In additional aspects, the scFv comprises the VH and the VL of gemtuzumab. In yet another example, the anti-CD33 antigen-binding domain is M195 or Hul95. or an antigen-binding fragment thereof, such as an scFv (Co et al., J. Immunol., 148: 1149-1154 (1992), incorporated by reference herein). In another example, the antigen-binding domain can be an scFv comprising a VH having the VH CDRs of Ml 95 or Hul95 and wherein the VL CDRs of M195 or Hul95. In additional aspects, the scFv comprises the VH and the VL of M195 or Hul95. In another example, the anti-CD33 antigen-binding domain is lintuzumab, or an antigen-binding fragment thereof, such as an scFv (Co et al., J. Immunol., 148: 1149-1154 (1992), incorporated by reference herein). For example, the antigen-binding domain can be an scFv comprising a VH having the VH CDRs of lintuzumab and wherein the VL CDRs of M195 or Hul95. In additional aspects, the scFv comprises the VH and the VL of lintuzumab.

[0114] Also specifically described herein are multifunctional, trispecific, or bispecific antigen-binding molecules that comprise an antigen-binding domain that binds to CD 19. In some examples, the antigen binding domain comprises an anti-CD19 scFv. For example, the anti-CD19 antigen-binding domain or scFv can be derived from FMC63 murine antibody.

[0115] The multifunctional, trispecific or bispecific antigen-binding molecules comprise an antigen-binding domain (e g., the second antigen-binding domain and / or the third domain) that specifically binds to y8 T cells or to ay8 TCR, for example, a human y8 TCR. In certain aspects, the y8 TCR is a human Vy9V82-TCR. In yet other aspects, the antigen-binding domain binds to a gamma variable 2 (Vy2) chain of a TCR. In yet further aspects, the y8 TCR that the antigen-binding domain binds to is other than a gamma variable 2 (Vy2) chain. In certain aspects, the antigen-binding domain that specifically binds to a region of a y8 TCR common to the TCR of more than one y8 T cell subtype or to an epitope expressed by more than one y8 T cell subtype, for example, an epitope expressed by both V81 and V82 subpopulations of y8 T cells. For example, the antigen-binding molecule can be a pan y8 TCR antigen-binding moiety7. The terms “pan y8 TCR antigen-binding moiety7’' and “pan y8 TCR antigen-binding molecule” and the like are used interchangeably herein and refer to an antigen-binding domain, moiety or molecule that specifically binds to an epitope expressed by the V81 subpopulation, the V82 subpopulation, as well as y8 T cells other than those of the V81 and V82 subpopulations. In further aspects, the antibody or antigen-binding domain binds a pan-TCR (e.g., pan-TCR epitope) expressed on multiple y8 T cell types, for example, it can bind both V81 and V82 TCRs. In some examples, the antigen-binding domain that binds to the y8 TCR comprises an scFv. Anti-y8 TCR antibodies, antigen-binding molecules, and / or scFvs are commercially available (e.g., IMMU510), and have been described, for CN103130894A, CN115724974A, US20220403025A1, CNA102295702, CN108350428, W02016081518, WO2017197347, WO2019099744, US20230159638, and US1167395B2; the contents of which are expressly incorporated by reference herein. In certain aspects, the anti-y8 TCR antigen-binding domain or anti-y8 TCR scFv is a pan y8 TCR antigen-binding molecule (e.g., has binding affinity for pan-y8 TCR or a pan-TCR epitope), for example an anti-y8 TCR antibody or scFv that can engage both V81 and V82 TCRs. The anti-y8 TCR antigen-binding domain or scFv can be a pan y8 TCR antigen-binding domain or scFv or derived from an pan y8 TCR antibody, including, for example, clone IMMU510 available from Immunotech. Beckman Coulter Life Sciences; TCR gamma / delta Monoclonal Antibody (5A6.E9) available from Thermo Fisher Scientific; ySTCR-APC (Clone: Bl available from BD Biosciences); pan-y8 antibody (Catalogue number Sc-100289 available from Santa Cruz Biotechnology); 11F2 available from ThermoFisher Scientific and von Lilienfeld-Toal et al. (2006). Clin Exp Immunol (2006) 144:528-33; 6TCS 1 described, for example, in Rivas et al. (1989), J. Immunol. 142: 1840-6; anti-human REafffinity TCRg / d antibody (Clone REA591 ) available from Miltenyi Biotec; monoclonal antibody clone G5-4 and is described in CN103130894A: anti-gamma delta T cell receptor (Pan-GDTCR) described US20250017966; the pan y8 TCR antibody described in US20230408523; Pan05 and Pan07 described, for example, in US20230009275 (see, for example, FIGs. 20, 21, 22, 23, 24, 25 of the published application); the contents of which are expressly disclosed herein. These antibodies, for example, have binding affinity for pan-y8 TCR (e.g., pan-TCR epitope) so can engage both V81 and V82 TCRs. In yet further aspects, the anti-y8 TCR antigen-binding domain or anti-y8 TCR scFv comprise at least the CDRs from clone IMMU510 available from Immunotech, Beckman Coulter Life Sciences; TCR gamma / delta Monoclonal Antibody (5A6.E9) available from Thermo Fisher Scientific; y8TCR-APC (Clone: Bl available from BD Biosciences); pan-y8 antibody (Catalogue number Sc-100289 available from Santa Cruz Biotechnology); 11F2 available from ThermoFisher Scientific and von Lilienfeld-Toal et al. (2006). Clin Exp Immunol (2006) 144:528-33; 6TCS 1 described, for example, in Rivas et al. (1989), J. Immunol. 142: 1840-6; anti-human REafffinity TCRg / d antibody (Clone REA591) available from Miltenyi Biotec; monoclonal antibody clone G5-4 and is described in CN103130894A: anti-gamma delta T cell receptor (Pan-GDTCR) described US20250017966; the pan y8 TCR antibody described in US20230408523; Pan05 and Pan07 described, for example, in US20230009275 (see, for example, FIGs. 20, 21, 22, 23, 24, and 25 of in US20230009275).

[0116] The multifunctional antigen-binding molecule, for example, a trispecific antigenbinding molecule, can further comprise a third domain, for example, a third antigen-binding domain. The third domain can, for example, promote expansion of y8 T cells. For example, it can promote expansion of the V81 and V82 subpopulations of y8 T cells, and optionally promote expansion of one or more other y8 T cell subtypes. Tn certain aspects, the third domain binds or specifically binds to an epitope expressed by y8 T cells (for example, to a y8 TCR), wherein the epitope is different from that which the second antigen-binding domain binds, and wherein the binding of the third domain to the different epitope promotes the expansion of the y8 T cells. In certain aspects, the binding of the third domain to the different epitope promotes the expansion of the V81 and V82 subpopulations of y8 T cells. In further aspects, the binding of the third domain to the different epitope promotes the expansion of the V81 and V82 subpopulations of y8 T cells and one or more other y8 T cell subtypes. The different epitope to which the third domain binds can be expressed by more than one y8 T cell subtype (for example, an epitope on the y8 TCR common to the TCR of more than one y8 T cell subtype). For example, the epitope can be expressed by the V81 and V82 subpopulations and optionally one more additional y8 T cell subtype. The binding of the third domain to the epitope expressed on more than on y8 T cell subt pe thus promotes expansion of more than one y8 T cell subtype as described herein. Various antigens are known to stimulate expansion of y8 T cells, including isopentenyl pyrophosphate (IPP), (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), zoledronate (Zometa), and bromohydrin pyrophosphate (BrHPP). In certain aspects, the third domain comprises an activation domain or a co-stimulatory domain. For example, the third domain can be derived from (e.g., can be a fragment of, or an activation or co-stimulatory or other domain of) IL-2, IL-12, IL-15. CD3, NKG2D, CD28, CD3. 4-1BB. DAP-10. CD80. CD86, 0X40, or IPP. In certain examples, the third domain is an activation domain of IL-2, IL- 12 or IL-15. In yet other examples, the third domain is the co-stimulatory domain of 4-1BB or CD28. In other aspects, the third domain targets the ySTCR, e.g., an epitope on the y8 TCR common to the TCR of more than one y8 T cell subtype. In further aspects, the third domain comprises an antibody or antibody fragment that that targets the y8 TCR or other epitope on y3 T cells that promotes expansion of y8 T cells. An exemplary anti-y8 TCR antibody that expands both V81 and V82 subpopulations of y8 T cells is IMMU510 or another antibody that binds more than one y8 T cell subpopulation as described herein.

[0117] A linker can be present between various domains of the multifunctional, trispecific or bispecific molecule described herein. In certain embodiments, a linker can be present that attaches the antigen-binding domains, e.g., the antigen binding domain that binds the TAA (e.g., the first antigen-binding domain) and the antigen binding domain that binds the y8 TCR (e.g., the second antigen-binding domain). For example, in a bispecific antigen-binding molecule, a linker attaches the antigen binding domain that binds the TAA (e.g., CD33) to the antigen binding domain that binds the y8 TCR. In a trispecific antigen-binding molecule, a linker can attach the first antigen-binding domain to the second antigen-binding domain, and / or can attach the second antigen-binding domain to the third antigen-binding domain, and / or can attach the first antigen-binding domain to the third antigen-binding domain. In certain aspects, the linker is a peptide linker from 1 to 30 amino acids. In yet other aspects, the peptide linker is less than 15 amino acids in length. A linker can also be present between some or all of the individual elements of the multifunctional antigen-binding molecule or bispecific antigen-binding molecule. For example, a linker can be present that attaches one of the antigen-binding domains to a hinge or transmembrane region (see, for example, FIG. 2A). Each linker peptide in the multifunctional antigen-binding molecule can be the same or can be different. An exemplary linker peptide can be 30 amino acids in length or less, 20 amino acids in length or less, or 15 amino acids in length or less. Non-limiting examples of such linker peptides are FLAG, influenza virus haemagglutinin (HA), c-myc, polyHis; Strep tags, Strep II tags, FLAG tags, glutathione S-transferase (GST) tags, green fluorescent protein (GFP) tags, hemagglutinin A (HA) tags, histidine (His) tags, luciferase tags, maltose-binding protein (MBP) tags, c-Myc tags, protein A tags, protein G tags, a human serum albumin (HSA), or influenza virus haemagglutinin. In certain aspects, the peptide linker is or comprises c-myc (for example, having the amino acid sequence of EQKLISEEDL (SEQ ID NO: 1) or FLAG (for example, having the amino acid sequence of DYKDDDDK (SEQ ID NO: 2). In some example, the linker comprises (GGGGS)n, wherein n is an integer from 1 to 10, for example, GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 14), GGGGSGGGGSGGGGS (SEQ ID NO: 10), or GGGGSGGGGS (SEQ ID NO: 16). Another example of a linker peptide is (GGGGS)n, wherein n is an integer from 1 to 10 (SEQ ID NO: 12), for example. GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 9). In yet another embodiment, the linker peptide is HA. for example, having an amino sequence of GLFGAIAGFIENG (SEQ ID NO: 3) or EGMIDGWYG (SEQ ID NO: 4). In yet further aspects, the linker comprises FLAG or c-myc and (GGGGS)n(SEQ ID NO: 10), for example (GGGGS)4 (SEQ ID NO: 14). In a specific example, the linker is c-myc-(GGGGS)4 or FLAG-(GGGGS)4.

[0118] In some embodiments, the multifunctional or bispecific antigen-binding molecule comprises a signal peptide N-terminal to the antigen-binding domain closest to the N- terminus. The terms “signal peptide” and “signaling peptide” are used interchangeably herein. A signal peptide is a peptide that directs the engager to proper folding, modification and / or transportation. For example, the signal peptide can be the signal peptide of a protein selected from the group consisting of CD8a, CD28, GM-CSF, CD4, CD137, or a combination thereof. In some examples, the signal peptide is from CD8a or CD28.

[0119] In certain examples, one of the antigen-binding domains (e.g.. the antigen-binding domain that binds the TAA or the antigen binding domain that binds a y5 TCR) can be linked (e.g., by a linker) to a transmembrane domain. In some examples, the transmembrane domain is a CD28 transmembrane domain. The antigen binding domain and the transmembrane domain can be linked by an extracellular hinge domain or an extracellular spacer sequence. For example, the extracellular hinge domain can be the CD28 hinge domain. In yet additional examples, the transmembrane domain is a CD28 transmembrane domain and the extracellular hinge domain is the CD28 hinge domain. In an additional example, the transmembrane domain and / or one of the antigen-binding domains is attached (directly or indirectly) to a co-stimulatory domain. For example, the co-stimulatory domain can be a CD28 co-stimulatory domain. In some examples, the transmembrane domain is a CD28 transmembrane domain, the extracellular hinge domain is the CD28 hinge domain, and the co-stimulatory domain can be a CD28 co-stimulatory domain.

[0120] As discussed herein, the transmembrane domain can be attached to an antigen-binding domain via an extracellular hinge region. In certain aspects, the extracellular spacer or extracellular hinge domain sequence comprises one or more of a hinge region and / or a portion of an immunoglobulin heavy chain constant region (which may comprise CHI, a linker region, CH2 and / or CH3 domains) or any combination thereof, of a human immunoglobulin, i.e., IgA. IgD, IgE, IgG, and IgM. In certain embodiments, extracellular spacer or hinge domain comprises all or a portion of the hinge region of human IgD. In certain embodiments, extracellular spacer or hinge comprises all or a portion of the hinge region of human IgGl. In certain embodiments, the extracellular spacer or hinge comprises all or a portion of the hinge region of human IgD and all or a portion of the hinge region of human IgGl. In certain embodiments, the extracellular spacer or hinge comprises all or a portion of the hinge region of human IgD and all or a portion of the CH2 and CH3 domains of the heavy- chain constant region of human IgGl. In certain embodiments, the extracellular spacer or hinge comprises all or a portion of the hinge region of human IgD, all or a portion of the hinge region of human IgGl and all or a portion of the CH2 and CH3 domains of the heavy chain constant region of human IgGl. In certain embodiments, the extracellular spacer or hinge comprises all or a portion of the hinge region of human IgGl and all or a portion of the CH2 and CH3 domains of the heavy chain constant region of human IgGl. In certain embodiments, extracellular spacer or hinge comprises all of the hinge region of human IgD, all or a portion of the hinge region of human IgGl and the heavy chain constant region comprises all or a portion of the CH2 and CH3 domains of human IgGl. Preferably the hinge region amino acid sequence comprises the hinge region amino acid sequence from an immunoglobulin, such from IgD or IgGl , wherein the amino acid sequence comprises from 1 to 5 amino acid modifications, which may be selected as discussed herein. Preferably, the CH2 and CH3 domains of the heavy chain constant region comprises the CH2 and CH3 domain immunoglobulin heavy chain constant region amino added sequence from an immunoglobulin, such from IgGl, wherein the amino acid sequence comprises from 1 to 5 amino acid modifications, which may be selected as discussed herein. In other aspects, the extracellular spacer or the extracellular hinge domain comprises the hinge region of a protein selected from the group consisting of CD8a, CD28, CD 137, or a combination thereof. In certain aspects, the extracellular spacer or the extracellular hinge domain comprises the hinge region of CD8a. In any of the foregoing, the extracellular spacer may further comprise a linker, such as a linker having the sequence of Ser-Gly-Gly-Gly (SEQ ID NO: 8), Ser-Gly- Gly-Gly-Gly (SEQ ID NO: 9), (Gly-Gly-Gly-Gly-Ser)4, (SEQ ID NO: 14), or (Gly-Gly-Gly- Gly-Ser)n(SEQ ID NO: 10) or wherein n is an integer from 1 to 10, linking the extracellular spacer to the extracellular antigen binding domain.

[0121] In certain embodiments, an antigen-binding domain can be linked to the transmembrane domain via a flexible linker. The flexible linker can be present in addition to the extracellular spacer or instead of the extracellular spacer described herein. In certain embodiments, the antigen-binding domain is linked to the extracellular spacer via a linker. For example, the linker can be a FLAG. In additional example, the linker can comprise glycine and serine. In certain aspects, the flexible linker is a linker as described herein, for example, comprised of a polypeptide having the sequence of SEQ ID NO: 10 (Ser-Gly-Gly- Gly)nor SEQ ID NO: 9 (Ser-Gly-Gly-Gly-Gly) wherein n is an integer from 1 to 10. The flexible linker can be a polypeptide comprising from about 1-25 amino acids, preferably about 1-15 amino acids, preferably about 1-10 amino acids, preferably about 4-24 amino acids, preferably about 5-20 amino acids, preferably about 5-15 amino acids and preferably about 5-12 amino acids. In certain aspects, the linker is (Ser-Gly-Gly-Gly)n wherein n is 3. Another example of a peptide linker comprises or is (GGGGS)n. wherein n is an integer from 1 to 10, for example, (GGGGS)2(SEQ ID NO: 16), (GGGGS)3(SEQ ID NO: 15), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 14) or (GGGGS)4. In yet further aspects, the linker comprises FLAG or c-myc and (GGGGS)n (SEQ ID NO: 10), for example (GGGGS)4(SEQ ID NO: 14). In yet additional aspects, the linker is c-myc-(GGGGS)4or FLAG- (GGGGS)4.

[0122] As discussed above, the multifunctional antigen-binding molecule, trispecific, or bispecific antigen-binding molecule can comprise a transmembrane domain that corresponds to, or is derived or obtained from, the transmembrane domain of any molecule known in the art. For example, the transmembrane domain can correspond to that of a CD8 molecule or a CD28 molecule. CD8 is a transmembrane glycoprotein that sen es as a co- receptor for the T- cell receptor (TCR) and is expressed primarily on the surface of cytotoxic T-cells. The most common form of CD8 exists as a dimer composed of a CD8 and CD8P chain. CD28 is expressed on T-cells and provides co-stimulatory signals required for T-cell activation. A transmembrane domain from a CD8 polypeptide may have the sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 11), particularly amino acids 1-21, 1- 23 or 1-24 of SEQ ID NO: 11). CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2). A transmembrane domain from a CD28 polypeptide may have the sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 12). Preferably, the CD8 and CD28 are human. Exemplary' transmembrane domains include, but are not limited to, all or a portion of a transmembrane domain from a polypeptide selected from: an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDIIa, CD18), ICOS (CD278), 4-IBB (CD137), GITR. CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1). CD160, CDI9. IL2Rp. !L2Ry. IL7Ra, ITGA1, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDIId, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CDllb, ITGAX, CD1 1c, ITGB1 , CD29, ITGB2, CD 18, LFA-1, ITGB7. TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4). CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD 160 (BY55), PSGLI, CD100 (SEMA4D), SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. One of skill in the art will be able to determine the corresponding transmembrane regions from these polypeptides.

[0123] The multifunctional antigen-binding molecule, trispecific antigen-binding molecule, or bispecific multifunctional antigen-binding molecule can include one or more costimulatory domains (e.g., CD28, 41BB, DAP10, 0X40 or ICOS). Non-limiting examples of co-stimulatory domains include CD28, CD27. 4-IBB. DAP- 10, 0X40, and combinations thereof, as well as other similar molecules and fragments as well as mutations to the foregoing, such as modifying the immunoreceptor tyrosine-based activation moti s) (IT AMs). In certain embodiments, the costimulatory domain is a functional signaling domain from 4 IBB, 0X40 and / or CD28. A costimulatory domain from 0X40 can, for example, have the sequence: ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 17). A costimulatory domain from CD28 can, for example, have the sequence. RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 18). A costimulatory domain from 41BB can. for example, have the sequence. KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 19). Preferably, the encoded costimulatory signaling domain comprises a functional signaling domain of a protein chosen from one or more of CD27, CD28, 4- IBB (CD 137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83. CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 160, CD 19, CD4, CD8cL CD8fi, IL2Rp, IL2Ry, IL7Ra, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA- 6, CD49f, ITGAD, CDIId, ITGAE, CD103, ITGAL, CD18, LFA-1, ITGAM, CDllb, ITGAX, CD1 1c, 1TGB I, CD29, ITGB2, CD18, LFA-1, ITGB7. TNFR2, TRANCE / RA KL, DNAM1 (CD226). SLAMF4 (CD 244. 2B4), CD84, CD96 (Tactile). CEACAM1. CRTAM. Ly9 (CD229), CD 160 (BY55), PSGLI, CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS. SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, orNKG2D. In additional aspects, the co-stimulatory domain is a co-stimulatory domain of CD28, CD28T, 0X40, 4-1BB / CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CDS, CD7, CD9, CD 16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, (1) 137, CD154, PD-1, ICOS, lymphocyte function- associated antigen- 1 (LFA-1 (CD1 la / CD 18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF, TNFr, integrin, signaling lymphocytic activation molecule, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR. BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, p80 (KLRF1). NKp44, NKp30. NKp46, CD 19. CD4. CD8alpha. CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA- 6, CD49f, ITGAD, CDlld, ITGAE, CD 103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2. TRANCE RANKL, DNAM1 (CD226), SLAMF4 D244. 2B4). CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGLL CDIOO (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, or CD83 ligand. Furthermore, any of the costimulaloiy domain sequences may contain from 1 to 5 amino acid modifications.

[0124] The multifunctional antigen-binding molecule, trispecific antigen-binding molecule, or bispecific multifunctional antigen-binding molecule can comprise any one of the aforementioned transmembrane domains and / or any of the aforementioned hinge domain and / or any of the aforementioned co-stimulatory domains, for example, a CD28 costimulatory domain. For example, the molecule can comprise a CD28 transmembrane domain and a CD28 co-stimulatory domain. Furthermore, any of the transmembrane domain sequences may contain from 1 to 5 amino acid modifications. In another example, the antigen-binding molecule can include a CD28 co-stimulatory domain.

[0125] The invention further includes a nucleic acid encoding the multifunctional antigenbinding molecule, trispecific antigen-binding molecule, or bispecific antigen-binding molecule described herein or a vector comprising the nucleic acid.

[0126] In some examples, the nucleic acid encodes a multifunctional antigen-binding molecule or bispecific multifunctional antigen-binding molecule comprises: i. a first antigen-binding domain that binds to a TAA, such as CD33 or CD19; ii. a second antigen-binding domain that binds to a yd TCR, for example, that specifically binds to an epitope expressed by more than one y8 T cell subtype; iii. a linker that attaches the first antigen-binding domain to the second antigenbinding domain. In certain aspects, the nucleic acid molecule further comprises a transmembrane domain and optionally, an extracellular hinge domain and / or a co-stimulalory domain. In additional examples, the nucleic acid molecule further comprises a fluorescent or detectable marker, for example, mCherry (a fluorescent protein derived from Dis cosoma sp.), and eGFP (green fluorescent protein). mCherry and eGFP / EGFP are selectable markers used for research. The nucleic acid or vector can further encode a ribosome-skipping / self-cleaving peptide between the marker and the other domains. Examples of ribosome-skipping / self- cleaving peptides include, for example, porcine teschovirus-1 2A (P2A) sequence, thosea asigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), cytoplasmic polyhedrosis virus (BmCPV 2A), and flacherie virus (BmIFV 2A) of B. mori. In certain aspects, the multifunctional antigen-binding domain comprises the third domain as described herein.

[0127] In yet additional aspects, the invention is directed to a vector (such as a lentiviral vector) comprising the nucleic acid that encodes a multifunctional antigen-binding molecule or bispecific multifunctional antigen-binding molecule.

[0128] The invention also encompasses a nucleic acid encoding a membrane-bound multifunctional or bispecific antigen-binding and immune cell engaging molecule, wherein the membrane-bound molecule comprises an extracellular domain and a transmembrane / intracellular region, wherein the nucleic acid or vector comprises: i. an antigen-binding domain that binds to a tumor-associated antigen (TAA); ii. an antigen-binding domain that binds to an immune cell antigen; iii. a linker that attaches the antigen-binding domain that binds to the TAA to the antigen-binding domain that binds to the T-cell antigen; and iv. a transmembrane domain; and wherein the extracellular domain comprises the antigen-binding domain that binds to the TAA, the antigen-binding domain that binds to the T-cell antigen, and the linker; and further wherein the transmembrane / intracellular region comprises the transmembrane domain. In certain aspects, the membrane-bound multifunctional antigen-binding molecule is a membrane-bound bispecific antigen-binding molecule. In additional examples, the nucleic acid molecule further comprises a fluorescent or detectable marker, for example. mCherry (a fluorescent protein derived from Discosoma sp.), and eGFP (green fluorescent protein). mCherry and eGFP / EGFP are selectable markers used for research. The nucleic acid or vector can further encode a ribosome-skipping / self-cleaving peptide between the marker and the other domains. Example of ribosome-skipping / self-cleaving peptides include, for example, porcine teschovirus-1 2A (P2A) sequence, thosea asigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), cytoplasmic polyhedrosis virus (BmCPV 2A), and flacherie virus (BmIFV 2A) of B. mori. In yet additional aspects the membrane-bound multifunctional antigen-binding molecule further comprises an extracellular hinge domain and / or an intracellular co-stimulatory domain. In certain specific aspects, the transmembrane domain, extracellular hinge, and co-stimulatory domains are CD28 transmembrane domain, CD28 extracellular hinge, and CD28 co-stimulatory domain, respectively. Also encompassed herein is a vector (such as a lentiviral vector) comprising the nucleic acid encoding the membranebound multifunctional or bispecific antigen-binding molecule. In certain aspects, the membrane-bound multifunctional or bispecific antigen-binding molecule is overexpressed. Also encompassed is a host cell comprising the vector.

[0129] Also described herein is a method for the production of a multifunctional antigenbinding and immune cell engaging molecule as a membrane-bound protein, wherein the extracellular domain of the membrane-protein is harvested, and can be recovered or isolated. Therefore, the invention encompasses a process for the production of a multifunctional antigen-binding and immune cell engaging molecule, the process comprising culturing the host cell comprising a vector comprising the nucleic acid that encodes the membrane-bound multifunctional or bispecific antigen-binding molecule as described herein. The ectodomain or extracellular domain of the membrane-bound multifunctional or bispecific antigen-binding molecule (which comprises the antigen binding domains) can be purified from the culture.

[0130] A multifunctional antigen-binding molecule, trispecific multifunctional antigenbinding molecule, or bispecific multifunctional antigen-binding molecule, or membranebound multifunctional antigen-binding molecule or bispecific multifunctional antigenbinding molecule according to the present invention can be produced by any means known in the art, for example, it can be produced using recombinant DNA techniques. A nucleic acid sequence encoding the several regions of the multifunctional antigen-binding molecule or bispecific multifunctional antigen-binding molecule can prepared and assembled into a complete coding sequence by standard techniques of molecular cloning (chemically synthesized DNA assembly, genomic library’ screening, PCR. primer-assisted ligation, site- directed mutagenesis, etc.). The resulting coding region is preferably inserted into an expression vector and used to transfect a suitable expression host-cell line or primary' cell, such as HEK-293T cells. As used herein, a "nucleic acid construct’' or "nucleic acid sequence" is intended to mean a nucleic acid molecule, such as a DNA molecule, that can be transformed or introduced into an expression host-cell line and be expressed to produce a product (e.g., a chimeric receptor). Therefore, the invention further provides an isolated or purified nucleic acid sequence encoding the multifunctional antigen-binding molecule or bispecific multifunctional antigen-binding molecule described herein. "Nucleic acid sequence" is intended to encompass a polymer of DNA or RNA, i.e., a polynucleotide, which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides. The terms "nucleic acid" and "polynucleotide" as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, and double- and single -stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to methylated and / or capped polynucleotides. In the nucleic acid construct employed in the present invention, the promoter is operably linked to the nucleic acid sequence encoding a multifunctional antigen-binding molecule or bispecific multifunctional antigen-binding molecule (including, for example, the membrane-bound molecules) of the present invention, i.e., they are positioned so as to promote transcription of the messenger RNA from the DNA encoding the chimeric receptor. The promoter can be of genomic origin or synthetically generated. A variety of promoters are known in the art. The promoter can be constitutive or inducible, where induction is associated with the specific cell type or a specific level of maturation, for example. Alternatively, a number of well-known viral promoters are also suitable. Promoters of interest include the EFla promoter. P-actin promoter, SV40 early and late promoters, immunoglobulin promoter, human cytomegalovirus promoter, retrovirus promoter, and the Friend spleen focus-forming virus promoter. The promoters may or may not be associated with enhancers, wherein the enhancers may be naturally associated with the particular promoter associated with a different promoter.

[0131] The various manipulations for preparing the multifunctional antigen-binding molecule or bispecific multifunctional antigen-binding molecule of the invention (including, for example, the membrane-bound molecules) can be carried out in vitro and the construct can be introduced into vectors for cloning and expression in an appropriate cell using standard transformation or transfection methods. Thus, after each manipulation, the resulting construct from joining of the DNA sequences is cloned, the vector isolated, and the sequence screened to ensure that the sequence encodes the desired chimeric receptor. The sequence can be screened by restriction analysis, sequencing, or the like. Therefore, the invention comprises vectors encoding the multifunctional antigen-binding molecule or bispecific multifunctional antigen-binding molecule described herein (including, for example, the membrane-bound molecules)or functional equivalents thereof.

[0132] The invention additionally comprises cells (e.g., host cells) containing (i.e., transformed or transduced with) vectors encoding multifunctional antigen-binding molecule, bispecific multifunctional antigen-binding molecule, membrane-bound multifunctional antigen-binding molecule or membrane-bound bispecific multifunctional antigen-binding molecule of the invention, as well as functional variants thereof. Examples of mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36: 59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23: 243- 251 (1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2,1413 8065); mouse mammary tumor (MMT 060562, ATCC CCL5 1 ); TRI cells (Mather et a / ., Annals N. Y Acad. Sci. (1982) 383: 44-68); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). In certain aspects, the mammalian host cell is a HEK293T cell, HEK293 cell, HEK293FT Cell, Hela cell and CHO (Chinese Hamster Ovary) cell.

[0133] Compositions and Methods of Treatment

[0134] As described herein, the multifunctional antigen-binding molecule, trispecific antigenbinding molecule, or bispecific antigen-binding molecule can be used in the treatment of cancer. For example, encompasses are methods of treating cancer in a subject in need thereof comprising administering an effective amount of a multifunctional antigen-binding molecule, trispecific antigen-binding molecule, or bispecific multifunctional antigen-binding molecule as described herein. In certain aspects, each antigen-binding domain is a single-chain variable fragment (scFv). Specifically, described herein are methods of treating cancer in a subject in need thereof, the method comprising administering to said subject an effective amount of a population of 78 T cells and a multifunctional antigen-binding molecule, trispecific antigenbinding molecule, or bispecific multifunctional antigen-binding molecule, wherein the molecule comprises i) a first antigen-binding domain that binds to a tumor-associated antigen (TAA) and ii) a second antigen-binding domain that binds to a y8 TCR; and optionally, iii) a third domain. In certain aspects, each antigen-binding domain is a single-chain variable fragment (scFv). In yet further aspects, the TAA is selected from the group consisting of CD19, CD33, CD123, CD20, CD22, EGFRvIII, IL13ra2, folate receptor 1 (FOLR1), and mesothelin. In certain additional embodiments, the TAA is CD33. For example, when the TAA is CD33, the cancer is a CD33-posititive cancer. In yet additional aspects, the TAA is CD19. For example, when the TAA is CD19, the cancer is a CD19-positive cancer.

[0135] There are several types of cancer, for example, carcinoma is cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is cancer that begins in bone, cartilage, fat. muscle, blood vessels, or other connective or supportive tissue. Leukemia is cancer that starts in blood-forming tissue such as the bone marrow and causes large numbers of abnormal blood cells to be produced and enter the bloodstream. Lymphoma is cancer that begins in the cells of the immune system.

[0136] In certain aspects, the invention is directed to the treatment of a hematologic or hematopoietic cancer. Non-limiting examples of cancers include acute hematopoietic cancers (myelodysplastic cancer), myelodysplastic syndromes, Lymphocytic Leukemia (ALL), Acute Myelogenous Leukemia (AML), adult B-cell malignancies, including CLL (chronic lymphocytic leukemia), CML (chronic myelogenous leukemia), non-Hodgkin lymphoma (NHL), pediatric B-cell malignancies, including B lineage ALL (acute lymphocytic leukemia), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological and solid tumors, or any combination thereof. Such cancers include hematopoietic cancers (myelodysplastic cancer), myelodysplastic syndromes, pancreatic cancer, head and neck cancer, skin tumors, Minimal Residual Disease (MRD) among: acute Lymphocytic Leukemia (ALL), Acute Myelogenous Leukemia (AML), adult B-cell malignancies, including CLL (chronic lymphocytic leukemia), CML (chronic my elogenous leukemia), non-Hodgkin lymphoma (NHL), pediatric B-cell malignancies, including B lineage ALL (acute lymphocytic leukemia), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological and solid tumors, or any combination thereof.

[0137] In yet additional aspects, the cancer is a B-cell lymphoma. Most non-Hodgkin lymphoma are B-cell lymphomas. Non-limiting examples of B-cell lymphoma include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic leukemia (SLL), mantle cell lymphoma, marginal zone lymphoma, Burkitt lymphoma, Burkitt-like lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), Hairy cell leukemia, primary central nervous system lymphoma, primary intraocular lymphoma.

[0138] In certain aspects, the cancer to tumor being treated can be an intracranial tumor. Intracranial tumors include, but are not limited to, gliomas, meningiomas, acoustic neuromas, pituitary adenomas, medulloblastomas, germ cell tumors and craniopharyngiomas. In some aspects, the cancer being treated in accordance with the invention is a CNS tumor including, but not limited to, intracranial and spinal ependymoma (excluding subependymoma); low grade infiltrative supratentorial astrocytoma / oligodendroglioma, medulloblastoma, anaplastic gliomas, glioblastoma, metastatic lesion of the CNS and primary CNS lymphoma.

[0139] In some aspects, the cancer being treated is a melanoma. In certain aspects, the cancer being treated is uveal melanoma.

[0140] In some aspects, the cancer being treated is a neuroendocrine or adrenal tumor. Examples include but are not limited to bronchopulmonary disease, GI tract, lung or thymus, pancreas, paraganglioma or pheochromocytoma.

[0141] In some aspects, the cancer being treated is non-Hodgkin’s lymphoma including but not limited to mycosis fungoides and Sezary syndrome.

[0142] In some aspects, the cancer being treated is a soft tissue sarcoma. Examples include angiosarcoma, unresectable or progressive retroperitoneal / intra-abdominal soft tissue sarcoma, rhabdomyosarcoma, extremity / superficial trunk and / or head and neck cancer, or solitary fibrous tumor / hemangiopericytoma.

[0143] In some aspects, the cancer being treated is bone cancer. Examples include Ewing’s sarcoma and mesenchymal chondrosarcoma.

[0144] In some aspects, the cancer being treated is uterine sarcoma, small cell lung cancer (SCLC) or Zollinger-Ellison syndrome.

[0145] In some aspects, the cancer being treated in accordance with the invention is a gynecologic cancer (e.g., cancers of the female reproductive system) including, but not limited to ovarian cancer, cancer of the fallopian tube(s), peritoneal cancer and breast cancer. In some aspects, the cancer being treated in accordance with the invention is ovarian cancer.

[0146] In some aspects, a cancer being treated in accordance with the invention is glioblastoma.

[0147] Brain tumors spread extensively within the brain but do not usually metastasize outside the brain. Gliomas are very invasive inside the brain, even crossing hemispheres. They do divide in an uncontrolled manner, though. Depending on their location, they can be just as life threatening as malignant lesions. An example of this would be a benign tumor in the brain, which can grow and occupy space within the skull, leading to increased pressure on the brain.

[0148] In certain aspects, the cancer is a CD33-associated cancer, for example, when the multifunctional antigen-binding molecule or bispecific multifunctional antigen-binding molecule comprises an antigen binding domain that specifically binds CD33. CD33- associated cancers include, but are not limited to, hematopoietic cancers, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, acute Lymphocytic Leukemia (ALL), Acute Myeloid Leukemia (AML), adult B-cell malignancies, including CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin's lymphoma (NHL), pediatric B-cell malignancies, including B lineage ALL (acute lymphocytic leukemia), multiple myeloma, lung, breast, ovarian, prostate, colon, melanoma, or other hematological and solid tumors, or any combination thereof. In certain aspects, the CD33-associated cancer is a leukemia such as AML, CLL, CML or ALL. In other aspects, the CD-33 associated cancer is AML, myelodysplastic syndromes, or CLL.

[0149] In yet additional aspects, the cancer is a CD19-associated cancer, for example, when the multifunctional antigen-binding molecule or bispecific multifunctional antigen-binding molecule comprises an antigen binding domain that specifically binds CD19. CD19- associated cancers include ALL, multiple myeloma, and non-Hodgkin's lymphoma.

[0150] In further aspects, the multifunctional antigen-binding molecule, trispecific antigenbinding molecule, or bispecific multifunctional antigen-binding molecule comprises an antigen binding domain that specifically binds CD19 can be used to treat B-cell related diseases or conditions. B-cells are known to play a role of the pathogenesis of numerous disorders including cancers, autoimmune diseases and certain inflammatory diseases. Because of this role, B-cells are a therapeutic target and the selective depletion of B cells can be used in the treatment of B-cell associated cancer, autoimmune disorders and inflammatory disease. Therefore, provided herein are methods for reducing the number of B cells in a patient in need thereof, for example, an autoimmune disease patient, comprising administering to said patient an effective amount of a multifunctional antigen-binding molecule or a bispecific antigen-binding molecule described herein, for example, comprising i. a first antigen-binding domain that specifically binds to CD19, and ii. a second antigenbinding domain that specifically binds to a y3 TCR (e g., a human y3 TCR). Also encompassed herein is a method of treating a B-cell mediated inflammatory disease or B-cell mediated autoimmune disease in a patient in need thereof comprising administering to said patient an effective amount of a multifunctional antigen-binding molecule or a bispecific antigen-binding molecule comprising i. a first antigen-binding domain that specifically binds to CD19, and ii. a second antigen-binding domain that specifically binds to a y8 TCR (e.g., a human y8 TCR). In certain aspects, the method is a method of treating a B-cell mediated autoimmune disease. In certain aspects, the second antigen-binding domain specifically binds to an epitope expressed by more than one y8 T cell subtype. In certain additional aspects, the second antigen-binding domain binds to an epitope expressed by both V81 and V82 subpopulations of y8 T cells. In yet additional aspects, the epitope is also expressed by another y8 T cell subty pes, or in other words, non-V81V82 subpopulations. In yet further aspects, the second antigen-binding domain is a pan y8 TCR antigen-binding molecule. In certain specific aspects, one or more antigen-binding domains is a single-chain variable fragment (scFv). B-cell mediated autoimmune diseases include diseases characterized by the loss of B-cell tolerance and / or the inappropriate production of autoantibodies by B cells or overexpansion of B cells. Non-limiting examples of a B-cell mediated autoimmune disease include systemic lupus erythematosus, lupus nephritis, multiple sclerosis, rheumatoid arthritis, antiphospholipid syndrome, pemphigus, cicatricial pemphigoid, myasthenia gravis, neuromyelitis optica, immune thrombocytopenia, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas' disease, Graves' disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, ANCA-associated vasculitis. Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia and rapidly progressive glomerulonephritis, heavy chain disease, primary or immunocyte-associated amyloidosis and monoclonal gammopathy of undetermined significance. Non-limiting examples of a B-cell mediated inflammatory’ disease are type 2 diabetes, periodontal disease, and graft-versus-host disease.

[0151] In certain specific aspects, the invention is a method for reducing the number of B cells in a systemic lupus erythematosus patient in need thereof comprising administering to said patient an effective amount of the multifunctional antigen-binding molecule or the bispecific antigen-binding molecule. Also encompassed herein is a method of treating a B- cell mediated inflammatory disease or B-cell mediated autoimmune disease in a patient in need thereof comprising administering to said patient an effective amount of the multifunctional antigen-binding molecule or the bispecific antigen-binding molecule. The multifunctional, trispecific or bispecific antigen-binding molecule and optionally, a composition comprising y5 T-cells, can be administered in combination with an additional therapeutic treatment for the treatment of cancer or B-cell mediated conditions described herein. Such additional therapeutic treatments include, but are not limited to, surgery, chemotherapy (e.g., an additional chemotherapeutic agent different from the chemotherapeutic agent to which the DR cells are resistant), checkpoint inhibitors. PARP inhibitors, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, my cophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, Cytoxan, fludarabine, FK506, rapamycin, mycophenolic acid, steroids, and cytokines. In yet additional aspects, the additional therapeutic agent is an immune checkpoint inhibitor, as described, for example, in WO2018 / 035413, the contents of which are expressly incorporated by reference herein. For example, immune checkpoint inhibitor can be one that targets CTLA-4, PDL1, PDL2, PD1, B7-H3. B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160 (also referred to as BY55), CGEN-15049. CHK 1 kinase. CHK2 kinase, A2aR, 0X40. or a B-7 family ligand. In further aspects, the additional therapeutic agent is a DDR inhibitor, including but not limited to PARP inhibitors as described, for example, in WO 2020 / 097306, the contents of which are expressly incorporated by reference herein. Non-limiting examples of PARP inhibitors are olaparib, rucaparib, niraparib, Talazoparib (Pfizer), veliparib (Abbvie), E7016 (Eisai), CEP-9722 (Teva), and BGB-290 (Pamiparib, BeiGene), as well as combinations thereof.

[0152] In certain aspects, the methods further comprise administration of a chemotherapeutic agent and / or a checkpoint inhibitor.

[0153] The term “chemotherapeutic agent7’ as used herein refers to a compound or a derivative thereof that can interact with a cancer cell, thereby reducing the proliferative status of the cell and / or killing the cell for example, by impairing cell division or DNA synthesis, or by damaging DNA, effectively targeting fast dividing cells. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., cyclophosphamide, ifosfamide. temozolomide); metabolic antagonists (e.g., methotrexate (MTX), 5-fluorouracil or derivatives thereof); a substituted nucleotide; a substituted nucleoside; DNA demethylating agents (also known as antimetabolites; e.g., azacitidine); antitumor antibiotics (e.g., mitomycin, adriamycin); plant-derived antitumor agents (e.g., vincristine, vindesine, TAXOL®, paclitaxel, abraxane); cisplatin; carboplatin; etoposide; and the like. Such agents may further include, but are not limited to, the anti-cancer agents trimethotrexate (TMTX); temozolomide (TMZ); raltitrexed; S-(4-Nitrobenzyl)-6-thioinosine (NBMPR); 6- benzyguanidine (6-BG); a nitrosoureas a nitrosourea (rabinopyranosyl-N-methyl-N- nitrosourea (Aranose), Carmustine (BCNU, BiCNU), Chlorozotocin, Ethylnitrosourea (ENU), Fotemustine, Lomustine (CCNU), Nimustine, N-Nitroso-N-methylurea (NMU), Ranimustine (MCNU), Semustine, and Streptozocin (Streptozotocin)); cytarabine; and camptothecin; or a therapeutic derivative of any thereof.

[0154] In certain aspects, the population of yd T cells administered to the subject comprises yd T cells that have been engineered to express a survival factor. The term ‘‘survival factor” refers to any agent now known or later discovered in the art that confers resistance to a chemotherapeutic agent, and / or to a chemotherapeutic agent treatment regimen and / or allows the cells comprising the survival factor to survive in a treatment environment (such as a chemotherapy treatment environment). Thus, when the population of yd T cells administered to the subject comprises yd T cells that express a survival factor, the chemotherapeutic agent to which the survival factor confers resistance can be co-administered. yd T-cells that express a survival factor can be used in methods of drug resistant immunotherapy as described, for example. U.S. Pat. No. 10.322,145, the contents of which are expressly incorporated by reference herein. The term “drug resistant immunotherapy” or DRI is a strategy for treating cancer whereby anti -cancer immune cells, preferably yd T-cells, are genetically engineered to resist the toxic effects of chemotherapy drugs which allows for the combined administration of chemotherapy and immunotherapy. Chemotherapy resistance or the acquisition of chemoresistance is a well-known phenomenon in the field of cancer treatment. Such resistance to chemotherapeutic agents can arise from the expression of certain DNA, RNA or polypeptides that impact drug resistance genes, expression of a gene that conveys drug resistance, the expression of a polypeptide that confers resistance to chemotherapeutic agents. The DRI strategy described herein uses chemoresistance to confer resistance to the immune cells that can be used in cancer immunotherapy. A polypeptide that confers resistance to a chemotherapeutic agent can be referred to herein as a “survival polypeptide” or a "survival factor”).

[0155] Exemplary survival factors have been described, for example, in U.S. Pat. Nos. 10,322,145 and 10,543,233 as well as WO2018 / 035413, WO2017041 106 and W02018107134, the contents of each of which are expressly incorporated by reference herein. The phrase “confers resistance” and the like encompasses the acquisition of resistance to a chemotherapeutic agent or improvement in resistance to a chemotherapeutic agent. The ‘"survival factor” includes an agent that confers resistance to a chemotherapeutic agent when it is expressed by the yb T-cell. The “survival factor” can thus be a DNA, RNA or polypeptide that is expressed by the yb T-cells (e.g., encoded by a drug resistance gene) and that confers resistance to a chemotherapeutic agent. As described herein, the yb T-cell can be engineered to express the DNA, RNA or polypeptide that confers resistance to a chemotherapeutic drug by including a vector which expresses a gene, a gene fragment, a DNA, an siRNA, or an mRNA, that encodes the survival factor that confers resistance to a chemotherapeutic agent. In yet other aspects, the survival factor is a DNA that confers resistance to a chemotherapeutic agent. In further aspects, the survival factor is an RNA (e.g., a RNAi, siRNA, micoRNA, or mRNA) that confers resistance to a chemotherapeutic agent.

[0156] In certain aspects, the survival factor is a polypeptide that confers resistance to a chemotherapeutic agent; for example, the polypeptide confers resistance when it is expressed by the yb T-cells. The survival polypeptide or polypeptide that confers resistance to a chemotherapeutic agent can be any polypeptide known in the art that provides resistance to a treatment regimen comprising a chemotherapeutic agent, and / or allows the cells comprising the survival polypeptide to survive in a treatment environment created by the chemotherapeutic agent. Exemplary chemotherapeutic agents are nucleoside-analog chemotherapy drug, alkylating agent, antimetabolite, antibiotic, topoisomerase inhibitor, mitotic inhibitor, differentiating agent, or hormone therapy agent and the survival factor provides resistance to the chemotherapeutic agent. In additional aspects, the chemotherapeutic agent is an alkylating agent. In certain embodiments, the survival polypeptide is MGMT, multidrug resistance protein 1 (MDRI), or 5' nucleotidase II (NT5C2). In yet further aspects, the survival polypeptide is MGMT and the chemotherapeutic agent is an alkylating agent such as carmustine (BCNU), lomustine (CCNU), and temozolomide. In certain aspects, the chemotherapeutic agent is temozolomide (TMZ). In additional aspects, the survival polypeptide is MDRI and the chemotherapeutic agent is an anthracycline, vinca alkaloids, epipodophyllotoxins, camptothecin, methotrexate (MTX), saquinavir, and mitoxantrone (MX) (Sodani et all. (2011). Multi drug resistance associated proteins in multidrug resistance. Chin J Cancer 31(2): 58-72). NT5C2 is a polypeptide known in the art to provide resistance to thiopurine chemotherapy (Tzoneva et al. (2013), Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL, Nat Med. 19(3): 368-371). Other survival polypeptides include, for example, a drug resistant variant of dihydrofolate reductase (L22Y-DHFR) and thymidylate synthase. In certain aspects, the survival polypeptide is MGMT. However, other survival factors may be used depending on the chemotherapeutic agent being co-administered, the nature of the treatment environment (i.e., what other treatment regimens are being given to the patient in combination with the cell compositions of the present disclosure). In certain embodiments, the survival factor is MGMT, multidrug resistance protein 1 (MDR1), or 5' nucleotidase II (NT5C2). Other survival factors include, for example, a drug resistant variant of dihydrofolate reductase (L22Y-DHFR) and thymidylate synthase. In certain aspects, the survival factor is MGMT. Other polypeptides that confer resistance may be used or expressed by the cell depending on the nature of the treatment environment (i.e., what other treatment regimens are being given to the patient in combination with the cells compositions of the present disclosure). MGMT repairs alkylating lesions of the DNA by removing mutagenic adducts from the 06 position of guanine. Such mutagenic adducts can be caused by alkylating agents (including, but not limited to, temozolomide). Thus, MGMT is a polypeptide that confers resistance to alkylating agents such as temozolomide. The survival factor can be a polypeptide that confers resistance to a chemotherapeutic agent, including, but not limited to, the specific chemotherapeutic agents described herein.

[0157] The chemotherapeutic agent to which the survival factor confers resistance can be any chemotherapeutic agent described herein. In additional aspects, the chemotherapeutic agent to which the survival factor confers resistance is an alkylating agent; a metabolic antagonist; a DNA demethylating agent; a substituted nucleotide; a substituted nucleoside; an antitumor antibiotic; a plant-derived antitumor agent or a nitrosourea. Preferably the chemotherapeutic agent is selected from cisplatin; carboplatin; cyclophosphamide; etoposide; fludarabine; methotrexate (MTX); trimethotrexate (TMTX); temozolomide; dacarbazine (DTIC), raltitrexed; S-(4-Nitrobenzyl)-6-thioinosine (NBMPR); 6-benzy guanidine (6-BG); a nitrosourea (rabinopyranosyl-N-methyl-N-nitrosourea (Aranose), Carmustine (BCNU, BiCNU), Chlorozotocin, Ethylnitrosourea (ENU). Fotemustine. Lomustine (CCNU), Nimustine. N-Nitroso-N-methylurea (NMU), Ranimustine (MCNU). Semustine. Streptozocin (Streptozotocin)); cytarabine; camptothecin; and a therapeutic derivative of any thereof. Preferably, the y5 T-cells have been genetically modified to encode alkyl guanine transferase (AGT), P140K-MGMT, O6methylguanine DNA methyltransferase (MGMT), L22Y-DHFR. thymidylate synthase, dihydrofolate reductase, or multiple drug resistance-1 protein (MDR1). In additional examples, the y5 T-cells can be genetically modified to be resistant to at least two chemotherapeutic agents selected from: an alkylating agent; a metabolic antagonist; a DNA demethylating agent; a substituted nucleotide; a substituted nucleoside; an antitumor antibiotic; a plant-derived antitumor agent and a nitrosurea. In some embodiments, the y5 T- cells are genetically modified to be resistant to at least two chemotherapeutic agents selected from cisplatin; carboplatin; etoposide; methotrexate (MTX); trimethotrexate (TMTX); temozolomide; dacarbazine (DTIC), raltitrexed; S-(4-Nitrobenzyl)-6-thioinosine (NBMPR); 6-benzy guanidine (6-BG); a nitrosourea (rabinopyranosyl-N-methyl-N-nitrosourea (Aranose), Carmustine (BCNU, BiCNU), Chlorozotocin, Ethylnitrosourea (ENU), Fotemustine, Lomustine (CCNU), Nimustine, N-Nitroso-N-methylurea (NMU), Ranimustine (MCNU), Semustine, Streptozocin (Streptozotocin)); cy tarabine; camptothecin; and a therapeutic derivative of any thereof. Preferably, the chemotherapeutic agent is TMZ, methotrexate, DTIC, BCNU, CCNU, MCNU, NMU or ENU. In additional aspects, the chemotherapeutic agent includes, but is not limited to: alkylating agents (e.g., cyclophosphamide, ifosfamide, melphalan); metabolic antagonists (e.g., methotrexate (MTX), 5 -fluorouracil or derivatives thereof); DNA demethylating agents (also known as antimetabolites; e.g., azacitidine): a substituted nucleotide; a substituted nucleoside; antitumor antibiotics (e.g., mitomycin, adriamycin); plant-derived antitumor agents (e.g., vincristine, vindesine, TAXOL®, paclitaxel, abraxane); cisplatin; carboplatin; etoposide; and the like. Such agents may further include, but are not limited to, the anti-cancer agents trimethotrexate (TMTX); temozolomide (TMZ); raltitrexed; S-(4-Nitrobenzyl)-6-thioinosine (NBMPR); 6-benzyguanidine (6-BG); nitrosoureas (for example, bis-chloroethylnitrosourea, also known as BCNU and carmustine, lomustine, also known as CCNU, + / - procarbazine and vincristine (PCV regimen) and fotemustine); doxorubicin; cytarabine; camptothecin; and a therapeutic derivative of any thereof.

[0158] In certain aspects, the population of y8 T cells administered to the subject comprises v5 T cells that have been engineered to express a chimeric antigen receptor (CAR). The term "chimeric antigen recep torts)" andCLCAR(s))," as used herein, refers to artificial T-cell receptors, T-bodies, single-chain immunoreceptors, chimeric T-cell receptors, or chimeric immunoreceptors, for example, and encompass engineered receptors that graft an artificial specificity (for example, an antigen recognition domain) onto a particular immune effector cell, for example, y3 T-cells. In some embodiments. CARs comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain that may vary in length and that comprises an antigen recognition domain. Also as described herein, a CAR can lack an intracellular signaling domain, for example, the CD3z intracellular signaling domain. Exemplary CARs have been described, for example, in W02017041106, W02018107134 and WO2022159582 (PCT Application No. PCT / US22 / 13130), the contents of which are expressly incorporated by reference herein. Additional examples of CARs are non-signaling CARs or nsCARs. An engineered y8 T-cell that expresses a CAR that does not comprise or include an intracellular T-cell activation domain such as CD3z signaling domain is referred to herein as a '‘non-signaling CAR y8 T-cell" or an “nsCAR.” Exemplary nsCARs have been described, for example, in PCT Application No. PCT / US24 / 16112; the contents of which are expressly incorporated by reference herein, flu some embodiments, the population of y5 T cells comprise y5 T cells that express a CAR or nsCAR directed to the same tumor antigen or TAA as the multifunctional antigen-binding molecule or bispecific antigen-binding molecule. In other aspects, the population of y3 T cells comprise y8 T cells that express a CAR or nsCAR is directed to a different tumor antigen or TAA on the cancer cell as the multifunctional antigen-binding molecule or bispecific antigen-binding molecule. In certain specific aspects, the population of y5 T cells comprise y8 T cells that express a CAR or nsCAR directed to CD33, and the multifunctional antigen-binding molecule or bispecific antigen-binding molecule comprises an antigen-binding domain that binds to CD33. In yet additional aspects, the population of yd T cells comprise y8 T cells that express a CAR or nsCAR directed to CD33, and the multifunctional antigen-binding molecule or bispecific antigen-binding molecule comprises an antigen-binding domain that binds to a tumor antigen other than CD33. In yet additional aspects, the population of y5 T cells comprise y§ T cells that express a CAR or nsCAR directed to CD 19, and the multifunctional antigen-binding molecule or bispecific antigen-binding molecule comprises an antigen-binding domain that binds to CD 19. In yet additional aspects, the population of y6 T cells comprise y8 T cells that express a CAR or nsCAR directed to CD 19, and the multifunctional antigen-binding molecule or bispecific antigen-binding molecule comprises an antigen-binding domain that binds to a tumor antigen other than CD 19.

[0159] The y5 T-cells and / or the multifunctional, trispecific, or bispecific antigen-binding molecule and / or additional agent used in the treatment method are used or administered in an effective amount or a therapeutically effective amount.

[0160] The compositions (e.g., the yS T cell product and / or the multifunctional, trispecific, or bispecific antigen-binding molecule) described herein can be delivered as a pharmaceutical composition, or made into an implant appropriate for administration in vivo, with appropriate carriers or diluents, which further can be pharmaceutically acceptable. The means of making such compositions or implants have been described in the art. Where appropriate, the compositions described herein can be formulated into a preparation in semisolid or liquid form, such as a capsule, solution, injection, inhalant, or aerosol, in the usual ways for their respective route of administration. Means know n in the art can be utilized to prevent or minimize release and absorption of the composition until it reaches the target tissue or organ, or to ensure timed- release of the composition. Desirably, however, a pharmaceutically acceptable form is employed which does not effectuate the cells. Thus, desirably the cells as described herein can be made into a pharmaceutical composition containing a balanced salt solution, for example, Hanks' balanced salt solution, or normal saline. Therefore, the invention includes pharmaceutical compositions comprising 78 T-cells of the present disclosure.

[0161] A pharmaceutical composition described herein can be used alone or in combination with other well-established agents useful for treating cancer or other condition to be treated. For example, an agent useful for treating cancer can include a chemotherapeutic agent as described herein. Whether delivered alone or in combination with other agents, the pharmaceutical composition of the present invention can be delivered via various routes and to various sites in a mammalian, particularly human, body to achieve a particular effect. One skilled in the art will recognize that, although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. For example, intradermal delivery may be advantageously used over inhalation for the treatment of melanoma. Local or systemic delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, intraportal, intrahepatic, peritoneal, subcutaneous, or intradermal administration.

[0162] The composition(s) described herein can be provided in unit dosage form wherein each dosage unit, e.g., an injection, contains a predetermined amount of the composition, alone or in appropriate combination with oilier active agents. The term unit dosage form as used herein refers to physically discrete units suitable as un it ary dosages for human and animal subjects, each unit containing a predetermined quantity7of the composition of the present invention, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle, where appropriate. The specifications for the unit dosage forms of the present invention depend on the particular pharmacodynamics associated with the pharmaceutical composition in the particular subject. For example, a therapeutically effective amount or sufficient number of the 78 T-cells, administered alone or in combination with an additional therapeutic agent, is introduced into the subject such that a long-term, specific, response is established. In another example, a therapeutically effective amount of the multifunctional, trispecific or bispecific antigen-binding molecule administered alone or in combination with an additional therapeutic agent, is introduced into the subject such that a long-term, specific, response is established. For example, in one embodiment, the response includes inhibition or treatment of cancer. In one embodiment, the response is the reduction in size of a tumor or elimination of tumor growth or regrowth or a reduction in metastasis to a greater degree than would otherwise result in the absence of the treatment with the y8 T-cells or composition thereof. In yet other aspects, the response is amelioration or treatment of a B- cell mediated disease or condition, such as a B-cell mediated inflammatory disease or a B- cell mediated autoimmune disease. In certain aspects, the therapeutically effective amount results in at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% decrease in tumor size. Accordingly, the therapeutically effective amount takes into account the route of administration and the number of cells and / or dose should be such that a sufficient number of so as to achieve the desired therapeutic response.

[0163] Furthermore, the amounts of the y8 T-cells of the present disclosure, the multifunctional or bispecific antigen-binding molecule and / or additional agent administered as described herein (e.g.. the amount per each cell to be contacted or the amount per certain body weight) can vary in different applications. In certain non-limiting examples, the concentration of the cells can be sufficient to provide in the subject being treated at least from about IxlO5to about IxlO10cells although any suitable amount can be utilized. The dosing schedule can be based on well-established cell-based therapies or an alternate continuous infusion strategy can be employed.

[0164] The amounts and dosages described herein provide general guidance to be utilized by the practitioner upon optimizing the method of the present invention for practice of the invention. The recitation herein of such ranges by no means precludes the use of a higher or lower amount of a component, as might be warranted in a particular application. For example, the actual dose and schedule can vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on inter- individual differences in pharmacokinetics, drug disposition, and metabolism. One skilled in the art can readily make any necessary adjustments in accordance with the exigencies of the particular situation. y8 cells, e.g., infused y8 T cells, are able to kill tumor cells in the recipient. Unlike antibody therapies, the y8 T cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control. In certain aspects, the cells administered to the patient, or their progeny, persist in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen months, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty -two months, twenty- three months, two years, three years, four years, or five years after administration of the cells to the patient. As described herein, the cancer to be treated can be a hematological cancer or a solid tumor.

[0165] In some aspects, the total amount of an agent to be administered in practicing a method of the invention can be administered to a subject as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol, in which multiple doses are administered over a prolonged period of time. One skilled in the art would know that the amount of the composition to treat a pathologic condition in a subject depends on many factors including the age and general health of the subject as well as the route of administration and the number of treatments to be administered. In view of these factors, the skilled artisan would adjust the particular dose, as necessary.

[0166] A pharmaceutical composition can be formulated to be compatible with the intended method or route of administration; exemplary routes of administration are set forth herein. Furthermore, the pharmaceutical compositions can be used in combination with other therapeutically active agents or compounds as described herein in order to treat or prevent the diseases, disorders and conditions as contemplated by the present disclosure.

[0167] Pharmaceutical compositions typically comprise a therapeutically effective amount of one or more agents and one or more pharmaceutically and physiologically acceptable formulation agents. Suitable pharmaceutically acceptable or physiologically acceptable diluents, carriers or excipients include, but are not limited to, antioxidants (e.g., ascorbic acid and sodium bisulfate), preservatives (e.g., benzyl alcohol, methyl parabens, ethyl or n-propyl, p-hydroxybenzoate), emulsifying agents, suspending agents, dispersing agents, solvents, fillers, bulking agents, detergents, buffers, vehicles, diluents, and / or adjuvants. For example, a suitable vehicle can be physiological saline solution or citrate buffered saline, possibly supplemented with other materials common in pharmaceutical compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary7vehicles. Those skilled in the art will readily recognize a variety of buffers that can be used in the pharmaceutical compositions and dosage forms contemplated herein. Typical buffers include, but are not limited to. pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. As an example, the buffer components can be water soluble materials such as phosphoric acid, tartaric acids, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and salts thereof. Acceptable buffering agents include, for example, a Tris buffer. N-(2-Hydroxyethyl)piperazine-N'-(2- ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N- Morpholinojethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), and N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).

[0168] After a pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form, a lyophilized form requiring reconstitution prior to use, a liquid form requiring dilution prior to use, or other acceptable form. Preferably, the pharmaceutical composition is provided in a single-use container (e g., a single-use vial, ampoule, syringe, or autoinjector (similar to, e.g.. an EPIPEN®), whereas a multi-use container (e.g., a multi-use vial) is provided in other embodiments. Any drug delivery apparatus can be used to deliver the composition, including implants (e.g., implantable pumps) and catheter systems, slow injection pumps and devices, all of which are well known to the skilled artisan. Depot injections, which are generally administered subcutaneously or intramuscularly, can also be utilized to release the polypeptides disclosed herein over a defined period of time. Depot injections are usually either solid- or oil-based and generally comprise at least one of the formulation components set forth herein. One of ordinary skill in the art is familiar with possible formulations and uses of depot injections.

[0169] The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents mentioned herein. The sterile injectable preparation can also be a sterile injectable solution or suspension in anon- toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1.3-butane diol. Acceptable diluents, solvents and dispersion media that can be employed include water, Ringer's solution, isotonic sodium chloride solution, CREMOPHOR EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS), ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed, including synthetic mono- or diglycerides. Moreover, fatly acids such as oleic acid, find use in the preparation of injectables. Prolonged absorption of particular injectable formulations can be achieved by including an agent that delays absorption (e.g., aluminum monostearate or gelatin).

[0170] The pharmaceutical compositions can be in a form suitable for oral use, for example, as tablets, capsules, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups, solutions, microbeads or elixirs. Pharmaceutical compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions can contain one or more agents such as, for example, sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets, capsules and the like contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients can be, for example, diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, com starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.

[0171] The tablets, capsules and the like suitable for oral administration can be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate can be employed. They can also be coated by techniques known in the art to form osmotic therapeutic tablets for controlled release. Additional agents include biodegradable or biocompatible particles or a polymeric substance such as polyesters, polyamine acids, hydrogel, polyvinyl pyrrolidone, polyanhydrides, polygly colic acid, ethylene-vinylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate, or lactide / glycolide copolymers, polylactide / glycolide copolymers, or ethylenevinylacetate copolymers in order to control delivery' of an administered composition. For example, the oral agent can be entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization, by the use of hydroxymethylcellulose or gelatin-mi crocapsules or poly (methylmethacrolate) microcapsules, respectively, or in a colloid drug delivery system. Colloidal dispersion systems include macromolecule complexes, nano-capsules, microspheres, microbeads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Methods for the preparation of the above-mentioned formulations will be apparent to those skilled in the art.

[0172] Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, kaolin or microcrystalline cellulose, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

[0173] Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture thereof. Such excipients can be suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, poly vinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents, for example a naturally-occurring phosphatide (e.g.. lecithin), or condensation products of an alkylene oxide with fatty acids (e.g., polyoxy-ethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., for heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene sorbitan monooleate). The aqueous suspensions can also contain one or more preservatives.

[0174] Oily suspensions can be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents can be added to provide a palatable oral preparation.

[0175] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified herein.

[0176] The pharmaceutical compositions can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example, liquid paraffin, or mixtures of these. Suitable emulsifying agents can be naturally occurring gums, for example, gum acacia or gum tragacanth; naturally occurring phosphatides, for example, soy bean, lecithin, and esters or partial esters derived from fatty acids; hexitol anhydrides, for example, sorbitan monooleate; and condensation products of partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate.

[0177] Formulations can also include carriers to protect the composition against rapid degradation or elimination from the body, such as a controlled release formulation, including implants, liposomes, hydrogels, prodrugs and microencapsulated delivers’ systems. For example, a time delay material such as glyceryl monostearate or glyceryl stearate alone, or in combination with a wax, can be employed.

[0178] Suppositories can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include, but are not limited to, cocoa butter and polyethylene glycols.

[0179] The pharmaceutical compositions suitable for use in accordance with the invention may be in any format (e.g., sprays for nasal or inhalation use) currently known or developed in the future.

[0180] Also provided are kits comprising the compositions typically comprise a therapeutically effective amount of one or more agents used in the combination therapies of the invention described herein. Kits typically include a label indicated the intended use of the contents of the kits and instructions for use.

[0181] Any of the compositions or a combination of the compositions described herein can be comprised in a kit. The kits may comprise one or more suitably aliquoted compositions of the present invention or reagents to generate compositions of the invention. The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits may include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. Such containers may include injection or blow molded plastic containers into which the desired vials are retained, for example.

[0182] The kits are generally in the form of a physical structure housing various components, as described below, and can be utilized, for example, in practicing the methods described above. A kit can include a composition comprising one or more of the therapeutic agents used in the combination therapy of the invention (e.g., a y5 T cell product) provided in, e.g., one or more sterile containers, which can be in the form of a pharmaceutical composition suitable for administration to a subject. The pharmaceutical composition can be provided in a form that is ready for use or in a form requiring, for example, reconstitution or dilution prior to administration. When the compositions are in a form that needs to be reconstituted by a user, the kit can also include buffers, pharmaceutically acceptable excipients, and the like, packaged with or separately the therapeutic agent. When combination therapy is contemplated, the kit can contain the several agents separately or they can already be combined in the kit.

[0183] A kit of the invention can be designed for conditions necessary to properly maintain the components housed therein (e.g., refrigeration or freezing). A kit can contain a label or packaging insert including identifying information for the components therein and instructions for their use (e.g., dosing parameters, clinical pharmacology of the active ingredient(s), including mechanism(s) of action, pharmacokinetics and pharmacodynamics, adverse effects, contraindications, etc.).

[0184] Each component of the kit can be enclosed within an individual container, and all of the various containers can be within a single package. Labels or inserts can include manufacturer information such as lot numbers and expiration dates. The label or packaging insert can be. e.g., integrated into the physical structure housing the components, contained separately within the physical structure, or affixed to a component of the kit (e.g., an ampule, syringe or vial).

[0185] Labels or inserts can additionally include, or be incorporated into, a computer readable medium, such as a disk (e.g., hard disk, card, memory disk), optical disk such as CD- or DVD-ROM / RAM. DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic / optical storage media, FLASH media or memory-type cards. In some embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via an internet site, are provided.

[0186] Enumerated Embodiments

[0187] A. Embodiment Set A

[0188] Embodiment A-l: A multifunctional antigen-binding molecule, wherein the molecule comprises i. a first antigen-binding domain that specifically binds to a tumor-associated antigen; ii. a second antigen-binding domain that specifically binds to a y5 TCR, wherein second antigen-binding domain binds to an epitope expressed by more than one yS T cell subtype; and iii. an optional third domain.

[0189] Embodiment A-2: The antigen-binding molecule of Embodiment A-l, wherein the second antigen-binding domain binds to an epitope expressed by both V51 and V62 subpopulations of y8 T cells.

[0190] Embodiment A-3: The antigen binding molecule of Embodiment A-2, wherein the epitope is expressed by another y<5 T cell subtype.

[0191] Embodiment A-4: The antigen-binding molecule of any one of the preceding Embodiments, wherein the second antigen-binding domain is a pan yd TCR antigen-binding molecule.

[0192] Embodiment A-5: The antigen-binding molecule of any one of Embodiments A-l to A-4, wherein the antigen-binding molecule is capable of expanding or enhancing the expansion of the V81 and V82 subpopulations of y8 T cells.

[0193] Embodiment A-6: The antigen-binding molecule of any one of Embodiments A-l to A-5, wherein the third domain is present and binds to a different epitope on y5 T cells than the second antigen-binding domain.

[0194] Embodiment A-7: The antigen-binding domain of Embodiment A-6, wherein the binding of the third antigen-binding domain to the different epitope promotes expansion of y5 T cells.

[0195] Embodiment A-8: The antigen-binding domain of Embodiment A-7, wherein the binding of the third antigen-binding domain to the different epitope promotes expansion of expansion of the V61 and V82 subpopulations of y6 T cells.

[0196] Embodiment A-9: The antigen-binding molecule of any one of the preceding embodiments, wherein each of the first and the second antigen-binding domains is independently selected from a Fab domain, a Fab', a single-chain variable fragment (scFv) or a single domain antibody (sdAb).

[0197] Embodiment A-9a: The molecule of Embodiment A-9, wherein the first antigenbinding domain is an scFv.

[0198] Embodiment A- 9b: The molecule of Embodiment A-9, wherein the second antigenbinding domain is an scFv.

[0199] Embodiment A-9c: The molecule of Embodiment A-9, wherein each of the first and the second antigen-binding domains is an scFv. Embodiment A-10: The antigen-binding molecule of any one of Embodiments A-l to A-4, wherein the first and the second antigen-binding domains are attached by a linker, and / or wherein the second antigen -binding domain and the third domain are attached by a linker.

[0200] Embodiment A-ll: The antigen-binding molecule of Embodiment A-10, wherein the linker is a peptide linker.

[0201] Embodiment A-l 2: The antigen-binding molecule of Embodiment A-9c, wherein the VH of the first scFv is attached to the VH of the second scFv by a linker, or wherein the VL of the first scFv is attached to the VL of the second scFv by a linker.

[0202] Embodiment A-l 3: The antigen-binding molecule of Embodiment A- 12, wherein the linker is a peptide linker.

[0203] Embodiment A-14: The antigen-binding molecule of any one of the preceding Embodiments, wherein the tumor-associated antigen (TAA) is selected from the group consisting of CD19, CD33, CD123, CD20, CD22, EGFRvIII, IL13ra2. folate receptor 1 (FOLR1), CLL-1, MMP-2, and mesothehn.

[0204] Embodiment A-l 5: The antigen-binding molecule of Embodiment A-14, wherein the TAA is CD33.

[0205] Embodiment A-l 6: The antigen-binding molecule of Embodiment A-14, wherein the TAA is CD 19.

[0206] B. Embodiment Set B

[0207] Embodiment B-l: A multifunctional antigen-binding molecule, wherein the molecule comprises i. a first antigen-binding domain that binds to CD33 (e g., that specifically binds to CD33), ii. a second antigen-binding domain that binds to a yb TCR, and iii. an optional third domain.

[0208] Embodiment B-2: The antigen-binding molecule of Embodiment B-l, wherein the second antigen-binding domain specifically binds to an epitope expressed by more than one yb T cell subtype.

[0209] Embodiment B-3: The antigen-binding molecule of Embodiment B-2, wherein the second antigen-binding domain binds to an epitope expressed by both Vbl and V52 subpopulations of yb T-cells.

[0210] Embodiment B-4: The antigen binding molecule of Embodiment B-3, wherein the epitope is expressed by another yb T cell subt pe. Embodiment B-5: The antigen-binding molecule of any one of the preceding Embodiments of Embodiment Set B, wherein the second antigen-binding domain is a pan y5 TCR antigen-binding molecule.

[0211] Embodiment B-6: The antigen-binding molecule of any one of preceding Embodiments of Embodiment Set B, wherein the third domain is present and binds to a different epitope on y3 T cells than the second antigen-binding domain.

[0212] Embodiment B-7 : The antigen-binding domain of Embodiment B-6, wherein the binding of the third domain to the different epitope promotes expansion of the y8 T cells.

[0213] Embodiment B-8: The antigen-binding domain of Embodiment B7. wherein the binding of the third domain to the different epitope promotes expansion of the V81 and V82 subpopulations of y8 T cells.

[0214] Embodiment B-9: The antigen-binding molecule of the preceding Embodiments of Embodiment Set B, wherein each of the first and second antigen-binding domains is independently selected from a Fab domain, a Fab', a single-chain variable fragment (scFv) or a single domain antibody (sdAb).

[0215] Embodiment B-10: The antigen-binding molecule of Embodiment B-9, wherein at least one antigen-binding domain is an scFv.

[0216] Embodiment B-l 1: The antigen-binding molecule of Embodiment B-9, wherein the first antigen-binding domain is an scFv.

[0217] Embodiment B-12: The antigen-binding molecule of Embodiment B-9, wherein the second antigen-binding domain is an scFv.

[0218] Embodiment B-l 3: The antigen-binding molecule of Embodiment B-9, wherein each of the first and the second antigen-binding domains is an scFv.

[0219] Embodiment B-14: The antigen-binding molecule of any one of the preceding Embodiments of Embodiment Set B, wherein the first and the second antigen-binding domains are attached by a linker, and / or wherein the second antigen-binding domain and the third domain are attached by a linker.

[0220] Embodiment B-15: The antigen-binding molecule of Embodiment B-14, wherein the linker is a peptide linker.

[0221] Embodiment B-l 6: The antigen-binding molecule of Embodiment B-l 3, wherein the VH of the first scFv is attached to the VH of the second scFv, or wherein the VL of the first scFv is attached to the VL of the second scFv. Embodiment B-17: The antigen-binding molecule of Embodiment B-16, wherein the linker is a peptide linker.

[0222] Embodiment B-18: The antigen-binding molecule of any one of preceding Embodiments of Embodiment Set B, wherein the first antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of gemtuzumab, vadastuximab talirine (SGN-CD33A), lintuzumab, BI 836858, IMGN779, AL003, M195, or Hui 95.

[0223] Embodiment B-19: The antigen-binding molecule of any one of the preceding Embodiments of Embodiment Set B, wherein the first antigen-binding comprises a VH and VL, wherein the VH and VL comprises the CDRs of the VH and VL of gemtuzumab, vadastuximab talirine (SGN-CD33A). lintuzumab, BI 836858, IMGN779, AL003, M195, or Hui 95.

[0224] C. Embodiment Set C

[0225] Embodiment C-L A multifunctional antigen-binding molecule, wherein the molecule comprises i. a first antigen-binding domain that binds to CD19 (e.g., that specifically binds to CD19), ii. a second antigen-binding domain that binds to a yb TCR, and iii. an optional third domain.

[0226] Embodiment C-2: The antigen-binding molecule of Embodiment C-l, wherein the second antigen-binding domain specifically binds to an epitope expressed by more than one yb T cell subtype.

[0227] Embodiment C-3: The antigen-binding molecule of Embodiment C-2, wherein the second antigen-binding domain binds to an epitope expressed by both V51 and Vb2 subpopulations of yd T-cells.

[0228] Embodiment C-4: The antigen binding molecule of Embodiment C-3, wherein the epitope is expressed by another yb T cell subtype.

[0229] Embodiment C-5: The antigen-binding molecule of any one of the preceding Embodiments of Embodiment Set C, wherein the second antigen-binding domain is a pan yb TCR antigen-binding moiety.

[0230] Embodiment C-6: The antigen-binding molecule of any one of the preceding Embodiments of Embodiment Set C, wherein the third domain is present and binds to a different epitope on yb T cells than the second antigen-binding domain. Embodiment C-7: The antigen-binding domain of Embodiment C-6, wherein the binding of the third antigen-binding domain to the different epitope promotes expansion of the yS T cells.

[0231] Embodiment C-8: The antigen-binding domain of Embodiment C-7, wherein the binding of the third antigen-binding domain to the different epitope promotes expansion of expansion of the V51 and V<52 subpopulations of y5 T cells.

[0232] Embodiment C-9: The antigen-binding molecule of the preceding Embodiments of Embodiment Set C, wherein each of the first and second antigen-binding domains is independently selected from a Fab domain, a Fab', a single-chain variable fragment (scFv) or a single domain antibody (sdAb).

[0233] Embodiment C-10: The antigen-binding molecule of Embodiment C-9, wherein at least one antigen-binding domain is an scFv.

[0234] Embodiment C-l l: The antigen-binding molecule of Embodiment C-9, wherein the first antigen-binding domain is an scFv.

[0235] Embodiment C-12: The antigen-binding molecule of Embodiment C-9, wherein the second antigen-binding domain is an scFv.

[0236] Embodiment C-13: The antigen-binding molecule of Embodiment C-9, wherein each of the first and the second antigen-binding domains is an scFv.

[0237] Embodiment C-14: The antigen-binding molecule of any one of the preceding Embodiments of Embodiment Set C, wherein the first and the second antigen-binding domains are attached by a linker, and / or wherein the second antigen-binding domain and the third domain are attached by a linker.

[0238] Embodiment C-15: The antigen-binding molecule of Embodiment C-14, wherein the linker is a peptide linker.

[0239] Embodiment C-16: The antigen-binding molecule of Embodiment C-13, wherein the VH of the first scFv is attached to the VH of the second scFv, or wherein the VL of the first scFv is attached to the VL of the second scFv.

[0240] Embodiment C-17: The antigen-binding molecule of Embodiment C-16, wherein the linker is a peptide linker.

[0241] D. Embodiment Set D

[0242] Embodiment D-l : A bifunctional antigen-binding molecule, wherein the molecule comprises i. a first antigen-binding domain that specifically binds to a tumor-associated antigen; and ii. a second antigen-binding domain that specifically binds to a y8 TCR, wherein second antigen-binding domain binds to an epitope expressed by more than one y8 T cell subtype.

[0243] Embodiment D-2: The antigen-binding molecule of Embodiment D-l, wherein the second antigen-binding domain binds to an epitope expressed by both V51 and V62 subpopulations of y8 T cells.

[0244] Embodiment D-3: The antigen binding molecule of Embodiment D2, wherein the epitope is expressed by another y8 T cell subtype.

[0245] Embodiment D-4: The antigen-binding molecule of any one of the preceding Embodiments of Embodiment Set D, wherein the second antigen-binding domain is a pan y8 TCR antigen-binding molecule.

[0246] Embodiment D-5: The antigen-binding molecule of any one of the preceding Embodiments of Embodiment Set D, wherein the antigen-binding molecule is capable of expanding or enhancing the expansion V81 and V82 subpopulations of y8 T cells.

[0247] Embodiment D-6: The antigen-binding molecule of any one of the preceding embodiments of Embodiment Set D. wherein each antigen-binding domain is independently selected from a Fab domain, a Fab', a single-chain variable fragment (scFv) or a single domain antibody (sdAb).

[0248] Embodiment D-7: The molecule of Embodiment D-6, wherein the first antigenbinding domain is an scFv.

[0249] Embodiment D-8: The molecule of Embodiment D-6, wherein the second antigenbinding domain is an scFv.

[0250] Embodiment D-9c: The molecule of Embodiment D-6, wherein each antigen-binding domain is an scFv.

[0251] Embodiment D-10: The antigen-binding molecule of any one of the preceding Embodiments of Embodiment Set D, wherein the two antigen-binding domains are attached by a linker.

[0252] Embodiment D-l 1 : The antigen-binding molecule of Embodiment D-10, wherein the linker is a peptide linker.

[0253] Embodiment D-l 2: The antigen-binding molecule of Embodiment D-9, wherein the VH of the first scFv is attached to the VH of the second scFv, or wherein the VL of the first scFv is attached to the VL of the second scFv. Embodiment D-13: The antigen-binding molecule of Embodiment D-9, wherein the VH of the first scFv is attached to the VH of the second scFv by a linker, or wherein the VL of the first scFv is attached to the VL of the second scFv by a linker.

[0254] Embodiment D-14: The antigen-binding molecule of Embodiment D-13, wherein the linker is a peptide linker.

[0255] Embodiment D-15: The antigen-binding molecule of any one of Embodiments the preceding Embodiments of Embodiment Set D, wherein the tumor-associated antigen (TAA) is selected from the group consisting of CD19, CD33, CD123, CD20, CD22, EGFRvIII, IL13ra2, folate receptor 1 (FOLR1), CLL-1, MMP-2, and mesothelin.

[0256] Embodiment D-17: The antigen-binding molecule of Embodiment D-15. wherein the TAA is CD33.

[0257] Embodiment D-18: The antigen-binding molecule of Embodiment D-15, wherein the TAA is CD 19.

[0258] E. Embodiment Set E

[0259] Embodiment E-l : A bispecific antigen-binding molecule, wherein the molecule comprises i. a first antigen-binding domain that binds to CD33 (e.g., that specifically binds to CD33). and ii. a second antigen-binding domain that binds to a y8 TCR.

[0260] Embodiment E-2: The antigen-binding molecule of Embodiment E-l. wherein the second antigen-binding domain specifically binds to an epitope expressed by more than one y8 T cell subtype.

[0261] Embodiment E-3 : The antigen-binding molecule of Embodiment E-2, wherein the second antigen-binding domain binds to an epitope expressed by both V81 and V82 subpopulations of y8 T-cells.

[0262] Embodiment E-4: The antigen binding molecule of Embodiment E-3, wherein the epitope is expressed by another y8 T cell subtype.

[0263] Embodiment E-5: The antigen-binding molecule of any one of the preceding Embodiments of Embodiment Set E , wherein the second antigen-binding domain is a pan y8 TCR antigen-binding molecule.

[0264] Embodiment E-6: The antigen-binding molecule of the preceding Embodiments of Embodiment Set E, wherein each antigen-binding domain is independently selected from a Fab domain, a Fab', a single-chain variable fragment (scFv) or a single domain antibody (sdAb). Embodiment E-7: The antigen-binding molecule of Embodiment E-6, wherein at least one antigen-binding domain is an scFv.

[0265] Embodiment E-8: The antigen-binding molecule of Embodiment E-6, wherein the first antigen-binding domain is an scFv.

[0266] Embodiment E-9: The antigen-binding molecule of Embodiment E-6, wherein the second antigen-binding domain is an scFv.

[0267] Embodiment E-10: The antigen-binding domain of Embodiment E-6, wherein each antigen-binding domain is an scFv.

[0268] Embodiment E-l l: The antigen-binding molecule of any one of Embodiments E-6, wherein the two antigen-binding domains are attached by a linker.

[0269] Embodiment E-12: The antigen-binding molecule of Embodiment E-l l, wherein the linker is a peptide linker.

[0270] Embodiment E-13: The antigen-binding molecule of Embodiment E-10, wherein the VH of the first scFv is attached to the VH of the second scFv, or wherein the VL of the first scFv is attached to the VL of the second scFv.

[0271] Embodiment E-14: The antigen-binding molecule of Embodiment E-10, wherein the VH of the first scFv is attached to the VH of the second scFv by a linker, or wherein the VL of the first scFv is attached to the VL of the second scFv.

[0272] Embodiment E-15: The antigen-binding molecule of Embodiment E-14, wherein the linker is a peptide linker.

[0273] Embodiment E-16: The antigen-binding molecule of any one of the preceding Embodiments of Embodiment Set E, wherein the antigen-binding domain that binds CD33 comprises a heavy chain variable region VH and a light chain variable region VL of gemtuzumab, vadastuximab talirine (SGN-CD33A), lintuzumab, BI 836858, IMGN779, AL003, M195, or Hui 95.

[0274] F. Embodiment Set F

[0275] Embodiment F-l: A bispecific antigen-binding molecule, wherein the molecule comprises i. a first antigen-binding domain that binds to CD19 (e.g., that specifically binds to CD19), and ii. a second antigen-binding domain that binds to a yb TCR.

[0276] Embodiment F-2: The antigen-binding molecule of Embodiment F-l, wherein the second antigen-binding domain specifically binds to an epitope expressed by more than one y8 T cell subtype. Embodiment F-3: The antigen-binding molecule of Embodiment F-2, wherein the second antigen-binding domain binds to an epitope expressed by both V51 and V62 subpopulations of y8 T cells.

[0277] Embodiment F-4: The antigen binding molecule of Embodiment F-3, wherein the second antigen-binding domain also binds to an epitope expressed by another yd T cell subtype.

[0278] Embodiment F-5: The antigen-binding molecule of any one of the Embodiment F-4, wherein the second antigen-binding domain is a pan y5 TCR antigen-binding molecule.

[0279] Embodiment F-6: The antigen-binding molecule of the preceding of Embodiment Set F, wherein each antigen-binding domain is independently selected from a Fab domain, a Fab', a single-chain variable fragment (scFv) or a single domain antibody (sdAb).

[0280] Embodiment F-7: The antigen-binding molecule of Embodiment F-6, wherein at least one antigen-binding domain is an scFv.

[0281] Embodiment F-8: The antigen-binding molecule of Embodiment F-6, wherein the first antigen-binding domain is an scFv.

[0282] Embodiment F-9: The antigen-binding molecule of Embodiment F-6, wherein the second antigen-binding domain is an scFv.

[0283] Embodiment F-10: The antigen-binding domain of Embodiment F-6, wherein each antigen-binding domain is an scFv.

[0284] Embodiment F-l 1: The antigen-binding molecule of any one of Embodiment the preceding embodiments of Embodiment Set F, wherein the two antigen-binding domains are attached by a linker.

[0285] Embodiment F-12: The antigen-binding molecule of Embodiment F-l 1, wherein the linker is a peptide linker.

[0286] Embodiment F-l 3: The antigen-binding molecule of Embodiment F-10, wherein the VH of the first scFv is attached to the VH of the second scFv. or wherein the VL of the first scFv is attached to the VL of the second scFv.

[0287] Embodiment F-14: The antigen-binding molecule of Embodiment F-10, w herein the VH of the first scFv is attached to the VH of the second scFv by a linker, or wherein the VL of the first scFv is attached to the VL of the second scFv by a linker.

[0288] Embodiment F-15: The antigen-binding molecule of Embodiment F-14, wherein the linker is a peptide linker. G. Embodiment Set G

[0289] Embodiment G-l : A method for treating cancer a subject in need thereof, the method comprising administering to said subject an effective amount of an antigen-binding molecule of any one of the preceding embodiments (Embodiment Set A to Embodiment Set F).

[0290] Embodiment G-2: The method of Embodiment G-l, wherein each of the first and the second antigen-binding domains is an scFv.

[0291] Embodiment G-3: The method of any one of Embodiments G- l and G-2. wherein the second antigen-binding domain specifically binds to an epitope expressed by more than one yd T cell subtype.

[0292] Embodiment G-4: The method of Embodiment G-3, wherein the second antigenbinding domain binds to an epitope expressed by both V51 and V82 subpopulations of y8 T cells.

[0293] Embodiment G-5: The method of Embodiment G-4, wherein the epitope is expressed by another y8 T cell subtype.

[0294] Embodiment G-6: The method of any one of the preceding Embodiments of Embodiment Set G, wherein the second antigen-binding domain is a pan y8 TCR antigenbinding molecule.

[0295] Embodiment G-7 : The method of any one of the preceding Embodiments of Embodiment Set G, wherein the antigen-binding molecule comprises a third domain, wherein the third domain binds to a different epitope on y8 T cells than the second antigen-binding domain, and wherein the binding of the third antigen-binding domain to the different epitope promotes expansion of y8 T cells.

[0296] Embodiment G-7a: The method of Embodiment G-7, wherein the binding of the third antigen-binding domain to the different epitope promotes expansion of expansion of the V81 and V82 subpopulations of y8 T cells.

[0297] Embodiment G-8: The method of any one of preceding Embodiments of Embodiment Set G, wherein the TAA is selected from the group consisting of CD19, CD33, CD123, CD20, CD22, EGFRvIII, IL13ra2, folate receptor 1 (FOLR1), CLL-1, MMP-2, and mesothelin.

[0298] Embodiment G-8a: The method of Embodiment G-8, wherein the TAA is CD33.

[0299] Embodiment G-9: The method of Embodiment G-8, wherein the TAA is CD19. Embodiment G-10: The method of any one of preceding Embodiments of Embodiment Set G, further comprising administration of an effective amount of a population of y8 T cells.

[0300] Embodiment G-l 1: The method of Embodiment G-10, wherein the population of y8 T cells is expanded and optionally activated ex vivo.

[0301] Embodiment G-12: The method of any one of Embodiments G-10 and G-l 1, wherein the y8 T cells are allogeneic or autologous.

[0302] Embodiment G-13: The method of any one of claim Embodiments G10-G12, wherein the y6 T cells are CAR y6 T cells.

[0303] Embodiment G-14: The method of Embodiment G-13, wherein the y8 T cells are nsCAR y8 T cells.

[0304] Embodiment G-l 5: The method of any one of Embodiments G-l to G9 . wherein the method does not comprise transplantation or administration of y8 T cells.

[0305] Embodiment G-16: The method of any one the preceding Embodiments of Embodiment Set G, wherein the cancer is a hematologic malignancy or a solid tumor.

[0306] Embodiment G-l 7: The method of Embodiment G-16, wherein the cancer is a hematologic cancer.

[0307] Embodiment G-l 8: The method of Embodiment G-16, wherein the cancer is a solid tumor.

[0308] Embodiment G-19: The method of Embodiment G-8, wherein the cancer is a CD33- associated cancer.

[0309] Embodiment G-20: The method of Embodiment G-19, wherein the CD33-associated cancer is myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, acute Lymphocytic Leukemia (ALL), Acute Myeloid Leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin's lymphoma (NHL), B lineage ALL (acute lymphocytic leukemia), multiple myeloma, lung, breast, ovarian, prostate, colon, and melanoma.

[0310] Embodiment G-21 : The method Embodiment G-20, wherein the cancer is AML.

[0311] Embodiment G-22: The method of Embodiment G-9, wherein the cancer is a CD 19- associated cancer.

[0312] Embodiment G-23: The method of Embodiment G-22, wherein the CD19-associated cancer is ALL. Embodiment G-24: The method of any one Embodiments G-l to G-23, wherein a population of the patient's yb T cells is expanded in vivo, and optionally activated, after administration of the antigen-binding molecule, or wherein expansion of a population of the patient's yb T cells is enhanced after administration of the antigen-binding molecule.

[0313] Embodiment G-24a: The method of G-24, wherein the method does not comprise transplantation or administration of yb T cells.

[0314] Embodiment G-25 : The method of Embodiment G-24 and G-24a, wherein the population of the patient’s yb T cells that is expanded includes Vbl and Vb2 subpopulations of yb T cells.

[0315] Embodiment G-26: The method of Embodiment G-24, wherein the expansion of a population of the patient’s yb T cells comprising Vbl and Vb2 subpopulations is enhanced after administration of the antigen-binding molecule.

[0316] Embodiment G-27: The method of any one of Embodiments G-10 to G-l 4, wherein the administered population of yb T cells is expanded after administration of the antigenbinding molecule, or wherein the expansion of the administered population of yb T cells is enhanced expanded after administration of the antigen-binding molecule.

[0317] H. Embodiment Set H

[0318] Embodiment H-l : A method of enhancing the cytotoxicity of a population of yb T cells, the method comprising administering to a subject suffering from cancer an effective amount of an antigen-binding molecule of any one the Embodiments of Embodiment Set A, Embodiment Set B, Embodiment Set C, Embodiment Set D, Embodiment Set E and Embodiment Set F.

[0319] Embodiment H-2: The method of Embodiment H-l, wherein the population of yb T cells is a population of the patient's yb T cells.

[0320] Embodiment H-3: The method of Embodiment H-l. further comprising administration of an effective amount of a population of yb T cells and wherein the cytotoxicity of the administered population of yb T cells is enhanced.

[0321] Embodiment H-4: The method of Embodiment H-3. wherein the population of yb T cells are allogeneic or autologous.

[0322] Embodiment H-5: The method of any one of claim Embodiments H-3 and H-4, wherein the yb T cells are CAR yb T cells. Embodiment H-5a: The method of Embodiment H-5, wherein the y8 T cells are nsCAR y5 T cells.

[0323] Embodiment H-6: The method of any one of the preceding Embodiments of Embodiment Set H, wherein each of the first and second antigen-binding domains is an scFv.

[0324] Embodiment H-7 : The method of any one of the preceding Embodiments of Embodiment Set H wherein the second antigen-binding domain specifically binds to an epitope expressed by more than one y8 T cell subtype.

[0325] Embodiment H-8: The method of Embodiment H-7, wherein the second antigenbinding domain binds to an epitope expressed by both V61 and V62 T-cells.

[0326] Embodiment H-9: The method of Embodiment H-8, wherein the epitope is expressed by another y5 T cell subty pe.

[0327] Embodiment H-10: The method of any one of the preceding Embodiments of Embodiment Set H, wherein the second antigen-binding domain is a pan y8 TCR antigenbinding molecule.

[0328] Embodiment H-lOa: The method of any one of the preceding Embodiments of Embodiment Set G, wherein the antigen-binding molecule comprises a third domain, wherein the third domain binds to a different epitope on y8 T cells than the second antigen-binding domain, and wherein the binding of the third antigen-binding domain to the different epitope promotes expansion of y8 T cells.

[0329] Embodiment H-lOb: The method of Embodiment H-lOa, wherein the binding of the third antigen-binding domain to the different epitope promotes expansion of expansion of the V81 and V52 subpopulations ofy5 T cells.

[0330] Embodiment H- 11 : The method of any one of the preceding Embodiments of Embodiment Set H, wherein the TAA is selected from the group consisting of CD19, CD33, CD123, CD20, CD22, EGFRvIII, IL13ra2, folate receptor 1 (FOLR1), CLL-1, MMP-2, and mesothelin.

[0331] Embodiment H-12: The method of Embodiment H-l 1, wherein the TAA is CD33.

[0332] Embodiment H-13: The method of Embodiment H-12, wherein the TAA is CD 19.

[0333] Embodiment H-14: The method of any one the preceding Embodiments of Embodiment Set H, wherein the cancer is a hematologic malignancy or a solid tumor.

[0334] Embodiment H-15: The method of Embodiment H-14, wherein the cancer is a hematologic cancer. Embodiment H-16: The method of Embodiment H-14, wherein the cancer is a solid tumor.

[0335] Embodiment H-17: The method of Embodiment H-12, wherein the cancer is a CD33- associated cancer.

[0336] Embodiment 14-18: The method of Embodiment H-17, wherein the CD33-associated cancer is myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, acute Lymphocytic Leukemia (ALL), Acute Myeloid Leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin's lymphoma (NHL), B lineage ALL (acute lymphocytic leukemia), multiple myeloma, lung, breast, ovarian, prostate, colon, or melanoma.

[0337] Embodiment H-19: The method Embodiment H-18. wherein the cancer is AML.

[0338] Embodiment H-20: The method of Embodiment H-13, wherein the cancer is a CD19- associated cancer.

[0339] Embodiment H-21 : The method of Embodiment H-20, wherein the CD19-associated cancer is ALL.

[0340] Embodiment H-22: The method of any one of the preceding Embodiments of Embodiment Set H, wherein a population of the patient’s y8 T cells is expanded in vivo, and optionally activated, after administration of the antigen-binding molecule, or wherein expansion of a population of the patient’s y8 T cells is enhanced after administration of the antigen-binding molecule.

[0341] Embodiment H-22a: The method of H-22, wherein the method does not comprise transplantation or administration of y5 T cells.

[0342] Embodiment H-23: The method of any one of Embodiments H-22 and H-22a, wherein the population of patients y8 T cells includes V81 and V82 subpopulations of y8 T cells.

[0343] Embodiment H-24: The method of any one of Embodiments H-3. H-4, H-5 and H-5a, wherein an administered population of y8 T cells is expanded after administration of the antigen-binding molecule, or wherein expansion of an administered population of y8 T cells is enhanced after administration of the antigen-binding molecule.

[0344] I. Embodiment Set I

[0345] Embodiment 1-1 : A method of treating a B-cell mediated autoimmune disease or B- cell mediated inflammatory disease in a patient in need thereof, the method comprising administering to said patient an effective amount of an antigen-binding molecule of any one of Embodiments of Embodiment Set C and Embodiment Set F.

[0346] Embodiment 1-2: The method of Embodiment 1-1, wherein the B-cell mediated autoimmune disease is selected from the group consisting of systemic lupus erythematosus, lupus nephritis, multiple sclerosis, rheumatoid arthritis, antiphospholipid syndrome, pemphigus, cicatricial pemphigoid, myasthenia gravis, neuromyelitis optica, immune thrombocytopenia, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas' disease, Graves' disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, ANCA-associated vasculitis, Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia and rapidly progressive glomerulonephritis, heavy chain disease, primary or immunocyte-associated amyloidosis and monoclonal gammopathy of undetermined significance.

[0347] Embodiment 1-3: The method of Embodiment 1-2, wherein the B-cell mediated autoimmune disease is systemic lupus erythematosus.

[0348] Embodiment 1-4: The method of Embodiment 1-2. wherein the B-cell mediated inflammatory condition is selected from the group consisting of type 2 diabetes, periodontal disease, and graft-versus-host disease.

[0349] J. Embodiment Set J

[0350] Embodiment J-l: A nucleic acid or vector encoding the antigen-binding molecule of any one of Embodiments of Embodiment Set A to Embodiment Set F.

[0351] Embodiment J-2: The nucleic acid or vector of Embodiment J-l, wherein the nucleic acid molecule further comprises a transmembrane domain and optionally, an extracellular hinge domain and / or a co-stimulatory domain.

[0352] Embodiment J-3: A host cell transformed or transfected with the nucleic acid or vector of any one of Embodiments J-l and J-2.

[0353] Embodiment J-4: A nucleic acid encoding a membrane-bound multifunctional antigen-binding and immune cell activating molecule, wherein the membrane-bound molecule comprises an extracellular domain and a transmembrane / intracellular region, wherein the nucleic acid comprises: i. a first antigen-binding domain that binds to a tumor-associated antigen (TAA); ii. a second antigen-binding domain that binds to an immune cell antigen; iii. a linker that attaches the first antigen-binding domain to the second antigenbinding domain; and iv. a transmembrane domain; and wherein the extracellular domain comprises the first antigen-binding domain, the second antigen-binding domain, and the linker; and further wherein the transmembrane / intracellular region comprises the transmembrane domain.

[0354] Embodiment J-5: The nucleic acid of Embodiment J-4, wherein the nucleic acid further comprises an extracellular hinge domain and / or an intracellular co-stimulatory domain.

[0355] Embodiment J-6: The nucleic acid of Embodiment J-5, wherein an extracellular hinge domain links the transmembrane domain to one of the antigen-binding domains.

[0356] Embodiment J-7: The nucleic acid of Embodiment J-5, further comprising an intracellular co-stimulatory domain.

[0357] Embodiment J-8: The nucleic acid of any one of Embodiments J-4 to J-7, wherein the transmembrane domain, extracellular hinge, and co-stimulatory domains are CD28 transmembrane domain, CD28 extracellular hinge, and CD28 co-stimulatory domain, respectively.

[0358] Embodiment J-9: The nucleic acid of any one of claims J-4 to J-8, wherein the immune cell antigen is a T-cell antigen.

[0359] Embodiment J-10: The nucleic acid of Embodiment J-9, wherein the T-cell antigen is CD3

[0360] Embodiment J-l 1 : The nucleic acid of Embodiment J-9, wherein the T-cell antigen is a y8 T-cell antigen.

[0361] Embodiment J- 12 : The nucleic acid of Embodiment J-l 1, wherein the y3 T-cell antigen is a y8 TCR.

[0362] Embodiment J-l 3 : The nucleic acid of Embodiment J-l 1, wherein the second antigenbinding domain binds to an epitope expressed by more than one y3 T cell subtype.

[0363] Embodiment J- 14: The nucleic acid of Embodiment J-l 3, wherein the second antigenbinding domain binds to an epitope expressed by both V81 and V82 subpopulations of y8 T cells.

[0364] Embodiment J- 15 : The nucleic acid of Embodiment J-l 4, wherein the epitope is expressed by another y8 T cell subtype.

[0365] Embodiment J-l 6: The nucleic acid of any one of the preceding Embodiments J-13 to J- 16, wherein the second antigen-binding domain is a pan y8 TCR antigen-binding molecule. Embodiment J- 16a: The method of any one of Embodiments J- 11 to J- 16, wherein the antigen-binding molecule further comprises a third domain, wherein the third domain is present binds to a different epitope on yb T cells than the second antigen-binding domain, and wherein the binding of the third antigen-binding domain to the different epitope promotes expansion of y<5 T cells.

[0366] Embodiment J- 16b: The method of Embodiment J- 16a, wherein the binding of the third antigen-binding domain to the different epitope promotes expansion of expansion of the V51 and V52 subpopulations of y5 T cells.

[0367] Embodiment J-17: The nucleic acid of any one of claims J-4 to J8, wherein the immune cell antigen is a NK cell antigen.

[0368] Embodiment J-17a: The nucleic acid of Embodiment J-17, wherein the NK cell antigen is CD16a, NKG2D, NKG2C, Nkp30, Nkp46, Nkp80 and CD160.

[0369] Embodiment J- 18 : The nucleic acid of any one of Embodiments J-4 to J- 16, J- 16a, J- 16b, J-17 and J-17a, wherein the TAA is wherein the TAA is selected from the group consisting of CD19, CD33, CD123, CD20, CD22, EGFRvIII, IL13ra2, folate receptor 1 (FOLR1), CLL-1, MMP-2, and mesothelin.

[0370] Embodiment J- 19: A vector comprising the nucleic acid of any one of claims J-4 to J-6, J-16a, J-l 6b, J-17 and J-17a and J-18.

[0371] Embodiment J-20: The vector of Embodiment J- 19, wherein the nucleic acid is overexpressed.

[0372] Embodiment J-21 : A host cell transformed or transfected with the nucleic acid or vector of any one of Embodiments J-l 9 and J-20.

[0373] Embodiment J-22: A process for the production of a multifunctional antigen-binding and immune cell engaging molecule, the process comprising culturing the host cell of Embodiment J-21 under conditions suitable for expression and harvesting of the engaging molecule; and further comprising purifying the engaging molecule from the culture.

[0374] Embodiment J-23: A nucleic acid or vector encoding the multifunctional antigenbinding molecule, wherein the multifunctional antigen-binding molecule comprises: i. an antigen-binding domain that binds to CD33; ii. an antigen-binding domain that binds to y5 TCR; iii. a linker that attaches the antigen-binding domain that binds to CD33 to the antigen-binding domain that binds to y5 TCR. Embodiment J-23a: The nucleic acid or vector of Embodiment J-23, wherein the multifunctional antigen-binding molecule further comprises a third domain, wherein the third binds to a different epitope on y8 T cells than the second antigen-binding domain, and wherein the binding of the third antigen-binding domain to the different epitope promotes expansion ofy8 T cells.

[0375] Embodiment J-24: The nucleic acid or vector of any one of Embodiments J-23 and J- 23a, the nucleic acid molecule further comprises a transmembrane domain and optionally, an extracellular hinge domain and / or a co-stimulatory domain.

[0376] Embodiment J-25: A vector comprising the nucleic acid of any one of claims Embodiments J-23, J-23a and J-24.

[0377] Embodiment J-26: A host cell transformed or transfected with the vector of Embodiment J-25.

[0378] Embodiment J-27 : A nucleic acid or vector encoding the bispecific antigen-binding molecule, wherein the bispecific antigen-binding molecule comprises: i. an antigen-binding domain that binds to CD 19; ii. an antigen-binding domain that binds to y8 TCR; iii. a linker that attaches the antigen-binding domain that binds to CD 19 to the antigen-binding domain that binds to y5 TCR.

[0379] Embodiment J-27a: The nucleic acid or vector of Embodiment J-27, wherein the multifunctional antigen-binding molecule further comprises a third domain, wherein the third binds to a different epitope on y8 T cells than the second antigen-binding domain, and wherein the binding of the third antigen-binding domain to the different epitope promotes expansion of y8 T cells.

[0380] Embodiment J-28: The nucleic acid or vector of any one of Embodiments J-27 and J- 27a, the nucleic acid molecule further comprises a transmembrane domain and optionally, an extracellular hinge domain and / or a co-stimulatory domain.

[0381] Embodiment J-29: A vector comprising the nucleic acid of any one of claims Embodiments J-27, J-27a and J-28.

[0382] Embodiment J-30: A host cell transformed or transfected with the vector of Embodiment J-29. EXAMPLES

[0383] The following examples are offered by way of illustration and are not to be construed as limiting the invention in any way.

[0384] Example 1 : 33XGD engager treatment significantly enhanced cytotoxicity of Y8 T cells Treatment of y8 T cells with a CD33 / y8 TCR multifunctional y8 T cell engager (33xGD engager) significantly enhanced y8 T cell cytotoxicity against CD33-positive AML cell lines in a dose dependent manner. The treatment with 33xGD engager also enhanced y8 T cells degranulation after coculture with CD33-positive MOLM-13 AML cells for 24 hours and triggered significant release of cytotoxic cytokines. Specifically, FIGs. 3A-3C shows cytotoxicity' (%) of y5 T cells (donor 1) in combination with crude comprising the 33xGD engager (“33xGD293) or medium control co-cultured with leukemia cell lines HL-60 (AML), KG-la (AML) and K562 cells (CML) at different effector to target ratios (E:T). 100 pl of 33xGD crude was added to 400 pl total of y8 T cell and target cell co-culture. The figure shows that the addition of the engager significantly enhanced the cytotoxicity of y8 T cells against AML cells HL-60 and KG-la, and had less effect on K562 CML cells likely because of low CD33 expression on K562. This effect was also confirmed by flow cytometric analysis of y8 T cells in combination with crude comprising the 33xGD engager ty33xGD293) or medium control co-cultured with leukemia cell lines HL-60 , KG-la and K562 cells at different effector to target ratios (E:T) (data not shown). The y8 T cell mediated cytotoxicity was assessed by flow cytometry after 24 hours of co-culture (CSFE+7AAD+ / total CSFE+ cells).

[0385] FIG. 4A shows cytotoxicity (%) of y8 T cells in combination with different clones of stable 33xGD expression HEK293T cells (cl-c6) or a control (null x y8 TCR-binding domain, GD293) after 24 hours of co-culture with HL-60, KG-la, MOLM-13 and K562 cells at an E:T of 1. 100 pl of crude was added to 400 pl total of y8 T cell and target cell coculture. The clones were established for optimal production of the 33xGD engager. The figure shows that crude from all of the different clones enhanced cytotoxicity of y8 T cells against the CD33-positive HL-60, KG-la and MOLM-13 cells, and that the cytotoxic effect of the engager was retained by different clones generated using the process described herein.

[0386] FIG. 5 shows cytotoxicity (%) of y8 T cells treated with the 33xGD engager (“33xGD”) at the indicated volumes of the engager after 24 hours co-culture with HL-60, KG-la. MOLM-13 and K-562 cells; the indicated volume of the engager was added to 400 pl total of y8 T cell and target cell co-culture with an E:T of 1. The figure shows that cytotoxicity against the AML cell lines increased in a dose-dependent manner. The cytotoxicity of the engager against target cells was also confirmed by flow cytometric analysis (data not shown).

[0387] FIG. 6 shows flow cytometric analysis of y8 T cells co-cultured with the AML cell lines HL-60, KG- la and MOLM-13 at E:T of 1 for 24 hours with or without the 33xGD engager and stained with anti-CD107A. CD107A is a marker of degranulation and release of cytotoxic granules (CGs). Treatment of y8 T cells with the 33xGD engager enhances y8 T cells degranulation. It was also shown that the 33xGD engager upregulates the levels of activation / degranulation markers CD107, CD69 and PD1 (degranulation / activation markers) after co-culture with y8 T cells in a dose-dependent manner (data not shown).

[0388] FIG. 7 shows cytokine release by 33xGD engager expanded and activated y3 T cells co-cultured with M0LM13 cells. The figure shows the concentration of cytokine released (pg / ml) after y8 T cell treated with indicated volumes of 33xGD crude were co-cultured with M0LM13 at E:T of 1 for 24 hours using a commercial assay. The concentration of cytokine released was highest for IFN-y, Granzyme A, Granzyme B. Perforin and Granzyme, which are involved in y8 T cells killing ability, showing the cells have enhanced potency after treatment with the 33xGD engager, which are expressed in a dose-dependent manner.

[0389] FIG. 10 shows percent cytotoxicity over increasing concentration of CD33xGD engager (pM) for MOLM-13 (CD33+) cells after co-culture with y8 T cells. The figure shows that the CD33xGD engager induces dose-dependent cytotoxicity of y8 T cells against MOLM-13 cells after co-culture. Experiments were performed with multiple y8 T cell donors (N=3), and donor variability is reflected in the differences in EC50 (82.7 pM, 169.43 pM and 66.36 pM). EC50 values define cytotoxic efficacy. This figure shows that the CD33xGD engager enhances y8 T cell cytotoxicity against MOLM-13 (CD33+) AML cells in a dosedependent manner.

[0390] FIG. 11A shows percent cytotoxicity over increasing concentration of CD33xGD engager (pM) (E:T=1; 24 h) for MOLM-13 (CD33+) cells and Raji (CD33-) cells co-cultured with y8 T cells. This figure shows that the engager induces dose-dependent, target-specific killing activity.

[0391] FIG. 1 I B shows activation / degranulation markers present in cultures of MOLM-13 (CD33+) cells and Raji (CD33-) cells co-cultured with y8 T cells in the presence and absence of CD33xGD engager (pM) (E:T=1; 24 h). It was also shown that the 33xGD engager enhanced y8 T cell degranulation in a dose-dependent manner (data not shown).

[0392] FIG. 12 shows flow cytometric analysis of healthy donor PBMCs alone (left panel), healthy PBMCs co-cultured with MOLM-13 cells at E:T=10 for 4 days alone (middle panel), healthy PBMCs co-cultured with MOLM-13 cells (E:T=10 for 4 days) with the CD33xGD engager (right panel). This figure demonstrates elimination of AML cells (MOLM-13 cells) that express the target CD33 by PBMCs with the addition of CD33xGD engager. Control: PBMC only at day 4 (left panel). It was also shown that treatment with the engager (“off-the- shelf’ and simply added to the y8 T cell culture) significantly enhanced cytotoxicity against MOLM-13 cells, even at the lower E:T ratios (data not shown). Specifically, cytotoxicity was enhanced up to 200-fold at the low E:T ratios. It was observed that the response was quick as y8 T cells demonstrated enhanced killing shortly after treatment.

[0393] The process for preparing a CD33 / y8 TCR multifunctional y8 T cell engager (33xGD engager) and using the engager in combination with expanded y8 T cells for targeted lysis of CD33-positive AML cells is shown in FIG. 2A. In summary', an exemplary construct comprising a CD33 binding domain and y8 TCR binding domain is cloned into a lentiviral vector and the vector is used to transfect HEK-293T cells. After integration and cloning, HEK293T cells expressing the 33xGD y8 T cell engager were isolated and expanded. The CD33 / y3 TCR engager was purified and concentrated. The addition of the engager activates and directs the y8 T cells to CD33 (activated and CD33-redirected y8 T cells) on the surface of tumor cells. The activated and CD33-redirected y8 T cells are contacted with AML cells and their interaction results in release of granzyme and perforin, and targeted cell lysis. The construct has the following elements (wherein the left of the construct corresponds to the N- terminal side): signal peptide— target binding domain— G4S linker— y8 T cell binding domaindetection tag-y8 T cell expansion domain (GDED)-P2A-selection marker. The represents direct or indirect attachment; for example, there can be a hinge, transmembrane and / or co-stimulatory domain between certain elements. In another example, there can be a detectable tag, e.g., a Myc-tag or Flag-tag between certain elements. A non-limiting example of a target-binding domain is CD33-binding domain, including, for example, an anti-CD33 antibody or fragment, e.g., an scFv. An example of a y6 T cell binding domain is an antibody or fragment thereof that selectively binds to a y8 T cell, for example, an anti -human pan-y8 TCR monoclonal antibody or scFv. “P2A” is the P2A peptide. A non-limiting example of a signal peptide is CD8a (amino acid sequence is MALPVTALLLPLALLLHAARP (SEQ ID NO: 13)). An exemplary “G4S linker” is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 14). z. Lentiviral vector construction and packaging

[0394] Synthesized gBlocks encoding the peptide was cloned, sequence verified and packaged into lentivirus (LV) in HEK293T cells with a 2ndgeneration lentiviral vector system. ii. Transduction ofHEK293T cells with 33xGD-selection marker lentiviral vector

[0395] The HEK293T cells were transduced with 33xGD-selection marker lentiviral vectors and the expression of the 33xGD and selection marker were verified by flow cytometry with staining of anti-Flag monoclonal antibody.

[0396] Hi. Establishment of stable 33xGD bi-specific engager producing cell

[0397] After the lentiviral transduction, the HEK293T cells transduced with 33xGD-selection marker were diluted and seeded into 96-well plates at 0.5 cell / well density. Single cell clones with positive selection marker expression were picked and the expression of engager was verified by flow cytometry'. The cloned 33xGD293 producing cells were further expanded for 33xGD engager production. iv. yd T mediated cytotoxicity assay

[0398] Activated and expanded V52+ y5 T cells from healthy donors were co-cultured with CFSE labeled target cells in a 48-well plate at effector to target ratio 1.0. Different amount of 33xGD engager crude was added to each well and y5 T cell mediated killing of target cell was assessed by flow cytometry co-culture. The cytotoxicity at E / T=X is normalized by the formula:

[0399] %cytotoxicity = (Killed target cell% at ET=X - Killed target cell% at E / T=0) / (l- Killed target cell% at E / T=0). v. Cytokine release assay

[0400] Activated and expanded V62+ y5 T cells from healthy donors were co-cultured with MOLM-13 cells in a 96-well round-bottom plate at effector to target ratio 1.0. Different amount of 33xGD engager crude was added to each well and the supernatant media was harvested after coculture. The cytokine released was assayed and analyzed using a commercially available assay kit.

[0401] Example 2: Binding of purified 33xGD engager to CD33+ AML cells and y5 T cells

[0402] A serum-free, suspension culture was optimized for larger-scale protein expression and purification. Briefly, cells were adapted from adherent culture to suspension culture in shaker flasks and then further adapted from serum-free media to serum- and protein-free medium (FreeStyle 293). After adaptation, cells demonstrated high viability and growth rate.

[0403] 33xGD TCE purified from the suspension culture was added to MOLM-13 cells (CD33+AML cells) and analyzed by flow cytometry'. FIG. 8 shows flow cytometric analysis of MOLM-13 cells without (left) and with (right) the purified 33xGD engager, and stained with anti-FLAG antibody and anti-CD33 antibody. The 33xGD engager was purified from a serum-free suspension culture. The figure shows that purified 33xGD engager binds to the CD33+ AML cells.

[0404] Binding of purified 33xGD TCE to y6 T cells in a population of cells enriched for y6 T cells but also including other immune cell types was also characterized. The other immune cell ty pes were present in an amount of about 10% of the cell population, and includes ap T cells and NK. cells.

[0405] FIG. 9 sho vs flow cytometric analysis of y5 T cells (top) and non-y5 T cells (bottom) in a population of cells enriched for y5 T cells (and containing other immune cell ty pes) without (left) and with (right) the 33xGD engager, and stained with anti-FLAG antibody and the anti-y8 TCR. The figure shows that the purified 33xGD engager specifically binds yS T cells. Example 3: CD19xGD engager treatment significantly enhanced expansion and cytotoxicity of yb T cells

[0406] The CD19xGD engager was prepared using a similar process to that described above for the CD33xGD engager. The vector map is shown in FIG. 15. Briefly, synthesized gBlocks encoding the peptide was cloned, sequence verified and packaged into lentivirus (LV) in HEK293T cells with a 2ndgeneration lentiviral vector system. The functionality of the CD19xGD engager was subsequently evaluated by assessing its ability to enhance the cytotoxic activity' of yb T cells against both CD 19-positive and CD 19-negative target cell lines. Verification of the transduction process for the engager was conducted.

[0407] FIGs. 16A and 16B show that the 19xGD engager does-dependently enhanced y5 T cell cytotoxicity against CD19+ Nalm-6 cells at a low' effector to target (E:T) ratio (E:T of 1 ). FIGs. 17A and 17B are microscopic images of Nalm6 cells co-cultured with yb T cells in the presence of the CD19xGD engager (200 pl crude. 50% vv) (FIG. 17B) versus that in the absence of the CD19xGD engager (FIG. 17A). FIG. 17A shows little or no cell death in the presence of yb T cells alone w'hereas FIG. 17B shows a dramatic increase in cell death in the presence of the 33xGD engager, which can be seen by the reduction in cell number which is shown by an increase in negative space in 17B.

[0408] FIGs. 18A and 18B shows flow cytometric analysis of purified CD19xGD incubated with 5x105Nalm-6, MOLM-13 (FIG. 18 A) and expanded yb TCR+cells and ybTCR’ cells (FIG. 18B). Cells were washed and resuspended in 100 ul FACS buffer and incubated with 5 pl 19xGD TCE for 30 minutes. Cells were then washed and stained with anti-FLAG mAb before flow' cytometric analysis. 19xGD engager showed strong and selective binding to Nalm-6 (CD19+) and ybTCR+ ybT cells, and did not bind to MOLM-13 (CD19) and ybTCR negative cells.

[0409] FIGs. 19A and 19B show that yb T cells expanded from PBMCs obtained from two separate donors and treated with increasing concentration of purified 19xGD engager. “No Txt” are control PBMCs not treated with the 19xGD engager or . “Zoledronate” are PBMCs treated with Zoledronate , which is known to expand the Vb2 subpopulations of yb T cells. The figures show' results on day 10, yb T cell absolute numbers out of total cell count and (FIG. 29A) and yb T cell frequency within the CD45+ population (FIG. 29B). The figure shows that CD19xGD engager expands yb T cells from PBMCs during and after the cytotoxic lysis of normal B cells, Vbl and Vb2 subpopulations of yb T cells as w ell as non-Vbl, Vb2 subpopulations had expanded. Zoledronate (positive control) expanded primarily Vb2+ cells from PBMCs, as expected. y8 T cells from PBMCs without the CD19xGD engager (“No Txt”; negative control) did not expand. No other T-cell engager has been shown to drive significant expansion and proliferation of y8 T cells. FIGs. 20A and 20B show that CD19xGD induces strong and target-specific y8 T cells cytotoxicity.

[0410] FIG. 21 shows upregulation of CD107a, CD69, and PD-1 for y8 T cells alone, no treatment (No Txt), and increasing concentration of the engager. The figure shows that the CD19xGD engager dose-dependently upregulates the activation markers CD69 and CD 107a, and also upregulates PD-1 to a lesser degree, after treatment (N=3). The combination with the engager induces expression of cellular degranulation and activation markers, and these figures show that the engager promotes y8 T cell activation and degranulation.

[0411] FIG. 22 is a heatmap showing the levels of selected secreted cytokines and cytotoxic molecules following 24-hour coculture of 19xGD-TCE-treated y5T Cells and Nalm6 (B- ALL) cells. Serial dilutions of 19xGD-TCE at picomolar (pM) concentrations were introduced into a coculture of y5T cells and Nalm6 (B-ALL) cells at an effector-to-target (E:T) ratio of 1 : 1 for 24 hours (N=3). The supernatants from these cocultures, exposed to various concentrations of 19xGD-TCE, were harvested and analyzed for cytokine and cytotoxic molecule secretion using the LEGENDplex™ Human CD8 / NK Panel (BioLegend). Control conditions include medium-only, y5T-only, Nalm6-only, and y6T + Nalm6 coculture without 19xGD-TCE treatment. Heatmap illustrating the secretion levels of selected cytokines and cytotoxic molecules in the supernatants of cocultures from 3 different y6T donors. The color scale represents loglO-transformed concentrations. As shown in the figure, little IL-6, IL 10 and IL 17 secretion was observed. In another experiment (data not shown), the fold-change of cytokine release was highest for TNF-a and IFN-y, and the engager enhanced cytokine release for these enzymes in a dose-dependent manner.

[0412] FIG. 30 shows that the CD19xGD engager expands and activates both V81+ and V82+ subsets from PBMCs.

[0413] In summary, the CD19xGD and CD33xGD engagers enhance specific and dosedependent anti-target activities of y6 T cells, including activation, cytotoxicity, and cytokine release, at low pico-molar EC50 levels against CD19+ ALL and CD33+ AML target cells, respectively. These engagers also exhibited a favorable cytokine profile, with no detectable or minimal release of cytokines associated with CRS and tumor promotion, such as IL-4, IL-6, IL-10, and IL-17a. CD19xGD also demonstrated a robust expansion of both V81 and V82 y8 T cells from PBMCs). Moreover, the CD19xGD engager broadly targets the B-cell compartment and could be repurposed for autoimmune indications with the potential to achieve deeper B cell depletion through y8 T cells that also present a lower risk of toxicities such as CRS or ICANS. In addition, the pan-y8 TCEs described herein demonstrate the ability to eliminate specific target cells in a dose-dependent manner and significantly expand both V81+ and V52+ T cells. V81+ cells proliferate and can overcome exhaustion and target tissue resident B cells for deeper B cell depletion. y8 T cells secrete less IL-6 and may reduce cytokine-release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) as compared with CAR-T and CD3 T-cell engagers.

[0414] Example 4: CD19xGD engager treatment provides robust B-cell depletion in PBMCs obtained from SLE patients and is therefore an approach for treatment of autoimmune disease The CD19xGD engager described above was shown to drive B-cell depletion and y8 T cell expansion in cultures prepared from peripheral blood samples obtained from SLE patients. Three SLE donors with active disease manifestation were chosen for the study. The timelines of y8 T cell expansion (squares) and depletion of B cell (circles) cross in all three SLE donors studied, indicating that the expansion of T cells is critical for the depletion of autoimmune B cells. 5 nM of the CD19xGD engager was used with all three donors.

[0415] FIG. 23 shows flow cytometric analysis of y8 T cells and B cells in peripheral blood samples obtained from systemic lupus erythematosus (SLE) donors with active disease. As shown in the figure, there were low levels of y8 T cells and high levels of B cells, representative of chronic autoimmune disease.

[0416] FIGs. 24A, 24B and 24C shows the relative percentages of live B cells and y8 T cells over time (days) for cultures prepared from three SLE donor PBMC samples treated with the 19xGD engager. Each donor sample showed y8 T cell expansion (squares) and depletion of B cells (circles). The depletion accelerates as the y8 T cells expand suggesting that the expansion is important for the ultimate near-complete depletion of the B cells. These figures show that the engager drives B cell depletion and y8 T cell expansion in SLE donor sample culture.

[0417] FIG. 25 shows the change (in folds) over no treatment for the y8 T cell cytotoxic activation marker CD107A over time (days) in 19xGD engager treated cultures prepared from three SLE donor peripheral blood samples. The figure shows the degranulation of y8 T cells upon treatment with the engager as measured by surface expression of CD107A on both V81T cells (green / gray squares) and V82T cells (blue / black squares activation markers increase during B cell depletion. V81+ (green / gray squares) and V52+ cells (blue / black squares), subtypes undergo degranulation / activation and this further demonstrates that the increases follow the trajectories of B cell depletion in all three donors.

[0418] FIGs. 26A and 26B are graphs showing expression (logio ng / ml; culture supernatant at Day 1 was compared to Day 8) of IgG and IgM antibodies in cultures prepared from three SLE donor peripheral blood samples treated with 19xGD engager (5 nM) and control cultures (no treatment: 0 nM). The depletion of B cells in all three donors by 19xGD engager treatment was further confirmed by this data showing the loss of IgG (left) and IgM (right) antibodies in an ELISA test performed with culture supernatants from Day 8.

[0419] FIGs. 27A and 27B are representative bar graphs showing the absolute number of 78 T cells expanded from PBMCs obtained from a healthy donor (left) and a SLE donor (right) treated with increasing concentration of purified 19xGD engager on Day 10. No engager (No Txt) and Zoledronate serve as controls. The figures shows that the engager induces pan -78 T cell expansion in both healthy and SLE donors.

[0420] FIG. 28 are graphs showing percent B cells for two donors treated with serial dilutions (7 total) of the CD19xGD engager, zoledronate, or no treatment. Serial dilutions of the CD19xGD engager showed dose-dependent B cell killing of PBMCs from two separate healthy donors using only the unmanipulated 78 T cells present in the PBMCs as compared to PBMC without the CD19xGD engager and PBMC with y8 T cell stimulation (Zoledronate) demonstrating complete ablation of the B cells only when in combination with the engager at day 8.

[0421] It was also shown that serial dilutions of purified 19xGD engager showed dosedependent killing of B cells, which express the target CD19, using PBMCs from the two separate donors. This experiment utilizes only the 78 T cells present in the PBMCs, which are expanded and activated by the 19xGD engager as compared to PBMCs without the engager (No Txt) and as compared to Zoledronate, which is known to expand the V82 subpopulations of y8 T cells. This demonstrates that the engager can be used to expand endogenous y8 T cells and direct them to kill the target cells efficiently, which does not happen in the absence of 19xGD engager.

[0422] FIG. 29 shows expansion and activation (CD95+) of y8 T cell subsets from PBMCs treated with 20 nM CD19xGD engager, 5 nM CD19xGD engager and no engager. Circles are total expanded V81 and V82 out of CD45 and rectangles are total CD95+ V81 and V82 out of CD45. This figure shows that the engager expands and activates both the V81+ and V82+ y8 T cell subsets from PBMCs. The CD19xGD engager activates both Vdl+ and Vd2+ T cells as shown by the expression of CD95 (FAS) from PBMCs. No significant expansion or activation is seen without the CD19xGD engager (“No Txt”) over 10-day culture of PBMCs.

[0423] FIG. 30 are bar graphs showing expansion and activation (CD95+) of V51+ and V82+ y8 T cell subsets from PBMCs. This figure shows that the CD19xGD engager expands V82+ cells within physiologic ranges. The CD19xGD engager expands V52+ cells within the same range as Zoledronate, which is known to expand V82+ cells and is approved for some medical uses in patients as a therapeutic and is known to be safe. Suggesting that the level of expansion from Zoledronate does not result in patient toxicities. The CD19xGD engager. The CD19xGD engager induces V82+ cell expansion without off-target effects that can occur from broad cytokine stimulation, and provides a potentially safer and targeted way to expand and activate y8 T cells.

[0424] FIG. 31 are graphs showing that the CD19xGD engager drives B cell depletion and 70 expansion in PBMCs from SLE donor with active disease. Each SLE donor demonstrates 76 T cell expansion (top) and depletion of B cells (bottom). The depletion accelerates as yS T cells expand suggesting that the expansion may be important for the efficient and ultimate near-complete depletion of the B cells.

[0425] As described in the example above, the CD19xGD engager enhances specific and dose-dependent anti-tumor activity of 78 T cells at low picomolar EC50 levels, indicating activation, cytotoxicity and cytokine release against CD19+ ALL target cells and normal B cells. The engager exhibited a favorable cytokine release profile, with no detectable or minimal release of cytokines associated with CRS and tumor promotion, such as IL-4, IL-6, IL-10 and IL-17a. In addition, the engager induces deep depletion of B cells in PBMCs from SLE donors and demonstrated a robust expansion of both V81 and V82 T cells from PBMCs of both healthy or SLE donors, offering a pow erful and integrated approach for B-cell mediated autoimmune indications. The expanded y8 T cells exhibit favorable phenotypic profiles, suggesting that the engager may also sen e as a novel method for in vitro expansion of y8 T cell for therapeutic use.

[0426] In summaiy. the data demonstrates that the engager binds, activates, and expands y8 T cells, and that the engager demonstrates specific binding to target cells, including SLE B cells from patient PBMCs, induces potent cytotoxicity, and supports robust expansion of V81 and V82 T cells. These findings position the CD19xGD engager as a promising therapeutic candidate with potential applications in both oncology', inflammatory and autoimmune indications.

[0427] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

[0428] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are ...

Claims

1. CLAIMSWhat is claimed is:

1. A multifunctional antigen-binding molecule, wherein the molecule comprises i. a first antigen-binding domain that specifically binds to a tumor-associated antigen; and ii. a second antigen-binding domain that specifically binds to a y8 TCR, wherein the second antigen-binding domain binds to an epitope expressed by more than one 78 T cell subtype.

2. The antigen-binding molecule of claim 1 , wherein the second antigen-binding domain binds to an epitope expressed by both V81 and V82 subpopulations of y8 T cells.

3. The antigen binding molecule of claim 2, wherein the epitope is expressed by another y8 T cell subtype.

4. The antigen-binding molecule of any one of claims 1 to 3, wherein the second antigenbinding domain is a pan y8 TCR antigen-binding moiety.

5. The antigen-binding molecule of any one of claims 1 to 3, wherein the antigen-binding molecule further comprises a third domain, wherein the third domain specifically binds to a different y8 T cell epitope than the second antigen-binding domain, and wherein the binding of the third antigen-binding domain to the different epitope promotes expansion of y8 T cells.

6. The antigen-binding molecule of claim 5, wherein the binding of the third domain to the different epitope promotes expansion of the V81 and V82 subpopulations of y8 T cells.

7. The antigen-binding molecule of any one of the preceding claims, w herein each antigenbinding domain is independently selected from a Fab domain, a Fab', a single-chain variable fragment (scFv), and a single domain antibody (sdAb).

8. The antigen-binding molecule of any one of claims 1 to 4. wherein the first and the second antigen-binding domains are attached by a linker.

9. The antigen-binding molecule of claim 8, wherein the linker is a peptide linker.

10. The antigen-binding molecule of claim 5. wherein the second antigen-binding domain and the third domain are attached by a linker.

11. The antigen-binding molecule of any one of the preceding claims, wherein the tumor- associated antigen (TAA) is selected from the group consisting of CD19, CD33. CD123, CD20, CD22, EGFRvIII, IL13ra2, folate receptor 1 (FOLR1), CLL-1 , MMP-2, and mesothelin.

12. The antigen-binding molecule of claim 11, wherein the TAA is CD33.

13. The antigen-binding molecule of claim 1 1, wherein the TAA is CD19.

14. A multifunctional antigen-binding molecule, wherein the molecule comprises i. a first antigen-binding domain that specifically binds to CD33, and ii. a second antigen-binding domain that specifically binds to a y8 TCR and wherein the second antigen-binding domain specifically binds to an epitope expressed by more than one y8 T cell subtype.

15. The antigen-binding molecule of claim 14, wherein the second antigen-binding domain binds to an epitope expressed by both V81 and V82 subpopulations of y8 T-cells.

16. The antigen-binding molecule of claim 15, wherein the epitope is also expressed by another subpopulation of y8 T cells.

17. The antigen-binding molecule of any one of claims 14 to 16, wherein the second antigenbinding domain is a pan y8 TCR antigen-binding moiety.

18. The antigen-binding molecule of any one of claims 14 to 17, wherein the antigenbinding molecule further comprises a third domain, wherein the third domain specifically binds to a different y8 T cell epitope than the second antigen-binding domain, and wherein the binding of the third antigen-binding domain to the different epitope promotes expansion of the y8 T cells.

19. The antigen-binding molecule of claim 18, wherein the binding of the third antigenbinding domain to the different epitope promotes expansion of the V81 and V82 subpopulations of y8 T cells.

20. The antigen-binding molecule of any one of claims 14 to 19, wherein each antigenbinding domain is independently selected from a Fab domain, a Fab', a single-chain variable fragment (scFv) or a single domain antibody (sdAb).

21. A multifunctional antigen-binding molecule, wherein the molecule comprises i. a first antigen-binding domain that that specifically binds to CD 19, and ii. a second antigen-bindingdomain that specifically binds to a y8 TCR and wherein the second antigen-binding domain specifically binds to an epitope expressed by more than one y8 T cell subtype.

22. The antigen-binding molecule of claim 21, wherein the second antigen-binding domain binds to an epitope expressed by both V51 and V82 subpopulations of y8 T cells.

23. The antigen binding molecule of claim 22, wherein the epitope is expressed by another y8 T cell subtype.

24. The antigen-binding molecule of claim 23, wherein the second antigen-binding domain is a pan y8 TCR antigen-binding moiety.

25. The antigen-binding molecule of any one of claims 21 to 24, wherein the antigen-binding molecule further comprises a third domain, wherein the third domain specifically binds to a different y8 T cell epitope than the second antigen-binding domain, and wherein the binding of the third antigen-binding domain to the different epitope promotes expansion of the y5 T cells.

26. The antigen-binding molecule of claim 25, wherein the binding of the third antigenbinding domain to the different epitope promotes expansion of the V81 and V82 subpopulations of y8 T cells.

27. The antigen-binding molecule of any one of claims 21 to 26, wherein each antigenbinding domain is independently selected from a Fab domain, a Fab', a single-chain variable fragment (scFv) or a single domain antibody (sdAb).

28. A method for treating cancer in a subject in need thereof, the method comprising administering to said subject an effective amount of an antigen-binding molecule of any one of claims 1, 14 and 21.

29. The method of claim 28, wherein the second antigen-binding domain specifically binds to an epitope expressed by more than one y8 T cell subty pe.

30. The method of claim 29, wherein the second antigen-binding domain binds to an epitope expressed by both V81 and V82 subpopulations of y8 T cells.

31. The method of claim 30, wherein the second antigen-binding domain is a pan y8 TCR antigen-binding moiety.

32. The antigen-binding molecule of claim 28, wherein the antigen-binding molecule further comprises a third domain, wherein the third domain specifically binds to a different yd T cell epitope than the second antigen-binding domain, and wherein the binding of the third antigen-binding domain to the different epitope promotes expansion of the y8 T cells.

33. The antigen-binding molecule of claim 32, wherein the binding of the third antigenbinding domain to the different epitope promotes expansion of the V81 and V82 subpopulations of y8 T cells.

34. The method of claim 28, wherein the TAA is selected from the group consisting of CD 19, CD33, CD123, CD20, CD22, EGFRvIII, IL13ra2, folate receptor 1 (FOLR1), CLL-1, MMP- 2, and mesothelin.

35. The method of any one of claims 28 to 34, further comprising administration of an effective amount of a population of y8 T cells.

36. The method of any one of claims 28 to 34. wherein the method does not comprise administration of a population of y8 T cells with the antigen-binding molecule.

37. The method of claim 28, wherein the cancer is a hematologic malignancy or a solid tumor.

38. The method of claim 28, wherein the TAA is CD33, and the cancer is a CD33-associated cancer.

39. The method of claim 38, wherein the cancer is AML.

40. The method of claim 28, wherein the TAA is CD19 and the cancer is a CD19-associated cancer.

41. The method of claim 40, wherein the CD19-associated cancer is ALL.

42. The method of claim 36, wherein the expansion of a population of the patient’s y8 T cells is enhanced, and optionally activated, after administration of the antigen-binding molecule.

43. The method of claim 42, wherein the population of the patient’s y8 T cells includes V81 and V82 subpopulations of y8 T cells.

44. The method of claim 42, wherein the method does not comprise transplantation or administration of y8 T cells.

45. The method of claim 35, wherein expansion of the administered population of 76 T cells is enhanced by administration of the antigen-binding molecule.

46. A method of treating a B-cell mediated autoimmune disease or a B-cell mediated inflammatory disease in a patient in need thereof, the method comprising administering to said patient an effective amount of an antigen-binding molecule of any one of claims 21 to 27.

47. The method of claim 46, wherein the B-cell mediated autoimmune disease is selected from the group consisting of systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, antiphospholipid syndrome, pemphigus, cicatricial pemphigoid, myasthenia gravis, neuromyelitis optica, and immune thrombocytopenia.

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