Method for selectively expanding a γδ T cell population and composition thereof

Abstract

Methods for selectively expanding γδ T cell populations, compositions and mixtures thereof, and methods for using same as therapeutic agents are provided. [Solution] We provide a method for producing an enriched γδ T cell population by contacting a mixed cell population with one or more agents, such as antibodies, that selectively proliferate δ1 T cells or δ2 T cells by binding to activation epitopes specific to δ1 TCR or δ2 TCR, and we also provide methods for using engineered γδ T cell populations that express unengineered and chimeric antigen receptors, etc., as therapeutic agents for cancer, infectious diseases, and immune disorders.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit under Section 119(e) of U.S. Provisional Patent Application No. 62 / 335,572, filed May 12, 2016, the entire disclosure of which is hereby incorporated by reference in its entirety for all purposes. [Background technology]

[0002] Antigen recognition by T lymphocytes can be achieved through a variety of heterodimeric receptors, the T cell receptor (TCR). Approximately 95% of human T cells in blood and lymphoid organs express heterodimeric αβ TCR receptors (αβ T cell lineage). Approximately 5% of human T cells in blood and lymphoid organs express heterodimeric γδ TCR receptors (γδ T cell lineage). These T cell subsets are sometimes referred to as "αβ" and "γδ" T cells, respectively. αβ and γδ T cells have distinct functions. αβ T cell activation occurs when antigen-presenting cells (APCs) present antigens in association with class I / II MHC. In contrast to αβ T cells, γδ T cells can recognize antigens independently of MHC restriction. Furthermore, γδ T cells are capable of both innate and adaptive immune recognition and response.

[0003] γδ T cells utilize a distinct set of somatically rearranged variable (V), diversity (D), joining (J), and constant (C) genes. γδ T cells contain fewer V, D, and J segments than αβ T cells. Although the number of germline Vγ and Vδ genes is more limited than the repertoire of Vα and Vβ TCR genes, the potential γδ TCR repertoire is broader than that of αβ TCRs due to a more extensive junctional diversification process during rearrangement of the TCR γ and δ chains (Carding and Egan, Nat Rev Immunol (2002) 2:336).

[0004] Human γδ T cells use three major Vδ (Vδ1, Vδ2, and Vδ3) region genes and up to six Vγ region genes to generate their TCR (Hayday AC., Annu Rev Immunol. 2000;18,975-1026). The two major Vδ subsets are Vδ1 and Vδ2 γδ T cells. Vδ1 T cells, which possess various Vγs, account for the majority of the intraepithelial subset of mucosal γδ T cells, where the TCR appears to recognize stress molecules on epithelial cells (Beagley KW, Husband AJ. Crit Rev Immunol. 1998;18(3):237-254). Vδ2 T cells, which commonly co-express Vγ9, are abundant in peripheral blood and lymphoid systems.

[0005] The ability of γδ T cells to directly recognize antigens on diseased cells and exert their innate ability to kill tumor cells makes them an attractive therapeutic tool. Adoptive transfer of Vγ9Vδ2 T cells has resulted in limited clinical responses in research-stage cancer treatments (Kondo et al., Cytotherapy, 10:842-856, 2008; Lang et al., Cancer Immunology, Immunotherapy: CII, 60:1447-1460, 2011; Nagamine et al., 2009; Nicol et al., British Journal of Cancer, 105:778-786, 2011; Wilhelm et al., Blood. 2003 Jul 1;102(1):200-6), suggesting the need to isolate new γδ T cell populations and test them in the clinic.

[0006] The ability to selectively expand γδ T cell subset populations with potent antitumor activity at improved purity and clinically relevant levels is highly desirable. While cocktails of antibodies and cytokines have been used to expand more diverse sets of γδ T cells, activation of specific γδ T cell subsets to sufficiently pure and clinically relevant levels has not been achieved (Dokouhaki et al., 2010; Kang et al., 2009; Lopez et al., 2000; Kress, 2006). Therefore, clinically relevant methods for expanding specific γδ T cell subsets in vitro and the cells produced thereby are greatly needed.

[0007] Sequence Listing This application contains a Sequence Listing, which has been submitted via EFS Web in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on March 3, 2016, is named 47165-701.201-SL.txt, and is 4,366 kilobytes in size. Summary of the Invention

[0008] In one aspect, the present invention provides an in vitro method for producing an enriched γδ T cell population, which comprises: (i) selectively expanding δ1 T cells by binding to epitopes unique to the δ1 TCR; (ii) selectively expanding δ2 T cells by binding to epitopes unique to the δ2 ​​TCR; (iii) selectively expanding δ1 and δ4 T cells by binding to epitopes unique to the δ1 and δ4 TCRs; or (iv) by a method comprising contacting the mixed cell population or a purified fraction thereof with one or more agents that selectively expand δ1, δ3, δ4 and δ5 T cells by binding to epitopes unique to δ1TCR, δ3TCR, δ4TCR and δ5TCR, resulting in an enriched γδ T cell population. In a preferred embodiment, the enriched γδ T cell population comprises clinically relevant levels of γδ T cells.

[0009] In another aspect, the present invention provides an in vitro method for producing an enriched γδ T cell population from an isolated mixed cell population, comprising: (i) selectively expanding δ1 T cells by binding to epitopes unique to the δ1 TCR; (ii) selectively expanding δ2 T cells by binding to epitopes unique to the δ2 ​​TCR; (iii) selectively expanding δ1 and δ4 T cells by binding to epitopes unique to the δ1 and δ4 TCRs; or (iv) directly contacting the mixed cell population with one or more agents that selectively expand δ1, δ3, δ4 and δ5 T cells by binding to epitopes unique to δ1TCR, δ3TCR, δ4TCR and δ5TCR to provide clinically relevant levels of an enriched γδ T cell population.

[0010] In certain embodiments, a clinically relevant level is at least about 10 8 or more γδ T cells, at least about 10 9 or more γδ T cells, at least about 10 10 or more γδ T cells, at least about 10 11 or more γδ T cells, or at least about 10 12 or more γδ T cells (e.g., about 10 8 pieces ~ about 10 12In one embodiment, the isolated mixed cell population is derived from a single donor, and the method provides a clinically relevant enriched γδ T cell population expanded from a single donor, e.g., a single sample from a single donor, or two or more samples from a single donor. In other embodiments, the isolated mixed cell population is derived from more than one or multiple donors. In some embodiments, after the first enrichment step of the present invention, the enriched γδ T cell population comprises clinically relevant levels of 10 8 In other embodiments, the enriched γδ T cell population after the second, third, fourth, fifth, etc. enrichment steps of the present invention comprises more than 10 γδ T cell subsets. 8 It contains more than one γδ T cell subset.

[0011] In certain embodiments, the agent that selectively expands δ1 T cells is selected from agents that bind to the same epitope as an antibody selected from TS-1 and TS8.2. In some embodiments, the agent that selectively expands δ1 T cells is an agent that binds to an epitope that is different from the epitope bound by the TS-1 and / or TS8.2 antibodies. In some embodiments, the agent that selectively expands δ1 T cells is an agent that binds to an epitope that does not overlap with the epitope bound by TS-1 or TS8.2 or that does not compete with TS-1 or TS8.2. In some embodiments, the agent that selectively expands δ1 T cells is selected from agents that specifically bind to an epitope comprising the δ1 variable region. In other embodiments, the agent binds to a δ1 TCR variable region comprising the amino acid sequence of the consensus sequence in Figure 24. In still other embodiments, the agent binds to an epitope comprising residue Arg71 or Asp72 of the δ1 variable region and / or residue Lys120 of the J1 or J2 region. In some embodiments the agent has reduced binding to a mutant δ1TCR polypeptide comprising a mutation at K120 of δ1J1 or δ1J2, for example K120A, K120G, K120P, K120V, K120E, K120D or K120S.

[0012] In certain embodiments, the agent that selectively expands δ1 T cells is selected from agents that bind to the same epitope as antibody R9.12. In some embodiments, the agent that selectively expands δ1 T cells is an agent that binds to an epitope that is different from the epitope bound by antibody R9.12. In some embodiments, the agent that selectively expands δ1 T cells is an agent that binds to an epitope that does not overlap with the epitope bound by antibody R9.12 or that does not compete with antibody R9.12.

[0013] In a particular embodiment, the agent that selectively expands δ1 T cells is an agent that selectively binds to and / or selectively expands δ1 and δ4 T cells, or δ1, δ3, δ4 and δ5 T cells. In a particular embodiment, the agent that selectively expands δ1 T cells is an agent comprising a complementarity determining region (CDR) of an antibody selected from the group consisting of δ1-05, δ1-08, δ1-18, δ1-22, δ1-23, δ1-26, δ1-35, δ1-37, δ1-39, δ1-113, δ1-143, δ1-149, δ1-155, δ1-182, δ1-183, δ1-191, δ1-192, δ1-195, δ1-197, δ1-199, δ1-201, δ1-203, δ1-239, δ1-253, δ1-257, δ1-278, δ1-282 and δ1-285. In a particular embodiment, the agent that selectively expands δ1 T cells is an agent comprising a variable region of an antibody selected from the group consisting of δ1-05, δ1-08, δ1-18, δ1-22, δ1-23, δ1-26, δ1-35, δ1-37, δ1-39, δ1-113, δ1-143, δ1-149, δ1-155, δ1-182, δ1-183, δ1-191, δ1-192, δ1-195, δ1-197, δ1-199, δ1-201, δ1-203, δ1-239, δ1-253, δ1-257, δ1-278, δ1-282 and δ1-285. In a particular embodiment, the agent that selectively expands δ1 T cells is an agent that binds to the same or essentially the same epitope as, or competes with, an antibody selected from the group consisting of δ1-05, δ1-08, δ1-18, δ1-22, δ1-23, δ1-26, δ1-35, δ1-37, δ1-39, δ1-113, δ1-143, δ1-149, δ1-155, δ1-182, δ1-183, δ1-191, δ1-192, δ1-195, δ1-197, δ1-199, δ1-201, δ1-203, δ1-239, δ1-253, δ1-257, δ1-278, δ1-282 and δ1-285.In a particular embodiment, the agent that selectively expands δ1 T cells is an antibody selected from the group consisting of δ1-05, δ1-08, δ1-18, δ1-22, δ1-23, δ1-26, δ1-35, δ1-37, δ1-39, δ1-113, δ1-143, δ1-149, δ1-155, δ1-182, δ1-183, δ1-191, δ1-192, δ1-195, δ1-197, δ1-197, δ1-199, δ1-201, δ1-203, δ1-239, δ1-253, δ1-257, δ1-278, δ1-282 and δ1-285. In certain embodiments, the agent that selectively expands δ1 T cells is an antibody that binds to a bin 1 δ1 epitope, a bin 1b δ1 epitope, a bin 2 δ1 epitope, a bin 2b δ1 epitope, a bin 2c δ1 epitope, a bin 3 δ1 epitope, a bin 4 δ1 epitope, a bin 5 δ1 epitope, a bin 6 δ1 epitope, a bin 7 δ1 epitope, a bin 8 δ1 epitope or a bin 9 δ1 epitope.

[0014] In some embodiments, the methods of the invention provide enriched γδ T cell population(s) comprising more than 60%, 70%, 80% or 90% δ1 cells from an isolated mixed cell population comprising T lymphocytes. In some embodiments, the methods of the invention provide enriched γδ T cell population(s) comprising more than 60%, 70%, 80% or 90% δ1 cells from an isolated, e.g. mixed, cell population comprising T lymphocytes, prior to a step of purification of the expanded γδ T cell population(s), e.g. by positive selection of γδ T cells from the enriched γδ T cell population(s) (e.g. positive selection of δ1 T cells, δ1 and δ3 γδ T cells; δ1 and δ4 γδ T cells; or δ1, δ3, δ4 and δ5 T cells (e.g. γδ T cells of the indicated δ subtype(s)); and / or positive selection of δ2 T cells) or by removal of non-γδ T cells (e.g. non-δ1 T cells such as αβ T cells) from the enriched γδ T cell population(s).

[0015] In some embodiments, the methods of the invention provide a γδ T cell population comprising δ1 γδ T cells and δ2 γδ T cells, wherein more than 60%, more than 70%, more than 80% or more than 90% of the population are δ1 γδ T cells. In some embodiments, the methods of the invention provide a γδ T-cell population comprising δ1 γδ T cells; δ2 γδ T cells; δ1 and δ4 γδ T cells; or δ1, δ3, δ4 and δ5 γδ T cells, wherein (i) more than 60%, more than 70%, more than 80% or more than 90% of the population are δ1 γδ T cells, (ii) more than 60%, more than 70%, more than 80% or more than 90% of the population are δ1 and δ3 γδ T cells, (iii) more than 60%, more than 70%, more than 80% or more than 90% of the population are δ1 and δ4 γδ T cells, or (iv) more than 60%, more than 70%, more than 80% or more than 90% of the population are δ1, δ3, δ4 and δ5 γδ T cells. In some embodiments, the methods of the invention provide compositions comprising a population of γδ T cells (e.g., δ1γδ T cells) that are free of αβ T cells or contain less than about 2%, about 1%, about 0.5%, about 0.4%, about 0.1%, about 0.05%, or about 0.01% αβ T cells.

[0016] In certain embodiments, the isolated mixed cell population from which γδ T cells are expanded in the methods described herein comprises about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1.5% or less, about 1.2% or less, about 1% or less, about 0.9% or less, about 0.8% or less, about 0.7% or less, about 0.6% or less, about 0.5% or less, about 0.4% or less, about 0.3% or less, about 0.2% or less, or about 0.1% or less γδ T cells (e.g., δ1, δ2, δ1 and δ2, δ1 and δ3, or δ1 and δ4 γδ T cells, or a combination thereof). In certain embodiments, the isolated mixed cell population from which γδ T cells are expanded using the methods described herein comprises between about 0.5% and about 5% γδ T cells (e.g., δ1, δ2, δ3, δ4, or δ8 γδ T cells, or a combination thereof).

[0017] In certain embodiments, the isolated mixed cell population is from one donor or multiple donors with greater than the median level of circulating γδ T cells or infiltrating γδ T cells within a sample (e.g., within a tumor or epithelial sample). Thus, for example, in some embodiments, the isolated mixed cell population from which γδ T cells are expanded using the methods described herein comprises between about 0.5% and about 10% γδ T cells (e.g., δ1, δ2, δ3, δ4, or δ8 γδ T cells, or a combination thereof). As another example, in some embodiments, the isolated mixed cell population from which γδ T cells are expanded using the methods described herein comprises between about 10% and about 30% γδ T cells (e.g., δ1, δ2, δ3, δ4, or δ8 γδ T cells, or a combination thereof). In some embodiments, the isolated mixed cell population from which γδ T cells are expanded using the methods described herein comprises about 1.5% or less γδ T cells, preferably less than about 1.2% γδ T cells, more preferably less than about 1% γδ T cells, even more preferably less than about 0.5% γδ T cells, for example, between about 0.1% and about 0.4% γδ T cells.

[0018] In certain embodiments, the isolated mixed cell population from which γδ T cells are expanded in the methods described herein comprises about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, or about 20% T lymphocytes and less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1.5%, less than about 1.2%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, or less than about 0.1% γδ T cells (e.g., δ1, δ2, δ1 and δ2, δ1 and δ3, or δ1 and δ4 γδ T cells, or a combination thereof). In certain embodiments, the isolated mixed cell population from which γδ T cells are expanded using the methods described herein comprises about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, or about 20% T lymphocytes and about 0.5% to about 5% γδ T cells (e.g., δ1, δ2, δ3, δ4, or δ8 γδ T cells, or a combination thereof).

[0019] In certain embodiments, the isolated mixed cell population is from one donor or multiple donors with greater than the median level of circulating γδ T cells or infiltrating γδ T cells within a sample (e.g., within a tumor or epithelial sample). Thus, for example, in some embodiments, the isolated mixed cell population from which γδ T cells are expanded using the methods described herein comprises about 80%, 70%, 60%, 50%, 40%, 30%, or 20% T lymphocytes and about 0.5% to about 10% γδ T cells (e.g., δ1, δ2, δ3, δ4, or δ8 γδ T cells, or a combination thereof). As another example, in some embodiments, the isolated mixed cell population from which γδ T cells are expanded using the methods described herein comprises about 80%, 70%, 60%, 50%, 40%, 30%, or 20% T lymphocytes and about 10% to 30% γδ T cells (e.g., δ1, δ2, δ3, δ4, or δ8 γδ T cells, or a combination thereof). In some embodiments, the isolated mixed cell population from which γδ T cells are expanded using the methods described herein comprises about 80%, 70%, 60%, 50%, 40%, 30%, or 20% T lymphocytes and about 1.5% or less γδ T cells, preferably less than about 1.2%, more preferably less than about 1%, and even more preferably less than about 0.5% γδ T cells, e.g., about 0.1% to about 0.4% γδ T cells.

[0020] In some embodiments, the isolated mixed cell population is selected from a peripheral blood sample (e.g., whole blood, PBMCs, or PBLs), a leukapheresis sample, a cord blood sample, a tumor sample, or a tissue sample (e.g., an epithelial sample). In some embodiments, the isolated mixed cell population is derived from a single donor. In other embodiments, the isolated mixed cell population is derived from more than one or multiple donors. In certain embodiments, the methods of the invention provide enriched γδ T-cell population(s) comprising polyclonal γδ TCR diversity.

[0021] In other embodiments, the agent that selectively expands 52 T cells is selected from agents that bind to the same epitope as an antibody selected from B6 and 15D. In yet other embodiments, the agent that selectively expands 52 T cells is selected from agents that bind to a different epitope than an antibody selected from B6 and 15D. In yet other embodiments, the agent that selectively expands 52 T cells is selected from agents that bind to an epitope that does not overlap with or compete with the epitopes bound by the B6 and 15D antibodies. In one embodiment, the agent that selectively expands 52 T cells is selected from agents that specifically bind to an epitope comprising the 52 variable region. In a specific embodiment, the agent has reduced binding to a mutant 52 TCR polypeptide comprising a mutation at G35 in the 52 variable region.

[0022] In a particular embodiment, the agent that selectively expands δ2 T cells is an agent comprising a complementarity determining region (CDR) of an antibody selected from the group consisting of δ2-14, δ2-17, δ2-22, δ2-30, δ2-31, δ2-32, δ2-33, δ2-35, δ2-36, and δ2-37 (or δ2-14, δ2-17, δ2-30, δ2-31, δ2-32, δ2-33, δ2-35, δ2-36, and δ2-37). In a particular embodiment, the agent that selectively expands δ2 T cells is an agent comprising a variable region of an antibody selected from the group consisting of δ2-14, δ2-17, δ2-22, δ2-30, δ2-31, δ2-32, δ2-33, δ2-35, δ2-36, and δ2-37 (or δ2-14, δ2-17, δ2-30, δ2-31, δ2-32, δ2-33, δ2-35, δ2-36, and δ2-37).

[0023] In a particular embodiment, the agent that selectively expands δ2 T cells is an agent that binds to the same or essentially the same epitope as or competes with an antibody selected from the group consisting of δ2-14, δ2-17, δ2-22, δ2-30, δ2-31, δ2-32, δ2-33, δ2-35, δ2-36, and δ2-37 (or δ2-14, δ2-17, δ2-30, δ2-31, δ2-32, δ2-33, δ2-35, δ2-36, and δ2-37). In certain embodiments, the agent that selectively expands 52 T cells is an antibody selected from the group consisting of 52-14, 52-17, 52-22, 52-30, 52-31, 52-32, 52-33, 52-35, 52-36, and 52-37 (or 52-14, 52-17, 52-30, 52-31, 52-32, 52-33, 52-35, 52-36, and 52-37). In certain embodiments, the agent that selectively expands 52 T cells is an antibody that binds to a bin 1 52 epitope, a bin 2 52 epitope, a bin 3 52 epitope, or a bin 4 52 epitope.

[0024] In some embodiments, the methods of the invention provide enriched γδ T cell population(s) comprising more than 60%, 70%, 80% or 90% δ2 cells from an isolated mixed cell population comprising T lymphocytes. In some embodiments, the methods of the invention provide enriched γδ T cell population(s) comprising more than 60%, 70%, 80% or 90% δ2 cells from an isolated, e.g., mixed, cell population comprising T lymphocytes, prior to a step of purification of the expanded γδ T cell population, e.g., by positive selection of γδ T cells from the enriched γδ T cell population(s) (e.g., positive selection of δ2 T cells) or by depletion of non-γδ T cells from the enriched γδ T cell population(s) (e.g., depletion of non-δ2 T cells or depletion of αβ T cells). In some embodiments, the methods of the invention provide a γδ T cell population comprising δ2 γδ T cells and δ1 γδ T cells, wherein more than 60%, more than 70%, more than 80% or more than 90% of the population are δ2 γδ T cells. In some embodiments, the methods of the invention provide compositions comprising a population of δ2γδ T cells that are free of αβ T cells or that contain less than about 2%, about 1%, about 0.5%, about 0.4%, about 0.1%, about 0.05%, or about 0.01% αβ T cells.

[0025] In certain embodiments, the isolated mixed cell population from which γδ T cells are expanded using the methods described herein comprises about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, or about 20% T lymphocytes and less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1%, less than about 0.05%, or less than about 0.02% δ2 cells. In some embodiments, the isolated mixed cell population from which γδ T cells are expanded in the methods described herein is selected from a peripheral blood sample (e.g., PBMCs or PBLs), a leukapheresis sample, an umbilical cord blood sample, a tumor, or a tissue. In certain embodiments, the methods of the invention provide enriched γδ T cell population(s) comprising polyclonal TCR diversity.

[0026] In certain embodiments, the enriched γδ T cell population has not been expanded by or in the presence of antigen-presenting cells (APCs), artificial antigen-presenting cells (aAPCs), an irradiated antigen-presenting cell population (e.g., irradiated PBMCs, irradiated immobilized cell lines, or irradiated aAPCs), an aminophosphonate, an aminophosphate, a bisphosphonate, or a combination thereof. In certain embodiments, the enriched γδ T cell population has not been expanded by or in the presence of irradiated PBMCs. In certain embodiments, the enriched γδ T cell population has not been expanded by or in the presence of irradiated aAPCs (e.g., engineered K562, RPMI8226, T2, or JVM-3). In certain embodiments, the enriched γδ T cell population has not been expanded by or in the presence of an irradiated immobilized cell line cell population. In a preferred embodiment, the one or more agents that selectively expand δ1 T cells, δ2 T cells, δ1 T cells and δ4 T cells, or δ1, δ3, δ4 and δ5 T cells (e.g., γδ T cells) are antibodies.

[0027] In some embodiments, the one or more agents that selectively expand δ1 T cells, δ2 T cells, δ1 T cells and δ4 T cells, or δ1, δ3, δ4 and δ5 T cells (e.g., γδ T cells of the indicated δ subtype(s)) are immobilized on a surface. In some embodiments, the one or more agents that selectively expand δ1 T cells, δ2 T cells, δ1 T cells and δ4 T cells, or δ1, δ3, δ4 and δ5 T cells (e.g., γδ T cells of the indicated δ subtype(s)) are immobilized on the surface of antigen-presenting cells (e.g., aAPCs) in the first and / or second or subsequent expansion. The agent immobilized on the surface of the antigen-presenting cell may, for example, bind to an Fc receptor expressed on the surface of the APC or may be expressed on the surface of the APC.

[0028] In some embodiments, the methods of the invention are carried out using culture medium supplemented with IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-18, IL-19, IL-21, IL-23, IL-33, IFNγ, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), a lectin (e.g., PHA-E, PHA-L, or ConA), or a combination of two or more or all of them. In some embodiments, the methods of the invention are carried out using culture medium that is not supplemented with IL-21. In some embodiments, the methods of the invention are practiced with first γδ T cell expansion using culture medium supplemented with IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-18, IL-19, IL-21, IL-23, IL-33, IFNγ, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), a lectin (e.g., PHA-E, PHA-L, or ConA), or a combination of two or more or all thereof. In some embodiments, the methods of the invention are practiced with first γδ T cell expansion using culture medium not supplemented with IL-21. In some embodiments, the methods of the invention are practiced with second γδ T cell expansion using culture medium supplemented with IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-18, IL-19, IL-21, IL-23, IL-33, IFNγ, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), a lectin (e.g., PHA-E, PHA-L, or ConA), or a combination of two or more or all thereof. In some embodiments, the methods of the invention are practiced with second γδ T cell expansion using culture medium not supplemented with IL-21.

[0029] In some embodiments, the methods of the invention are carried out using a first γδ T cell expansion comprising any of the γδ T cell expansion methods or compositions described herein, followed by a second γδ T cell expansion using culture media that does not contain one or more agents that selectively expand δ1 T cells; δ2 T cells; δ1 T cells and δ3 T cells; δ1 T cells and δ4 T cells; or δ1, δ3, δ4 and δ5 T cells (e.g. γδ T cells of the indicated δ subtype(s)). In some embodiments, the methods of the invention employ a first γδ T cell expansion comprising any of the γδ T cell expansion methods described herein comprising contacting engineered or non-engineered γδ T cells, engineered or non-engineered γδ T cell populations and / or isolated mixed cell populations with a fixed agent that selectively expands δ1 T cells; δ2 T cells; δ1 T cells and δ4 T cells; or δ1, δ3, δ4 and δ5 T cells (e.g. γδ T cells of the indicated δ subtype(s)), or a composition (e.g. activator) described herein, followed by: i) selecting δ1 T cells; δ2 T cells; δ1 T cells and δ4 T cells; or δ1, δ3, δ4 and δ5 T cells. ii) contains a structurally distinct (e.g. immobilized) agent that selectively expands δ1 T cells; δ2 T cells; δ1 T cells and δ4 T cells; or δ1, δ3, δ4 and δ5 T cells; iii) contains a non-immobilized (soluble) agent that selectively expands δ1 T cells; δ2 T cells; δ1 T cells and δ4 T cells; or δ1, δ3, δ4 and δ5 T cells; or iv) is performed with a second γδ T cell expansion using culture medium that contains an agent that expands T cells (e.g. expands αβ and γδ T cells) or that specifically expands γδ T cells compared to αβ T cells.

[0030] In some cases, the agent that expands T cells is an agent that binds to CD3, e.g., an anti-CD3 antibody (e.g., OKT3). In some cases, the agent that selectively expands γδ T cells is an agent that binds to the gamma chain constant region or the delta chain constant region, or an agent that binds to the γδ TCR, e.g., an antibody that binds to the γδ TCR (e.g., IMMU510). In some cases, the γδ T cell population is enriched by positive selection between the first and second cell expansions and / or after the second cell expansion. In some cases, the γδ T cell population is enriched by depletion of αβ T cells between the first and second cell expansions and / or after the second cell expansion.

[0031] In certain embodiments of the methods of the invention, e.g., enriched, γδ T cell populations are expanded in a first, second, or subsequent expansion in culture medium containing antigen-presenting cells (APCs). In some embodiments, the culture medium contains one or more agents (e.g., antibodies binding to the constant or variable region of the γδ TCR, antibodies binding to CD3, and / or aminophosphonates) that expand T cells, expand γδ T cells, selectively expand γδ T cells, or selectively expand δ1 T cells, δ2 T cells, δ1 T cells and δ3 T cells, or δ1 T cells and δ4 T cells. In certain embodiments, enriched γδ T cell populations are expanded in culture medium containing irradiated PBMCs. In certain embodiments, enriched γδ T cell populations are expanded in culture medium containing irradiated artificial APCs (aAPCs). In certain embodiments, enriched γδ T cell populations are expanded in culture medium containing a cell population of an irradiated immortalized cell line (e.g., K562 APCs). In some cases, the APCs do not express or exhibit reduced expression of HLA class I, HLA class II, HLA class II invariant chain, and / or HLA-DM. In some cases, the APCs express adhesion or costimulatory molecules, such as intercellular adhesion molecule-1, CD11a, CD18, CD54, CD80, CD86, 4-1BBL, OX-40L, CD70, or one or more membrane-anchored γδ T cell activators and / or leukocyte function-associated antigen-3. In some cases, the APCs express Fc receptors, e.g., Fc receptors specific for the isotype of an activator used in the γδ T cell expression methods described herein. In some cases, the APC expresses one or more Fc receptors selected from the group consisting of CD64, CD32A, CD32B, CD32C, CD16A, CD16B, FcRn, TRIM21, or CD307, or engineered variants thereof with higher affinity or altered specificity.

[0032] In some embodiments, the culture medium containing antigen-presenting cells (APCs), artificial antigen-presenting cells (aAPCs), or irradiated antigen-presenting cell populations (e.g., PBMCs, immobilized cell lines, or aAPCs) further contains one or more agents that selectively expand δ1 T cells; δ2 T cells; δ1 T cells and δ3 T cells; δ1 T cells and δ4 T cells; or δ1, δ3, δ4, and δ5 T cells. In particular embodiments, the cell populations, e.g., irradiated PBMCs, APCs, aAPCs, or immobilized cell lines, express on their cell surface one or more agents that selectively expand δ1 T cells; δ2 T cells; δ1 T cells and δ4 T cells; or δ1, δ3, δ4, and δ5 T cells. In some preferred embodiments, the one or more agents that selectively expand δ1 T cells; δ2 T cells; δ1 T cells and δ4 T cells; or δ1, δ3, δ4, and δ5 T cells are antibodies. In some embodiments, the one or more agents that selectively expand δ1 T cells; δ2 T cells; δ1 T cells and δ4; or δ1, δ3, δ4 and δ5 T cells are antibodies that are expressed on the surface of APCs or that bind to Fc receptors (e.g. Fcγ receptors) expressed on the surface of APCs.

[0033] In some embodiments, the present invention provides an ex vivo method for producing an enriched γδ T cell population from an isolated, e.g., mixed, cell population, comprising: (i) in a first γδ T cell expansion, (a) expanding γδ T cells (e.g., by binding to a γδ TCR); or (b) - selectively expand δ1 T cells by binding to activation epitopes unique to the δ1 TCR; - selectively expand δ2 T cells by binding to activation epitopes unique to the δ2 ​​TCR; - selectively expanding δ1 and δ4 T cells by binding to activation epitopes unique to the δ1 and δ4 TCRs; or - directly contacting, e.g., a mixed cell population, with one or more agents that selectively expand δ1, δ3, δ4 and δ5 T cells by binding to activation epitopes unique to the δ1, δ3, δ4 and δ5 TCRs, thereby producing a first enriched γδ T cell population, and then (ii) in a second γδ T cell expansion, (a) expanding T cells, (b) expanding γδ T cells (e.g., by binding to the γδ TCR), or (c) - selectively expand δ1 T cells by binding to activation epitopes unique to the δ1 TCR; - selectively expand δ2 T cells by binding to activation epitopes unique to the δ2 ​​TCR; - selectively expanding δ1 and δ4 T cells by binding to activation epitopes unique to the δ1 and δ4 TCRs; or - directly contacting at least a portion of the first enriched γδ T cell population with antigen presenting cells (APCs), optionally in the presence of one or more soluble or immobilized agents that selectively expand δ1, δ3, δ4 and δ5 T cells by binding to activation epitopes unique to the δ1, δ3, δ4 and δ5 TCRs, thereby producing a second enriched γδ T cell population, wherein the second enriched γδ T cell population is a clinically relevant number (e.g. 10 8 δ1 T cells; δ2 T cells; δ1 T cells and δ4 T cells; or δ1, δ3, δ4 and δ5 T cells.

[0034] In some cases, a clinically relevant number (e.g., 10 8 In some cases, a clinically relevant number (e.g., 10 or more) of γδ T cells can be obtained from a single donor, e.g., a single sample from a single donor, or two or more samples. 8 The γδ T cells (more than 100 cells / ml) are obtained from a single donor, e.g., a single sample or two or more samples from a single donor, within less than 30 days of expansion, preferably less than 21 days of expansion, more preferably within 19 days of expansion.

[0035] In some cases, the γδ T cell population is enriched by positive selection between (i) and (ii) or after (ii). In some cases, the γδ T cell population is enriched by depletion of αβ T cells between (i) and (ii) or after (ii). In some cases, the agent that expands T cells is an agent that binds to CD3, e.g., an anti-CD3 antibody. In some cases, the agent that selectively expands γδ T cells is an agent that binds to a gamma chain constant region or a delta chain constant region, or an agent that binds to a γδ TCR, e.g., an antibody that binds to a γδ TCR (e.g., IMMU510).

[0036] In certain embodiments of the methods of the invention, the enriched γδ T cell population is (i) a primary γδ T cell expansion, in which (a) γδ T cells are expanded (e.g., by binding to a γδ TCR), or (b) - selectively expand δ1 T cells by binding to activation epitopes unique to the δ1 TCR; - selectively expand δ2 T cells by binding to activation epitopes unique to the δ2 ​​TCR; - selectively expanding δ1 and δ4 T cells by binding to activation epitopes unique to the δ1 and δ4 TCRs; or - contacting the isolated, e.g. mixed, cell population with one or more first agents that selectively expand δ1, δ3, δ4 and δ5 T cells by binding to activation epitopes unique to the δ1, δ3, δ4 and δ5 TCRs to provide a first enriched γδ T cell population; and thereafter (ii) in a second γδ T cell expansion, (a) expanding T cells, (b) expanding γδ T cells (e.g. by binding to the γδ TCR), or (c) - selectively expand δ1 T cells by binding to activation epitopes unique to the δ1 TCR; - selectively expand δ2 T cells by binding to activation epitopes unique to the δ2 ​​TCR; - selectively expanding δ1 and δ4 T cells by binding to activation epitopes unique to the δ1 and δ4 TCRs; or - contacting the first enriched γδ T cell population, or a portion thereof, with one or more second agents that selectively expand δ1, δ3, δ4 and δ5 T cells by binding to activation epitopes unique to the δ1, δ3, δ4 and δ5 TCRs, wherein the one or more second agents are structurally distinct from the one or more first agents, thereby providing a second enriched γδ T cell population. In some cases, the one or more second agents bind to different γδ TCR epitopes compared to the one or more first agents.

[0037] In some cases, the γδ T cell population is enriched by positive selection between (i) and (ii) or after (ii). In some cases, the γδ T cell population is enriched by depletion of αβ T cells between (i) and (ii) or after (ii). In some cases, the agent that expands T cells is an agent that binds to CD3, e.g., an anti-CD3 antibody. In some cases, the agent that selectively expands γδ T cells is an agent that binds to a gamma chain constant region or a delta chain constant region, or an agent that binds to a γδ TCR, e.g., an antibody that binds to a γδ TCR (e.g., IMMU510).

[0038] In some cases, the first γδ T cell expansion is performed in culture medium containing antigen-presenting cells (APCs). In some cases, the second γδ T cell expansion is performed in culture medium containing antigen-presenting cells (APCs). In some cases, the first γδ T cell expansion is performed in culture medium without antigen-presenting cells (APCs). In some cases, the first γδ T cell expansion is performed in culture medium without antigen-presenting cells (APCs), and the second γδ T cell expansion is performed in culture medium containing antigen-presenting cells (APCs). In some cases, the first and second γδ T cell expansions are performed in culture medium containing antigen-presenting cells (APCs). In some cases, the first and second γδ T cell expansions are performed in culture medium containing an aminophosphate, an aminophosphonate, a bisphosphonate, or a combination thereof.

[0039] In another embodiment, the enriched γδ T cell population(s) of the present invention can be further formulated for administration to a subject. The enriched γδ T cell population(s) of the present invention comprise a therapeutically effective amount of γδ T cells. In certain embodiments, the γδ T cell population(s) have been engineered to stably express one or more structurally distinct tumor recognition moieties. In certain embodiments, the engineered γδ T cell(s) have been expanded and / or further expanded as described herein.

[0040] In some of the above aspects, embodiments, cases, and examples, the one or more agents stimulate the proliferation of γδ T cells at an average rate of one cell division per 30 hours or less, e.g., between about 17 hours and about 30 hours or less. In some embodiments, the average rate of division is for 0-4, 0-5, 0-7 consecutive days of γδ T cell proliferation, for 0-13 consecutive days of γδ T cell proliferation, for 0-19 consecutive days of γδ T cell proliferation, for 0-21 consecutive days of γδ T cell proliferation, or for at least 3, 4, 5, 6, 7, 10, 13, 19, or 21 consecutive days of γδ T cell proliferation. In other embodiments, the one or more agents stimulate the proliferation of γδ T cells at an average rate of one cell division per 24 hours or less, e.g., between about 17 hours and about 24 hours or less. In other embodiments, the one or more agents stimulate the proliferation of γδ T cells at an average rate of one cell division per 18 hours or less, or about 18 hours or less.

[0041] It will be appreciated that one or more of the above aspects, embodiments, cases and examples can be readily adapted for γδ T cell expansion (e.g., selective γδ T cell expansion) from a substantially homogeneous cell population, for example, after establishment of a γδ T cell engineered clone or cell line, a population containing one or more (e.g., structurally distinct) engineered γδ T cells, or after one or more steps of negative or positive selection of a cell population containing γδ T cells. Accordingly, one or more of the above methods, or a combination thereof, can be used to expand engineered γδ T cells by contacting the engineered γδ T cells or populations thereof with any one or more activating agents described herein, including one or more antibodies described herein, in soluble or immobilized form (e.g., immobilized on the surface of an APC).

[0042] Thus, in some embodiments, an isolated mixed cell population is replaced in a method or composition described herein with a γδ T cell engineered clone or cell line, or a population containing one or more (e.g., structurally distinct) engineered γδ T cells.

[0043] In other aspects, the invention provides selectively expanded γδ T cell population(s), wherein more than 60%, more than 70%, more than 80% or more than 90% of the expanded γδ T cells are δ1 T cells; δ1 T cells and δ4 T cells; or δ1 T cells, δ3 T cells, δ4 T cells and δ5 T cells. In particular embodiments, the selectively expanded γδ T cell population(s) comprises δ1 T cells and δ2 T cells, and more than 60%, more than 70%, more than 80% or more than 90% of the expanded γδ T cells are δ1 T cells, δ1 T cells and δ4 T cells; or δ1 T cells, δ3 T cells, δ4 T cells and δ5 T cells. In certain embodiments, the selectively expanded γδ T cell population(s) have not been purified by positive selection of γδ T cells from an enriched γδ T cell population (e.g. positive selection of δ1 T cells, δ1 T cells and δ3 T cells, or δ1 T cells and δ4 T cells) or by removal of non-γδ T cells from an enriched γδ T cell population (e.g. removal of non-δ1 T cells, such as αβ T cells).

[0044] In certain embodiments, the selectively expanded γδ T cell population(s) have been expanded directly from an isolated mixed cell population comprising T lymphocytes. In certain embodiments, the isolated mixed cell population from which γδ T cells are expanded using the methods described herein comprises about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, or about 20% T lymphocytes and less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1.5%, less than about 1.2%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, or less than about 0.1% δ1 cells. In certain embodiments, the isolated mixed cell population from which γδ T cells are expanded using the methods described herein comprises less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1.5%, less than about 1.2%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, or less than about 0.1% δ1 cells. In some embodiments, the isolated mixed cell population is selected from a peripheral blood sample, an umbilical cord blood sample, a tumor, an epithelial tissue, or a biopsy of skin, liver, or other tissue. In some embodiments, the isolated mixed cell population is derived from a single donor. In other embodiments, the isolated mixed cell population is derived from more than one or multiple donors. In one embodiment, the expanded γδ T cell population(s) are derived from intratumoral infiltrating lymphocytes, which may be isolated, for example, from colon adenocarcinoma metastases, liver, ovarian, head and neck or kidney cancers.

[0045] In some embodiments, the selectively expanded γδ T cell population(s) of the invention comprise more than 60% or 70% δ1 T cells; δ1 T cells and δ4 T cells; or δ1 T cells, δ3 T cells, δ4 T cells and δ5 T cells expressing naive or T central memory (TCM) phenotypes, respectively, CD45RA+ / CD27+ and / or CD45RA- / CD27+. In some embodiments, the expanded γδ T cell population comprises polyclonal γδ TCR diversity.

[0046] In other aspects, the invention provides selectively expanded γδ T cell population(s), wherein more than 80% or more than 90% of the expanded γδ T cells are δ2 T cells. In certain embodiments, the selectively expanded γδ T cell population(s) have not been purified by positive selection of γδ T cells from an enriched γδ T cell population (e.g., positive selection of δ2 T cells) or by removal of non-γδ T cells from an enriched γδ T cell population (e.g., removal of non-δ2 T cells, such as αβ T cells). In certain embodiments, the selectively expanded γδ T cell population(s) have been expanded directly from an isolated mixed cell population comprising T lymphocytes. In certain embodiments, the isolated mixed cell population from which the γδ T cell population(s) are expanded comprises about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, or about 20% T lymphocytes and less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1.5%, less than about 1.2%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, or less than about 0.1% δ2 cells. In certain embodiments, the isolated mixed cell population from which γδ T cells are expanded using the methods described herein comprises less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1.5%, less than about 1.2%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, or less than about 0.1% δ2 cells. In some embodiments, the isolated mixed cell population is selected from a peripheral blood sample, umbilical cord blood sample, or tumor. In one embodiment, the expanded mixed cell population(s) is derived from tumor-infiltrating lymphocytes, which may be isolated, for example, from colon adenocarcinoma metastasis, liver, ovarian, head and neck, or kidney cancer.

[0047] In another aspect, the invention provides selectively expanded γδ T cell population(s), wherein 10-90% of the expanded γδ T cells are δ1 T cells and 90-10% of the expanded γδ T cells are δ2 T cells.

[0048] In certain embodiments, the populations have been expanded together, e.g., by contacting the mixed cell population with an agent that selectively expands δ1 cells; δ1 T cells and δ3 T cells; δ1 T cells and δ4 T cells; or δ1 T cells, δ3 T cells, δ4 T cells and δ5 T cells, and an agent that selectively expands δ2 cells, either simultaneously or in separate contacting steps. In other embodiments, the population is a mixture of δ1 and δ2 T cell populations that have been selectively expanded separately from an isolated mixed cell population. In all embodiments, the expanded γδ T cell population(s) of the invention, or a mixture thereof, can be further formulated for administration to a subject.

[0049] In certain embodiments, the γδ T-cells or γδ T-cell population(s) of the invention are engineered to stably express one or more structurally distinct tumor recognition moieties encoded by an expression cassette. In one embodiment, the γδ T-cells are engineered to stably express two or more structurally distinct tumor recognition moieties encoded by an expression cassette. In some embodiments, the two or more structurally distinct tumor recognition moieties recognize different epitopes of the same antigen or different epitopes of different antigens. The tumor recognition moiety may be selected from the group consisting of an αβ TCR, a γδ TCR, a fragment of an αβ TCR or a γδ TCR, a chimeric antigen receptor (CAR), a whole antibody or antigen-binding fragment thereof, a single-chain variable fragment (scFv), a heavy or light chain single-domain antibody (sdAb), a Fab, a F(ab)2, or any combination thereof, that binds to (i) a cell surface tumor antigen or (ii) a peptide derived from a tumor antigen expressed on the cell surface in a complex with MHC (peptide-MHC complex). In a specific embodiment, the tumor recognition moiety is a γδ TCR of tumor-infiltrating lymphocytes that recognizes tumor-specific antigens in a non-MHC-restricted manner.

[0050] In another aspect, the present invention provides expanded γδ T cell population(s) obtained by the method of the present invention. In some embodiments, the γδ T cell population has been expanded in vitro without antigen stimulation with antigen-presenting cells or aminophosphonates. In certain embodiments, the γδ T cell population has been activated in vitro with a monoclonal antibody, antibody fragment, or fusion protein. The γδ T cell population(s) of the present invention may further be formulated for administration to a subject without co-administration of IL-2. Alternatively, the γδ T cell population(s) of the present invention may further be formulated for administration to a subject with co-administration of IL-2.

[0051] In another aspect, the present invention provides a method for treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an expanded γδ T-cell population according to the present invention. In one embodiment, the expanded γδ T-cell population is allogeneic with respect to the subject's MHC locus. In a specific embodiment, the expanded γδ T-cell population has been engineered to stably express one or more tumor recognition moieties encoded by one or more expression cassettes. In a further embodiment, the expanded γδ T-cell population stably expresses at least two structurally distinct recognition moieties, each of which recognizes (i) a different epitope of the same antigen, or (ii) a different epitope of a different antigen. The tumor recognition moiety may be selected from the group consisting of: αβTCR, γδTCR, chimeric antigen receptor (CAR) (including whole antibodies or antigen-binding fragments thereof, single-chain variable fragments (scFv), heavy or light chain single-domain antibodies (sdAbs), Fab, F(ab)2, or any combination thereof), which bind to (i) cell surface tumor antigens, or (ii) peptides derived from tumor antigens expressed on the cell surface in complexes with MHC (peptide-MHC complexes).

[0052] In another aspect, the invention provides engineered γδ T-cells, wherein the engineered γδ T-cells have been engineered to express a tumor recognition moiety that recognizes a tumor antigen, and / or wherein the engineered γδ T-cells are proliferated in vitro in the presence of (e.g., by contacting in vitro with) one or more agents that bind to the same epitope as the TS-1, TS8.2, B6, or 15D antibody. In another aspect, the invention provides engineered γδ T-cells, wherein the engineered γδ T-cells have been engineered to express a tumor recognition moiety that recognizes a tumor antigen, and / or wherein the engineered γδ T-cells are proliferated in vitro in the presence of (e.g., by contacting in vitro with) one or more agents that bind to a different or non-overlapping epitope than the epitope bound by the TS-1, TS8.2, B6, or 15D antibody. In some cases, the engineered γδ T cells are expanded in vitro in the presence of (e.g., in vitro contacted with) one or more agents that do not compete with binding of the TS-1, TS8.2, B6, or 15D antibody to the γδ T cells. In one embodiment, the agent is any one or more of the soluble or immobilized activators described herein. In one embodiment, the engineered γδ T cells are δ1, δ2, δ3, or δ4 engineered T cells.

[0053] In certain embodiments, the engineered γδ T cells are further engineered to lack gene expression from at least one HLA locus. In one embodiment, the one or more agents stimulate the proliferation of the engineered γδ T cells at an average rate of cell division less than 30 hours, e.g., between about 17 hours and about 30 hours. In some embodiments, the average rate of division is over 0-4, 0-5, 0-7 consecutive days of γδ T cell proliferation, 0-13 consecutive days of γδ T cell proliferation, 0-19 consecutive days of γδ T cell proliferation, 0-21 consecutive days of γδ T cell proliferation, or over at least 3, 4, 5, 6, 7, 10, 13, 19, or 21 consecutive days of γδ T cell proliferation. In other embodiments, the one or more agents stimulate the proliferation of the engineered γδ T cells at an average rate of cell division less than 24 hours, e.g., between about 17 hours and about 24 hours. In other embodiments, the one or more agents stimulate the proliferation of the engineered γδ T-cells at an average rate of cell division less than or about once every 18 hours. In certain embodiments, the tumor recognition moiety is derived from tumor-infiltrating lymphocytes.

[0054] In another aspect, the invention provides an expanded γδ T cell population, wherein the γδ T cell population comprises anti-tumor cytotoxicity. In some cases, the γδ T cell population comprises anti-tumor cytotoxicity independent of NKp30 activity, NKp44 activity, and / or NKp46 activity. In some cases, the γδ T cell population comprises anti-tumor cytotoxicity, wherein the anti-tumor cytotoxicity consists of or consists essentially of anti-tumor activity independent of NKp30 activity, NKp44 activity, and / or NKp46 activity. In some cases, the γδ T cell population comprises anti-tumor cytotoxicity, wherein at least 50%, 60%, 75%, 80%, 90%, or 99% of the anti-tumor cytotoxicity is independent of NKp30 activity, NKp44 activity, and / or NKp46 activity.

[0055] In some cases, the γδ T cell population is a population of engineered γδ T cells. In some cases, the γδ T cell population is a population of non-engineered γδ T cells. In some cases, the γδ T cell population does not comprise NKp30 activity-dependent anti-tumor cytotoxicity, NKp44 activity-dependent anti-tumor cytotoxicity, and / or NKp46 activity-dependent anti-tumor cytotoxicity. In some cases, the γδ T cell population does not comprise NKp30 activity-dependent anti-tumor cytotoxicity. In some cases, the γδ T cell population does not comprise NKp44 activity-dependent anti-tumor cytotoxicity. In some cases, the γδ T cell population is a population of engineered γδ T cells comprising an anti-CD20 chimeric antigen receptor (CAR). In some cases, less than 90%, less than 80%, less than 75%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the γδ T cells in the population express detectable levels of NKp30, NKp44, and / or NKp46.

[0056] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0057] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "figure" and "FIG."). [Brief explanation of the drawings]

[0058] [Figure 1] Schematic representation of engineered γδ T cells. Panel A shows an engineered γδ T cell expressing one tumor recognition moiety. Panel B shows an engineered γδ T cell expressing two structurally distinct tumor recognition moieties. [Figure 2] 1 illustrates a schematic diagram of a method for treating a subject. [Figure 3] Schematic of a method for administering a population of engineered γδ T cells to a subject. [Figure 4] 1 depicts a graph showing the growth of γδ1 and γδ2 lymphocytes isolated from a colon adenocarcinoma metastasis to the liver (TIL1) and a renal tumor (TIL2), which have been shown to express CCR4 and CCR7. [Figure 5] 1 depicts a graph showing γδ T cell growth in serum-containing and serum-free medium. [Figure 6] 1 depicts a graph showing an anti-γδ TCR antibody blocking experiment using 5A6.E9, B1, TS8.2, 15D, B3, B6, TS-1, γ3.20, IMMU510, or 11F2. [Figure 7] 1 depicts a graph showing an anti-γδ TCR antibody blocking experiment using 5A6.E9, B1, TS8.2, 15D, B3, B6, TS-1, γ3.20, IMMU510, or 11F2. [Figure 8] 1 depicts a competition experiment using anti-TCR V51 TS-1 antibody. [Figure 9] 1 depicts a competition experiment using anti-TCR V51 TS8.2 antibody. [Figure 10] 1 depicts a graph showing activation and proliferation of δ1 T cells from PBMCs. [Figure 11] 1 depicts a graph showing activation and proliferation of δ2 T cells from PBMCs. [Figure 12] 1 depicts a graph showing fold expansion of δ1 T cells from PBMCs. [Figure 13] 1 depicts a graph showing fold expansion of δ2 T cells from PBMCs. [Figure 14] The human V51 amino acid sequence is depicted, with CDR1, CDR3 and CDR3 regions underlined. [Figure 15] The human V52 amino acid sequence is depicted. CDR1, CDR3 and CDR3 regions are underlined. [Figure 16]This figure depicts the activation of PBMCs with TS-1 or TS8.2 antibodies, resulting in a significant and specific expansion of V51 T cells. (A) MAbs (TS1, TS8.2, B6, and 15D) were either directly coated (1 μg / mL) or captured (0.1 μg / mL) with goat anti-mouse Fc (5 μg / mL) (TS1Fc, TS8.2Fc, B6Fc, and 15Dfc) in 24-well plates. PBMCs were seeded at 10 cells / mL in RPMI containing 10% FBS and 100 IU / mL IL-2. On day 7, cells were transferred to new plates without antibody and further expanded until day 14. Culture medium was replenished every 2–3 days. Data depict the expansion of V51 cells over 14 days. (B) The same culture as in A, showing the percentage of V51 cells at day 14 and day 0 (d0). (C) Vδ1 cells were expanded from PBMCs isolated from different donors. MAbs (TS1, TS8.2, B6, and 15D) were either directly coated (1 μg / mL) or captured (0.1 μg / mL) with goat anti-mouse Fc (5 μg / mL) (TS1Fc, TS8.2Fc, B6Fc, and 15DFc) in 24-well plates. PBMCs were seeded at 106 cells / mL in RPMI containing 10% FBS and 100 IU / mL IL-2. On day 7, cells were transferred to a new plate without antibody and adjusted to 106 cells / mL with fresh medium. Culture medium was replenished every 2–3 days and adjusted to 106 cells / mL. Data depict Vδ1 cell expansion over 14 days. (D) PBMCs were seeded at 106 cells / mL in RPMI containing 10% FBS and 100 IU / mL IL-2. On day 7, cells were transferred to a new plate without antibody, adjusted to 106 cells / mL, and further expanded until day 23. Culture medium was replenished every 2–3 days. [Figure 17-1]This figure depicts activation of PBMCs with B6 and 15D antibodies, resulting in significant and specific activation and expansion of V52 T cells. (A) In 24-well plates, MAbs (TS1, TS8.2, B6, and 15D) were either directly coated (1 μg / mL) or captured (0.1 μg / mL) with goat anti-mouse Fc (5 μg / mL) (TS1Fc, TS8.2Fc, B6Fc, and 15DFc). PBMCs were seeded at 10 cells / mL in RPMI containing 10% FBS and 100 IU / mL IL-2. On day 7, cells were transferred to new plates without antibody and further expanded until day 14. Culture medium was replenished every 2–3 days. Data depict V52 T cell expansion over 14 days. (B) The same culture as in A, showing the percentage of V52 cells on days 14 and 0 (d0). (C) MAbs (15D and pan-γδ TCR MAb Immu510) were directly coated (1 μg / mL) in 24-well plates. Isolated PBMCs from different donors were seeded at 10 cells / mL in RPMI containing 10% FBS and 100 IU / mL IL-2. On day 7, cells were transferred to new plates without antibody. Cultures were replenished every 2–3 days and adjusted to 10 cells / mL with fresh medium. Data depict Vδ2 T cell expansion over 14 days. (D) Same culture as in C, showing the percentage of Vδ2 T cells on day 14 and day 0 (d0). (E) MAbs (TS1, TS8.2, B6, and 15D) were either directly coated (1 μg / mL) or captured (0.1 μg / mL) with goat anti-mouse Fc (5 μg / mL) (TS1Fc, TS8.2Fc, B6Fc, and 15DFc) in 24-well plates. PBMCs from another donor were seeded at 10 cells / mL in RPMI containing 10% FBS and 100 IU / mL IL-2. On day 7, cells were transferred to a new plate without antibody and adjusted to 10 cells / mL with fresh medium. Culture medium was replenished every 2–3 days and adjusted to 10 cells / mL. Data depict Vδ2 T cell expansion over 14 days. [Figure 17-2]This figure depicts activation of PBMCs with B6 and 15D antibodies, resulting in significant and specific activation and expansion of V52 T cells. (A) In 24-well plates, MAbs (TS1, TS8.2, B6, and 15D) were either directly coated (1 μg / mL) or captured (0.1 μg / mL) with goat anti-mouse Fc (5 μg / mL) (TS1Fc, TS8.2Fc, B6Fc, and 15DFc). PBMCs were seeded at 10 cells / mL in RPMI containing 10% FBS and 100 IU / mL IL-2. On day 7, cells were transferred to new plates without antibody and further expanded until day 14. Culture medium was replenished every 2–3 days. Data depict V52 T cell expansion over 14 days. (B) The same culture as in A, showing the percentage of V52 cells on days 14 and 0 (d0). (C) MAbs (15D and pan-γδ TCR MAb Immu510) were directly coated (1 μg / mL) in 24-well plates. Isolated PBMCs from different donors were seeded at 10 cells / mL in RPMI containing 10% FBS and 100 IU / mL IL-2. On day 7, cells were transferred to new plates without antibody. Cultures were replenished every 2–3 days and adjusted to 10 cells / mL with fresh medium. Data depict Vδ2 T cell expansion over 14 days. (D) Same culture as in C, showing the percentage of Vδ2 T cells on day 14 and day 0 (d0). (E) MAbs (TS1, TS8.2, B6, and 15D) were either directly coated (1 μg / mL) or captured (0.1 μg / mL) with goat anti-mouse Fc (5 μg / mL) (TS1Fc, TS8.2Fc, B6Fc, and 15DFc) in 24-well plates. PBMCs from another donor were seeded at 10 cells / mL in RPMI containing 10% FBS and 100 IU / mL IL-2. On day 7, cells were transferred to a new plate without antibody and adjusted to 10 cells / mL with fresh medium. Culture medium was replenished every 2–3 days and adjusted to 10 cells / mL. Data depict Vδ2 T cell expansion over 14 days. [Figure 18]This figure depicts a significant decrease in the percentage of αβ T cells in PBMCs cultured with the γδ-specific antibodies TS1, TS8.2, B6, and 15D. (A) MAbs (TS1, TS8.2, B6, and 15D) were either directly coated (1 μg / mL) or captured (0.1 μg / mL) with goat anti-mouse Fc (5 μg / mL) (TS1Fc, TS8.2Fc, B6Fc, and 15DFc) in 24-well plates. PBMCs were seeded at 10 cells / mL in RPMI containing 10% FBS and 100 IU / mL IL-2. On day 7, cells were transferred to new plates without antibody and further expanded until day 14. Culture medium was replenished every 2–3 days. Data depict αβ T cell expansion over 14 days. (B) The same culture as in A, but showing the percentage of αβ T cells at day 14 and day 0 (d0). [Figure 19] This figure depicts the shortening of γδ TCR doubling time. MAbs (TS1, TS8.2, B6, and 15D) were either directly coated (1 μg / mL) or captured (0.1 μg / mL) by goat anti-mouse Fc (TS1Fc, TS8.2Fc, B6Fc, and 15DFc) in 24-well plates. PBMCs were seeded at 10 cells / mL in RPMI containing 10% FBS and 100 IU / mL IL-2. On day 7, cells were transferred to a new plate without antibody and adjusted to 10 cells / mL with fresh medium. Cultures were replenished every 2–3 days and adjusted to 10 cells / mL with fresh medium. The doubling times of Vδ1, Vδ2, and αβ T cells are shown in (A), (B), and (C), respectively. [Figure 20] We demonstrate that Vδ1 activation by TS8.2 and TS1 primarily results in naive and central memory phenotypes. Antibodies (TS8.2, R9.12, and pan-γδImmu510) were directly coated (1 μg / mL) in 24-well plates. PBMCs were seeded at 106 cells / mL in RPMI containing 10% FBS and 100 IU / mL IL-2. The medium was replenished every 2–3 days until day 23, except on day 15, when it was diluted 1:2 with fresh medium. Cell phenotypes on days 14 and 23 were determined by CD45RA and CD27 expression using flow cytometry analysis. [Figure 21] This figure depicts that activation of PBMCs with TS8.2 and MICA enhances the proliferation of Vδ1 T cells but not αβ T cells. Antibody TS8.2 (1 μg / mL) or TS8.2 and MICA-Fc (1 and 5 μg / mL) were directly coated in 24-well plates. PBMCs were seeded at 106 cells / mL in RPMI containing 10% FBS and 100 IU / mL IL-2. The medium was replenished every 2–3 days. (A) Vδ1 T cell proliferation, and (B) αβ T cell proliferation. [Figure 22] Figure 1 depicts that activation of cord blood mononuclear cells with δ1- and δ2-specific antibodies results in significant and specific expansion of V51+ and V52+ T cells. MAbs TS8.2, R9.12, B6, and 15D were directly coated at 1 μg / mL in 24-well plates. Cord blood mononuclear cells were activated at 106 cells / mL in RPMI containing 10% FBS and 100 IU / mL IL-2. V51 and V52 T cell expansion are shown in (A) and (B), respectively. [Figure 23] Figure 1 depicts the binding of soluble TCRs generated by pairing of the V51J1, V51J2, and V51J3 chains with the γ8 chain. The data show that TS1 and TS8.2 recognize soluble TCRs generated from the V51J1 and V51J2 chains but fail to bind to the V51J3 chain, suggesting that the J1 and J2 gene segments are essential for TS1 and TS8.2 binding. R9.12, which binds to V51, and the pan-antibody Immuno510, which binds to the delta constant region, are not affected by specific J regions. [Figure 24]Figure 1 depicts a sequence alignment of the J1, J2, and J3 regions of human V51 and mutations made at selected positions in the V51J1 chain. BE-13 refers to the 51J region from a T-cell leukemia cell line (DSMZ accession number ACC396) expressing the 51γ8 TCR. Replacement of the single amino acid at position Lys120 in the V51J1 and V51J2 regions with Thr or Ala completely abolished binding of TS-1 and TS8.2 MAbs, suggesting that this amino acid in the V51J1 and V51J2 regions contributes to TS-1 and TS8.2 binding. Replacement of the single amino acid at position Thr120 in the V51J3 region with Lys resulted in the acquisition of binding of TS-1 and TS8.2 MAbs, further suggesting that this amino acid in the V51J1 and V51J2 regions contributes to TS-1 and TS8.2 binding. [Figure 25] 1 depicts that point mutation of Lys120 to Thr or Ala in Vδ1J1 leads to loss of binding by TS-1 and TS8.2 MAbs. [Figure 26] Figure 1 depicts the human V51 protein sequence and six mutant human V51 sequences based on differences between the human and bovine V51 amino acid sequences (GenBank: AFP25162.1). Mutations are shown in bold. [Figure 27] 1 depicts the loss of binding of TS-1 and TS8.2 to mutant human Vδ1 chains. [Figure 28] Binding of B6 MAb to various Vδ2 / γ chain pairings (γ3, γ8, γ9) is depicted. [Figure 29] Protein sequence alignment of the human V52 variable region (IMGT human TRDV2) with the rhesus V52 variable region (GenBank: AY190028.1) and depicting the changes made in CDR1 (G35S), CDR2 (D65G) and CDR3 (C104S) of V52. [Figure 30] 1 depicts the loss of binding of 15D to Vδ2 mutated in CDR1 (G35S). [Figure 31]Figure 1 depicts the characterization of highly enriched V51+, V52+, and αβ T cell cultures using TS8.2, B6, and IP26 for δ1, δ2, and αβ, respectively. PBMC-derived populations were expanded using a combination of TS-1 and TS8.2 to expand δ1 cells, or 15D and B6 to expand δ2 cells. αβ T cells were depleted from expanded cultures using IP26 microbeads. Positively selected αβ T cells were also harvested (enriched for αβ). [Figure 32] Depicts the cytotoxicity of V51+ and V52+ populations against solid tumor (BxPC3, SKMEL5) and plasmacytoma (RPMI8226) cell lines. [Figure 33-1] 1 depicts the amino acid sequences of the framework and complementarity determining regions of the heavy chain of a δ1-specific MAb. [Figure 33-2] 1 depicts the amino acid sequences of the framework and complementarity determining regions of the heavy chain of a δ1-specific MAb. [Figure 34-1] 34 depicts the amino acid sequences of the framework and complementarity determining regions of the light chains of the δ1-specific MAbs described in FIG. 33. [Figure 34-2] 34 depicts the amino acid sequences of the framework and complementarity determining regions of the light chains of the δ1-specific MAbs described in FIG. 33. [Figure 35-1] 1 depicts the amino acid sequences of the framework and complementarity determining regions of the heavy chain of a δ2-specific MAb. [Figure 35-2] 1 depicts the amino acid sequences of the framework and complementarity determining regions of the heavy chain of a δ2-specific MAb. [Figure 36-1] 1 depicts the amino acid sequences of the framework and complementarity determining regions of the light chain of a δ2-specific MAb. [Figure 36-2] 1 depicts the amino acid sequences of the framework and complementarity determining regions of the light chain of a δ2-specific MAb. [Figure 37] Figure 1 depicts the cross-reactivity of the indicated δ1-specific antibodies with other γδ TCRs as determined by ELISA. [Figure 38] 1 depicts the alignment of human and dolphin Vδ1 deduced amino acid sequences. [Figure 39] Depicts the percentage of proliferating V51 cells (A) or V52 cells (B) obtained by the γδ T cell expansion method described herein. [Figure 40] Depicts the fold expansion of V51 cells (A) or V52 cells (B) from isolated mixed cell populations obtained by the γδ T cell expansion method described herein. [Figure 41]

[0023] Figure 1 depicts phenotypic data from isolated mixed cell populations obtained by the γδ T cell expansion method described herein. Cell phenotype was determined by CD45RA and CD27 expression using flow cytometry analysis. [Figure 42] We demonstrate that δ1 T cells activated and expanded by the γδ T cell expansion method described herein can exhibit robust cytotoxicity against tumor cell lines. [Figure 43] Figure 1 illustrates the V51 fold expansion and purity at day 19 achieved by the methods of the invention. [Figure 44] 1 illustrates the fold expansion and cumulative doubling time of Vδ1 at day 19 achieved by the methods of the invention. [Figure 45] 1 illustrates an example of Vδ1 expansion fold and cumulative doubling time from day 14 to day 19 achieved by the methods of the present invention. [Figure 46] Figure 1 illustrates the expression of anti-CD20 chimeric antigen receptor (CAR) on V51 T cells. Seven days after transduction with the CAR retroviral construct, expanded cells were stained with a V51-specific antibody (R9.12) and anti-rituximab FITC conjugate. Up to 35% of V51 cells were CD20CAR+. [Figure 47-1] AB depict epitope binding specificity data for δ-1-specific γδ T cell activators. [Figure 47-2] AB depict epitope binding specificity data for δ-1-specific γδ T cell activators. [Figure 47-3] AB depict epitope binding specificity data for δ-1-specific γδ T cell activators. [Figure 48] 1 depicts epitope binding specificity data for delta-2 specific gamma delta T cell activators. DETAILED DESCRIPTION OF THE INVENTION

[0059] Detailed Description of the Invention While various embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the present invention may be employed.

[0060] definition Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention described herein belongs. For purposes of interpreting this specification, the following definitions shall apply, and where appropriate, terms used in the singular shall include the plural and vice versa. In the event that any explicit definition conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0061] As used herein, the term "γδ T cells (gamma delta T cells)" refers to a subset of T cells that express a unique T cell receptor (TCR) on their surface, the γδ TCR, which is composed of one γ chain and one δ chain. The term "γδ T cells" specifically includes all subsets of γδ T cells, including, but not limited to, Vδ1, Vδ2, and Vδ3 γδ T cells, as well as naive, effector memory, central memory, and terminally differentiated γδ T cells. By way of further example, the term "γδ T cells" includes Vδ4, Vδ5, Vδ7, and Vδ8 γδ T cells, as well as Vγ2, Vγ3, Vγ5, Vγ8, Vγ9, Vγ10, and Vγ11 γδ T cells.

[0062] As used herein, the term "T lymphocyte" or "T cell" refers to an immune cell that expresses CD3 (CD3+) and the T cell receptor (TCR+). T cells play a central role in cell-mediated immunity.

[0063] As used herein, the term "TCR" or "T cell receptor" refers to heterodimeric cell surface signaling proteins forming alpha-beta or gamma-delta receptors. αβTCRs recognize antigens presented by MHC molecules, whereas γδTCRs recognize antigens independent of MHC presentation.

[0064] The term "MHC" (major histocompatibility complex) refers to a subset of genes that encode cell surface antigen-presenting proteins. In humans, these genes are called human leukocyte antigen (HLA) genes. The abbreviations MHC or HLA are used interchangeably herein.

[0065] As used herein, the term "peripheral blood lymphocyte(s)" or "PBL(s)" is used in the broadest sense to refer to white blood cell(s) including T and B cells, plasma cells, monocytes, macrophages, natural killer cells, basophils, eosinophils, etc. at a wide range of differentiation and functional stages. T lymphocytes in peripheral blood range from approximately 20-80%.

[0066] As used herein, the term "cell population" refers to a number of cells obtained by direct isolation from a suitable source (usually from a mammal). The isolated cell population can then be cultured in vitro. Those skilled in the art will be familiar with the various methods of isolating and culturing cell populations for use with the present invention, as well as the various numbers of cells in a cell population suitable for use in the present invention. The cell population can be, for example, a mixed heterogeneous cell population derived from a peripheral blood sample, umbilical cord blood sample, tumor, stem cell precursor, tumor biopsy, tissue, lymph, or from an epithelial site of a subject in direct contact with the external environment, or derived from stem progenitor cells. Alternatively, the mixed cell population can be derived from an in vitro culture of mammalian cells established from a peripheral blood sample, umbilical cord blood sample, tumor, stem cell precursor, tumor biopsy, tissue, lymph, or from an epithelial site of a subject in direct contact with the external environment, or from stem progenitor cells.

[0067] An "enriched" cell population or preparation refers to a cell population derived from a starting mixed cell population, containing a higher percentage of a particular cell type than the percentage of that cell type in the starting population. For example, a starting mixed cell population can be enriched for a particular γδ T cell population. In one embodiment, the enriched γδ T cell population contains a higher percentage of δ1 cells than the percentage of that cell type in the starting population. As another example, an enriched γδ T cell population can contain a higher percentage of both δ1 cells and δ3 cells than the percentage of that cell type in the starting population. As yet another example, an enriched γδ T cell population can contain a higher percentage of both δ1 cells and δ4 cells than the percentage of that cell type in the starting population. As yet another example, an enriched γδ T cell population can contain a higher percentage of δ1 T cells, δ3 T cells, δ4 T cells, and δ5 T cells than the percentage of that cell type in the starting population. In another embodiment, the enriched γδ T cell population contains a higher percentage of δ2 cells than the percentage of that cell type in the starting population. In yet another embodiment, the enriched γδ T cell population contains a higher percentage of both δ1 cells and δ2 cells than the percentage of that cell type in the starting population. In all embodiments, the enriched γδ T cell population contains a lower percentage of αβ T cell population.

[0068] As used herein, "expanded" means that the number of desired or target cell types (e.g., δ1 and / or δ2 T cells) in the enriched preparation is greater than the number in the initial or starting cell population. "Selectively expanding" means preferentially expanding target cell types (e.g., δ1 and / or δ2 T cells) over other non-target cell types, such as αβ T cells or NK cells. In certain embodiments, an activating agent of the invention selectively expands δ1 T cells, e.g., engineered or non-engineered, without significant expansion of δ2 T cells. In other embodiments, an activating agent of the invention selectively expands δ2 T cells, e.g., engineered or non-engineered, without significant expansion of δ1 T cells. In certain embodiments, an activating agent of the invention selectively expands δ1 and δ3 T cells, e.g., engineered or non-engineered, without significant expansion of δ2 T cells. In certain embodiments, an activating agent of the invention selectively expands δ1 and δ4 T cells, e.g., engineered or non-engineered, without significant expansion of δ2 T cells. In a particular embodiment, the activating agent of the invention selectively expands, e.g., engineered or non-engineered, δ1, δ3, δ4 and δ5 T cells without significant expansion of δ2 T cells. In this context, the term "without significant expansion" means that the preferentially expanded cell population is expanded at least 10-fold, preferably 100-fold, more preferably 1,000-fold more than the reference cell population.

[0069] As used herein, the term "mixture" refers to a combination of two or more isolated enriched cell populations derived from a mixed heterogeneous cell population. According to certain embodiments, the cell population of the present invention is an isolated γδ T cell population.

[0070] The term "isolated" as applied to a cell population refers to a cell population isolated from the human or animal body that is substantially free of one or more cell populations that are associated with said cell population in vivo or in vitro.

[0071] The term "contacting," as used herein in the context of a cell population, refers to incubating an isolated cell population with a reagent, e.g., an antibody, cytokine, ligand, mitogen, or costimulatory molecule, which can be tethered to either the beads or the cells. The antibody or cytokine may be in soluble form or may be immobilized. In one embodiment, the immobilized antibody or cytokine is tightly or covalently bound to the bead or plate. In one embodiment, the antibody is immobilized to an Fc-coated well. In desirable embodiments, the contacting occurs in vitro (e.g., in a test tube) or in vivo.

[0072] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts" or "directed against" means that the antibody reacts with one or more antigenic determinants of the desired antigen but does not react with, or with a much lower affinity (K) with, other polypeptides. D >10 -6 The antibody may be, but is not limited to, a polyclonal, monoclonal, chimeric, sdAb (single domain heavy or light chain antibody), single chain F ab , F ab’ and F (ab’)2 fragments, scFv, diabodies, minibodies, nanobodies, and F ab Expression libraries are included.

[0073] As used herein, the term "chimeric antigen receptor (CAR)" may refer to an artificial T cell receptor, T body, single-chain immunoreceptor, chimeric T cell receptor, or chimeric immunoreceptor, and may encompass, for example, engineered receptors that fuse artificial specificity to specific immune effector cells. CARs can be employed to confer the specificity of a monoclonal antibody to T cells, thereby generating large numbers of specific T cells, for example, for adoptive cell therapy. In specific embodiments, CARs direct the specificity of cells to, for example, a tumor-associated antigen. In some embodiments, a CAR comprises an intracellular activation domain (which activates the T cell upon engagement of the targeting moiety with a target cell, such as a target tumor cell), a transmembrane domain, and an extracellular domain, which may be of various lengths and includes a disease- or disorder-associated antigen-binding region, e.g., a tumor antigen-binding region. In a particular aspect, a CAR comprises a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a CD3-zeta transmembrane domain and an intracellular domain. Other CAR design specificity can be derived from receptor (e.g., peptide) ligands or pattern recognition receptors such as dectin. In certain cases, the spacing of antigen recognition domains can be adjusted to reduce activation-induced cell death. In certain cases, CARs include domains for additional costimulatory signaling, such as CD3-zeta, FcR, CD27, CD28, CD137, DAP10 / 12, and / or OX40, ICOS, TLR, etc. In some cases, molecules including costimulatory molecules, reporter genes for imaging (e.g., positron emission tomography), gene products that conditionally eliminate T cells by the addition of prodrugs, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors can be co-expressed with the CAR.

[0074] The basic antibody structural unit is known to comprise a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Antibody molecules obtained from humans generally belong to one of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other in the nature of the heavy chains present in the molecule. Specific classes also have subclasses, such as IgG1 and IgG2. Furthermore, in humans, light chains can be kappa or lambda chains.

[0075] The term "Fab" refers to the entire light chain (V L and C L ) plus the variable region domain of the H chain (V H Fab refers to an antibody fragment consisting of a single constant domain (C1) of one heavy chain and the first constant domain (CH1) of one heavy chain. Papain digestion of an intact antibody can be used to generate two Fab fragments, each of which contains one antigen-binding site. Typically, the light and heavy chain fragments of the Fab produced by papain digestion are held together by an interchain disulfide bond.

[0076] The term "Fc" refers to an antibody fragment that contains the carboxy-terminal portions (CH2 and CH3) of both H chains and part of the hinge region held together by sulfide bonds. The effector functions of an antibody are determined by the sequence of the Fc region, and this region is also recognized by Fc receptors (FcRs) found on certain types of cells. An Fc fragment can be obtained by papain digestion of a whole antibody.

[0077] The term "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole antibody. The F(ab')2 fragment contains two Fab fragments and part of the hinge region held together by disulfide bonds. The F(ab')2 fragment has bivalent antigen-binding activity and is capable of cross-linking antigen.

[0078] The term Fab' refers to an antibody fragment that is the product of reduction of an F(ab')2 fragment. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group.

[0079] The term "Fv" refers to an antibody fragment consisting of a dimer of one heavy-chain and one light-chain variable domain in tight, non-covalent association. The folding of these two domains generates six hypervariable loops (three loops from each of the H and L chains) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to antigen, although usually with lower affinity than the entire binding site.

[0080] The term "single-chain Fv," also abbreviated as "sFv" or "scFv," refers to an antibody fragment comprising the VH and VL antibody domains linked in a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For reviews of scFvs, see, e.g., Plückthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994), and Malmborg et al., J. Immunol. Methods 183:7-13, 1995.

[0081] The term "linear antibody" refers to a pair of tandem V-chains that form a pair of antigen-binding regions. H -C H 1 segment (V H -C H 1-V H -C H The term "linear antibody" is used to refer to a polypeptide comprising a polypeptide of the present invention. Linear antibodies can be bispecific or monospecific and are described, for example, in Zapata et al., Protein Eng. 8(10):1057-1062 (1995).

[0082] The term "variable" refers to the fact that certain portions of the variable domains have extensive sequence differences among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Each naturally occurring heavy and light chain variable domain contains four FRs that generally adopt a beta-sheet configuration, connected by three hypervariable regions that form loops that connect, and in some cases form part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0083] The term "antigen-binding site" or "binding portion" refers to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues from the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent regions within the V regions of heavy and light chains are called "hypervariable regions," and are flanked by more conserved regions known as "framework regions" or "FRs." Thus, the term "FR" refers to the amino acid sequences naturally found adjacent to and between the hypervariable regions of immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged in three-dimensional space relative to each other to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each heavy and light chain are called "complementarity-determining regions" or "CDRs." The amino acid assignments for each domain are based on the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or the definitions of Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989).

[0084] The term "hypervariable region," "HVR," or "HV" refers to the region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops. Antibodies generally contain six HVRs: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). Among the six HVRs, H3 and L3 exhibit the greatest diversity in natural antibodies, and H3 in particular is thought to play a unique role in conferring subtle specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies composed only of heavy chains are functional and stable in the absence of light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0085] "Framework regions" (FRs) are variable domain residues other than the CDR residues. Each variable domain typically has four FRs identified as FR1, FR2, FR3, and FR4. When CDRs are defined according to Kabat, light chain FR residues are located at about residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4); heavy chain FR residues are located at about residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4). When a CDR comprises amino acid residues from a hypervariable loop, the light chain FR residues are located at about residues 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) of the light chain, and the heavy chain FR residues are located at about residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) of the heavy chain. In some cases, when a CDR comprises amino acids from both a CDR and a hypervariable loop according to the Kabat definition, the FR residues will be adjusted accordingly. For example, if CDRH1 comprises amino acids H26-H35, the heavy chain FR1 residues are located at positions 1-25 and the FR2 residues are located at positions 36-49.

[0086] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues among alternative human immunoglobulin VL or VH framework sequences. Typically, the alternative human immunoglobulin VL or VH sequences are from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a Kabat subgroup. In particular cases, for VL, the subgroup is Kabat subgroup kappa I. In particular cases, for VH, the subgroup is Kabat subgroup III.

[0087] As used herein, "Kd" or "Kd value" refers to the dissociation constant measured at 25°C using a surface plasmon resonance assay, e.g., a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ) on a CM5 chip with approximately 10 response units (RU) of antigen or antibody immobilized thereon. In the case of bivalent or other multivalent antibodies, the antibody is typically immobilized to avoid avidity-induced interference with the dissociation constant measurement. For further details, see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).

[0088] The term "epitope" includes any protein, lipid, or carbohydrate determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants often consist of active surface groupings of molecules such as amino acids, lipid, or sugar side chains and often have specific three-dimensional structural characteristics, as well as specific charge characteristics. Antibodies are characterized by an equilibrium dissociation constant (K D ) is 10 -6 ~10 -12 An "activating epitope" is capable of activating a specific γδ T cell population upon binding.

[0089] An antibody binds to "essentially the same epitope" as a reference antibody if the two antibodies recognize the same or sterically overlapping epitopes. The most widely used rapid method for determining whether two epitopes bind to the same or sterically overlapping epitopes is a competition assay, which can be configured in a variety of ways, using either labeled antigen or labeled antibody. In some embodiments, the antigen is immobilized on a 96-well plate, and the ability of an unlabeled antibody to block the binding of the labeled antibody is measured using a radioactive or enzyme label. Alternatively, competition tests using labeled and unlabeled antibodies are performed using flow cytometry on antigen-expressing cells.

[0090] "Epitope localization" is the process of identifying the binding site, or epitope, of an antibody on its target antigen. Antibody epitopes can be linear or conformational epitopes. Linear epitopes are formed by a contiguous sequence of amino acids in a protein. Conformational epitopes are formed from amino acids that are discontinuous in the protein sequence but brought together by the folding of the protein into its three-dimensional structure.

[0091] As defined herein, "epitope binning" is the process of classifying antibodies based on the epitopes they recognize. More specifically, epitope binning includes methods and systems for distinguishing the epitope recognition characteristics of different antibodies combined with computational processes for clustering antibodies based on their epitope recognition characteristics and for identifying antibodies with distinct binding specificities.

[0092] An "agent" or "compound" according to the present invention includes a small molecule, a polypeptide, a protein, an antibody or an antibody fragment. Small molecule, in the context of the present invention, in one embodiment means a chemical substance with a molecular weight of less than 1000 daltons, particularly less than 800 daltons, more particularly less than 500 daltons. The term "therapeutic agent" refers to an agent with biological activity. The term "anti-cancer agent" refers to an agent with biological activity against cancer cells.

[0093] As used herein, the term "cell culture" refers to any in vitro culture of cells. This term includes continuous cell lines (e.g., with an immortalized phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and other cell populations maintained in vitro, such as stem cells, blood cells, embryonic umbilical cord blood cells, tumor cells, transduced cells, etc.

[0094] The term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures aimed at preventing or slowing (reducing) an undesirable physiological change or disorder. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, attenuation of disease extent (e.g., reduction in tumor size, tumor burden, or tumor distribution), stabilized (i.e., not worsening) disease state, delay or slowing of disease progression, improvement or palliation of disease state, and remission (whether partial or complete). "Treatment" can also mean prolonging survival beyond that expected in the absence of treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in whom the condition or disorder is to be prevented.

[0095] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0096] The term "identical," as used herein, refers to two or more sequences or subsequences that are the same. Additionally, the term "substantially identical," as used herein, refers to two or more sequences that have the same percentage of sequence units when compared and aligned for maximum correspondence over a specified region, as determined using a comparison window or algorithm or by manual alignment and visual inspection. By way of example only, two or more sequences may be "substantially identical" if the sequence units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. Such percentages express the "percent identity" of two or more sequences. Sequence identity can exist over a region that is at least about 75-100 sequence units in length, a region that is at least about 50 sequence units in length, or, if not specified, over the entire sequence. This definition also refers to the complement of a test sequence. Further, by way of example only, two or more polynucleotide sequences are identical if the nucleic acid residues are the same, but two or more polynucleotide sequences are "substantially identical" if the nucleic acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. Identity can exist over a region that is at least about 75 to about 100 nucleic acids in length, over a region that is about 50 nucleic acids in length, or, if not specified, over the entire sequence of the polynucleotide sequences.

[0097] The term "pharmaceutically acceptable," as used herein, refers to a material, such as, but not limited to, a salt, carrier, or diluent, that does not abrogate the biological activity or properties of a compound and that is relatively non-toxic, i.e., that may be administered to an individual without causing undesired biological effects or adversely interacting with any of the components contained in the composition.

[0098] The term "subject" or "patient," as used herein, refers to a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, humans, non-human primates, livestock animals (e.g., cows), sport animals, and pets (e.g., cats, dogs, and horses). In certain embodiments, the mammal is a human.

[0099] The term "therapeutically effective amount," as used herein, refers to an amount of a composition and / or mixture containing an expanded cell population of the present invention administered to a subject, e.g., a human patient, already suffering from a disease, condition, or disorder sufficient to cure, at least partially arrest, or alleviate to some extent, one or more symptoms of the disease, disorder, or condition being treated. The effectiveness of such a composition will depend on the condition, including, but not limited to, the severity and course of the disease, disorder, or condition, previous therapy, the patient's health status and response to drugs, and the judgment of the treating physician. By way of example only, a therapeutically effective amount may be determined by routine experimentation, including, but not limited to, a dose escalation trial.

[0100] The term antigen-presenting cells (APCs) refers to wild-type APCs or engineered or artificial antigen-presenting cells (aAPCs). APCs can be provided as irradiated populations of APCs. APCs can be provided from immortalized cell lines (e.g., K562 or engineered aAPCs derived from immortalized cell lines), or as a fraction of cells from a donor (e.g., PBMCs).

[0101] As used herein, the terms "structurally distinct" and "structurally distinct," with respect to a protein or polypeptide fragment thereof, or epitope, refer to a covalent (i.e., structural) difference between at least two different proteins, polypeptide fragments thereof, or epitopes. For example, two structurally distinct proteins (e.g., antibodies) can refer to two proteins that have different primary amino acid sequences. In some cases, structurally distinct activators bind to structurally distinct epitopes, e.g., epitopes that have different primary amino acid sequences.

[0102] As used herein, the term "anti-tumor cytotoxicity" "independent" of a particular receptor activity (e.g., NKp30 activity, NKp44 activity, and / or NKp46 activity) refers to anti-tumor cytotoxicity that is exerted regardless of whether a particular receptor or a particular combination of receptors is expressed or functional in a cell. Thus, γδ T cells that exhibit anti-tumor cytotoxicity independent of NKp30 activity, NKp44 activity, and / or NKp46 activity may also exhibit NKp30 activity-dependent anti-tumor cytotoxicity, NKp44 activity-dependent anti-tumor cytotoxicity, and / or NKp46 activity-dependent anti-tumor cytotoxicity.

[0103] As used herein, the terms "NKp30 activity-dependent antitumor cytotoxicity," "NKp44 activity-dependent antitumor cytotoxicity," and "NKp46 activity-dependent antitumor cytotoxicity" refer to antitumor cytotoxicity that requires the functional expression of specific receptors. The presence or absence of such receptor-dependent antitumor cytotoxicity can be determined by performing a standard in vitro cytotoxicity assay, such as that performed in Example 48, in the presence or absence of an antagonist for the specific receptor. For example, the presence or absence of NKp30 activity-dependent antitumor cytotoxicity can be determined by comparing the results of an in vitro cytotoxicity assay, such as that performed in Example 48, in the presence of an anti-NKp30 antagonist with the results obtained in the absence of the anti-NKp30 antagonist.

[0104] As used herein, a γδ T cell population containing anti-tumor cytotoxicity, in which at least a specified "%" of the anti-tumor cytotoxicity is "independent" of a specific receptor activity (e.g., NKp30 activity, NKp44 activity, and / or NKp46 activity), refers to cells in which blockade of a specific receptor does not reduce measured anti-tumor cytotoxicity by more than that percentage. Thus, a γδ T cell population containing anti-tumor cytotoxicity, in which at least 50% of the anti-tumor cytotoxicity is independent of NKp30 activity, will exhibit no more than a 50% reduction in anti-tumor cytotoxicity in vitro in the presence of an NKp30 antagonist compared to the absence of the NKp30 antagonist.

[0105] Overview In humans, γδ T cells (γδ T cells) are a subset of T cells that provide a link between innate and adaptive immune responses. These cells undergo V-(D)-J segment rearrangement to generate antigen-specific γδ T cell receptors (γδ TCRs). γδ T cells can be directly activated by antigen recognition either by the γδ TCR or by other non-TCR proteins that act independently or in concert to activate γδ T cell effector function. γδ T cells represent a small fraction of the total T cell population in mammals, accounting for approximately 1–5% of T cells in peripheral blood and lymphoid organs. They appear to reside primarily in epithelial cell-rich compartments such as the skin, liver, gastrointestinal tract, respiratory tract, and reproductive tract. Unlike αβ TCRs, which recognize antigens bound to major histocompatibility complex molecules (MHC), γδ TCRs can directly recognize bacterial antigens, viral antigens, stress antigens expressed on diseased cells, and tumor antigens in the form of whole proteins or non-peptide compounds.

[0106] TS-1, TS8.2, B6, and 15D can activate γδ T cells. Without being bound by theory, the different levels of activation and proliferation of cultures arising from different donors may be due to the donor's γδ variable TCR repertoire and antibody binding epitopes. It is believed that all agents that bind to specific γδ T cell subsets can activate specific γδ T cells, particularly those that can increase specific γδ T cell populations in enriched cultures to clinically relevant levels, i.e., 10 8 It was found that not all binding epitopes of a γδ T cell population are activating epitopes, i.e., capable of activating a specific γδ T cell population upon binding.

[0107] The present inventors have identified specific γδ variable TCR binding regions associated with potent activation of specific γδ T cell subtypes, thus enabling the specific activation of γδ T cell subtypes to generate clinically relevant levels of highly enriched γδ T cell populations and mixtures thereof with improved purity that can be administered to patients. Novel activating ligands, including antibodies, that specifically bind to activation epitopes capable of inducing enhanced activation and proliferation of γδ T cell subtypes are also contemplated and are further described herein.

[0108] In some cases, clinically relevant levels (i.e., 10 8Production of γδ T cells (more than 100 cells / ml) can be achieved using relatively small amounts of culture medium. For example, in some embodiments, clinically relevant levels of γδ T cells can be obtained by expansion of a cell population (e.g., an isolated mixed cell population) in a harvest culture volume of approximately 25 L, 20 L, 10 L, 5 L, 3 L, 2 L, 1,5000 mL, 1,000 mL, 500 mL, 200 mL, 150 mL, 100 mL, or less (e.g., about 10 mL to about 100 mL, about 100 mL to about 500 mL, about 500 mL to about 5,000 mL, or about 5 L to about 25 L). As another example, in some embodiments, clinically relevant levels of γδ T cells can be obtained under conditions such that the total volume of culture medium used to expand an isolated mixed cell population obtained from a single donor or multiple donors is less than about 50 L, less than about 25 L, less than about 20 L, less than about 10 L, less than about 5 L, less than about 1 L, or less than about 750 mL (e.g., between about 750 mL and less than about 50 L, between about 100 mL and about 750 mL, between about 750 mL and about 5 L, between about 1 L and about 10 L, between about 10 L and about 50 L, or between about 10 L and about 25 L).

[0109] Described herein are methods for the selective activation and expansion of γδ T cell subtypes from isolated mixed cell populations, providing clinically relevant levels of enriched γδ T cell population(s) with cytotoxic properties, e.g., without prior removal of non-target cell types. Activating γδ variable TCR epitopes of specific γδ cell population(s) is also described. The invention further provides methods of treatment with compositions comprising the enriched γδ T cell population(s) of the invention.

[0110] In the present invention, clinically relevant levels (10 8 Methods for producing or providing engineered or non-engineered γδ T cells are described. Such methods can be used to produce such clinically relevant levels from a single donor, e.g., from a single sample of a single donor. Furthermore, such methods can be used to produce or provide γδ T cells in a single donor sample. 8For example, in some embodiments, the methods described herein can produce about or at least about 10 engineered or non-engineered γδ T-cells comprising one or more specific subsets of γδ T-cells. 9 pieces, about or at least about 10 10 pieces, about or at least about 10 11 or about or at least about 10 12 In some cases, such population sizes can be achieved in as little as 19-30 days and / or using less than about 1 L of total culture medium volume.

[0111] Isolation of γδ T cells In some aspects, the invention provides in vitro methods for expanding engineered or non-engineered γδ T cells. In some cases, the methods employ one or more (e.g., first and / or second) expansion steps that do not include cytokines that favor the expansion of specific populations of γδ T cells, such as IL-4, IL-2, or IL-15, or combinations thereof. In some embodiments, the invention provides in vitro methods for producing an enriched γδ T cell population from an isolated mixed cell population, comprising contacting the mixed cell population with one or more agents that selectively expand δ1 T cells; δ1 T cells and δ3 T cells; δ1 T cells and δ4 T cells; or δ1, δ3, δ4, and δ5 T cells by binding to epitopes unique to the δ1 TCR; δ1 and δ4 TCR; or δ1, δ3, δ4, and δ5 TCR, respectively, to result in an enriched γδ T cell population. In another aspect, the invention provides an in vitro method for producing an enriched population of γδ T cells from an isolated mixed cell population, the method comprising contacting the mixed cell population with one or more agents that selectively expand δ2 T cells by binding to epitopes unique to the δ2 ​​TCR, resulting in an enriched population of γδ T cells.

[0112] In another aspect, the present disclosure provides methods for genetically engineering γδ T cells isolated from a subject. The enrichment, activation, expansion, or genetic engineering methods can be performed alone or in combination, in any order. In one embodiment, γδ T cells can be isolated, genetically engineered, and then activated and expanded. In a preferred embodiment, γδ T cells can be isolated, activated, and expanded, and then optionally genetically engineered. In some embodiments, such activation and expansion can be followed by further activation and expansion of the genetically engineered γδ T cells.

[0113] For example, a non-manipulated γδ T cell population can be expanded from a composite sample of a subject. The composite sample can be from a peripheral blood sample (e.g., PBL or PBMC), a leukapheresis sample, a cord blood sample, a tumor, stem cell precursors, a tumor biopsy, a tissue, lymph, or an epithelial site of a subject in direct contact with the external environment, or derived from stem progenitor cells. In some cases, the present disclosure provides methods for the expansion of γδ T cell populations, including Vδ1 T cell populations. + cell, Vδ2 + cell, Vδ1 + cells and Vδ3 + cell, Vδ1 + cells and Vδ4 + cell, Vδ1 + cells, Vδ3 + cells, Vδ4 + cells and Vδ5 + The present invention provides a method for selectively expanding cells, or any combination thereof.

[0114] Peripheral blood mononuclear cells can be collected from a subject using an apheresis machine, for example, a Ficoll-Paque™ PLUS (GE Healthcare) system or another suitable device / system. γδ T cell(s), or a desired subpopulation of γδ T cell(s), can be purified from the collected sample, for example, by flow cytometry techniques. Cord blood cells can also be obtained from umbilical cord blood during the subject's birth. See WO2016 / 081518, which is incorporated by reference herein for all purposes, including but not limited to, methods and compositions for PBMC isolation, γδ T cell activation, and the manufacture and use of γδ T cell activators.

[0115] γδ T cells can be expanded from isolated complex samples or mixed cell populations cultured in vitro, e.g., by contacting the mixed cell population with one or more agents that expand γδ T cells by specifically binding to an epitope of the γδ TCR in a first expansion step, resulting in an enriched γδ T cell population. In some embodiments, γδ T cells contained in a total PBMC population can be activated and expanded without prior removal of one or more specific cell populations, e.g., one or more or all of the following non-γδ T cell monocytes: αβ T cells, B cells, and NK cells, resulting in an enriched γδ T cell population. In some aspects, activation and expansion of γδ T cells is performed without the presence of natural or engineered APCs. In some aspects, isolation and expansion of γδ T cells from tumor specimens can be performed using immobilized γδ T cell mitogens that bind to activating epitopes of the γδ TCR provided herein, as well as other activating agents, including antibodies and lectins specific for activating epitopes of the γδ TCR.

[0116] In certain embodiments, the isolated mixed cell population is contacted with one or more agents that expand γδ T cells for about or at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 17 days, about 19 days, about 21 days, about 25 days, about 29 days, about 30 days, or any range therein. For example, the isolated mixed cell population is contacted with one or more agents that expand γδ T cells for about 1 day to about 4 days, about 2 days to about 4 days, about 2 days to about 5 days, about 3 days to about 5 days, about 5 days to about 21 days, about 5 days to about 19 days, about 5 days to about 15 days, about 5 days to about 10 days, or about 5 days to about 7 days to produce a first enriched γδ T cell population. In another example, the isolated mixed cell population is contacted with one or more agents that expand γδ T cells for about 7 days to about 21 days, about 7 days to about 19 days, about 7 days to about 23 days, or about 7 days to about 15 days to produce a first enriched γδ T cell population.

[0117] In some cases, a purification or isolation step is performed between the first and second expansion steps. In some cases, the isolation step includes removal of one or more activating agents. In some cases, the isolation step includes specific isolation of γδ T cells or subtypes thereof. In some cases, one or more (e.g., all) activating agents (e.g., all activating agents that are not common components of cell culture medium, e.g., serum components and / or IL-2) are removed between the first and second expansion steps, but γδ T cells are not specifically isolated from other cell types (e.g., αβ T cells).

[0118] In some embodiments, after activating and expanding γδ T cells using an activating agent that binds to an activating epitope of the γδ TCR in a first enrichment step and optionally a second enrichment step, e.g., a first enriched γδ T cell population(s) of the invention may be further enriched or purified in a second, third, fourth, fifth, etc. enrichment step using techniques known in the art to obtain second or further enriched γδ T cell population(s). For example, αβ T cells, B cells, and NK cells may be removed from, e.g., the first enriched γδ T cell population(s). γδ T cells or populations of γδ T cell(s) expressing similar cell surface markers can be directly isolated from, e.g., the first enriched γδ T cell population(s) using positive and / or negative selection of cell surface markers expressed on the collected γδ T cell(s). For example, γδ T cells can be isolated from the enriched γδ T cell population (e.g., after the first and / or second expansion steps) based on positive or negative expression of markers such as CD2, CD3, CD4, CD8, CD24, CD25, CD44, Kit, TCRα, TCRβ, TCRγ (including one or more TCRγ subtypes), TCRδ (including one or more TCRδ subtypes), NKG2D, CD70, CD27, CD28, CD30, CD16, OX40, CD46, CD161, CCR7, CCR4, NKp30, NKp44, NKp46, DNAM-1, CD242, JAML, and other suitable cell surface markers.

[0119] In some embodiments, after the first expansion step (e.g., after an isolation step performed subsequent to the first expansion step), the expanded cells are optionally diluted and cultured in a second expansion step. In preferred embodiments, the second expansion step is performed under conditions in which the culture medium is replenished about every 1-2 days, about every 1-3 days, about every 1-4 days, about every 1-5 days, about every 2-5 days, about every 2-4 days, or about every 2-3 days during the second expansion step. In some embodiments, the second expansion step is performed under conditions in which the cells are diluted or adjusted to a density that supports an additional 1x, 2x, 3x, 4x, 5x, 6x, or more expansion of γδ T cells. In some cases, the cell density adjustment is performed concurrently (i.e., on the same day or simultaneously) with the replenishment of the culture medium. For example, the cell density can be adjusted every 1-2 days, 1-3 days, 1-4 days, 1-5 days, 2-5 days, 2-4 days, or 2-3 days during the second expansion step. Typical cell densities that support further expansion of γδ T cells include, but are not limited to, about 1×10 for culture. 5 , 2 × 10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 cells / mL, 10×10 6 cells / mL, 15×10 6 cells / mL, 20×10 6 cells / mL, or 30 x 10 6 cells / mL.

[0120] In some embodiments, the cell density is about 0.5 x 10 6 ~Approx. 1×10 6 cells / mL, approximately 0.5×10 6 ~Approx. 1.5×10 6 cells / mL, approximately 0.5×10 6 ~Approx. 2×10 6cells / mL, approximately 0.75×10 6 ~Approx. 1×10 6 cells / mL, approximately 0.75×10 6 ~Approx. 1.5×10 6 cells / mL, approximately 0.75×10 6 ~Approx. 2×10 6 cells / mL, approximately 1×10 6 ~Approx. 2×10 6 cells / mL, or approximately 1 x 10 6 ~Approx. 1.5×10 6 cells / mL, approximately 1×10 6 ~Approx. 2×10 6 cells / mL, approximately 1×10 6 ~Approx. 3×10 6 cells / mL, approximately 1×10 6 ~Approx. 4×10 6 cells / mL, approximately 1×10 6 ~Approx. 5×10 6 cells / mL, approximately 1×10 6 ~About 10×10 6 cells / mL, approximately 1×10 6 ~Approx. 15×10 6 cells / mL, approximately 1×10 6 ~Approx. 20×10 6 cells / mL, or approximately 1 x 10 6 ~Approx. 30×10 6 Adjust to a density of 100 cells / mL.

[0121] In some embodiments, the second expansion step is carried out under conditions that track and maintain the cells at a predetermined cell density (or density interval) and / or maintain the cells in a culture medium having a predetermined glucose content. For example, the cells are maintained at a density of about 0.5×10 6 ~Approx. 1×10 6 cells / mL, approximately 0.5×10 6 ~Approx. 1.5×10 6 cells / mL, approximately 0.5×10 6 ~Approx. 2×10 6 cells / mL, approximately 0.75×10 6 ~Approx. 1×10 6 cells / mL, approximately 0.75×10 6 ~Approx. 1.5×10 6 cells / mL, approximately 0.75×10 6~Approx. 2×10 6 cells / mL, approximately 1×10 6 ~Approx. 2×10 6 cells / mL, or approximately 1 x 10 6 ~Approx. 1.5×10 6 cells / mL, approximately 1×10 6 ~Approx. 3×10 6 cells / mL, approximately 1×10 6 ~Approx. 4×10 6 cells / mL, approximately 1×10 6 ~Approx. 5×10 6 cells / mL, approximately 1×10 6 ~About 10×10 6 cells / mL, approximately 1×10 6 ~Approx. 15×10 6 cells / mL, approximately 1×10 6 ~Approx. 20×10 6 cells / mL, approximately 1×10 6 ~Approx. 30×10 6 The viable cell density can be maintained at 1000 cells / mL.

[0122] In some cases, cells may be maintained at a higher concentration throughout at least a portion of the expansion. For example, cell viability may be enhanced at a higher cell concentration during the first portion of the first or second expansion. As another example, a higher cell concentration during the final portion of the first or second expansion may provide the most efficient utilization of culture volume. Thus, in some embodiments, cells are maintained at a concentration of about 1 x 10 cells throughout at least a portion of the first or second expansion culture or throughout the first or second expansion culture. 6 ~Approx. 20×10 6 The cells can be maintained at a viable cell density of 1000 cells / mL.

[0123] For another example, cells can be maintained in a culture medium having a glucose content of about 0.5 g / L to about 1 g / L, about 0.5 g / L to about 1.5 g / L, about 0.5 g / L to about 2 g / L, about 0.75 g / L to about 1 g / L, about 0.75 g / L to about 1.5 g / L, about 0.75 g / L to about 2 g / L, about 1 g / L to about 1.5 g / L, about 1 g / L to about 2 g / L, about 1 g / L to about 3 g / L, or about 1 g / L to about 4 g / L. In some embodiments, cells can be maintained in a culture medium having a glucose content of about 1.25 g / L. In some cases, for example, when maintaining high cell density cultures, cells may be maintained in a culture medium having a glucose content of about 1 g / L to about 5 g / L, about 1 g / L to about 4 g / L, about 2 g / L to about 5 g / L, or about 2 g / L to about 4 g / L.

[0124] Typically, glucose content is maintained by adding fresh serum-containing or serum-free culture medium to the culture. In some embodiments, cells can be maintained in culture medium having a predetermined glucose content interval at a predetermined viable cell density interval, e.g., by tracking each parameter and adding fresh medium to maintain the parameter within predetermined limits. In some embodiments, glucose content is maintained by adding fresh serum-containing or serum-free culture medium to the culture while removing spent medium in a perfusion bioreactor while retaining the cells therein. In some embodiments, additional parameters are tracked and / or maintained during γδ T cell expansion (e.g., selective γδ T cell expansion) or during the first or second γδ T cell expansion (e.g., selective γδ T cell expansion) steps described herein, including, but not limited to, one or more of pH, O partial pressure, O saturation, CO partial pressure, CO saturation, lactate, glutamine, glutamate, ammonium, sodium, potassium, and calcium.

[0125] For example, in the first expansion step, the γδ T cell subtype i) selectively expanding δ1 T cells by specifically binding to an epitope of the δ1 TCR; ii) selectively expanding δ2 T cells by specifically binding to an epitope of the δ2 ​​TCR; iii) selectively expanding δ1 and δ4 T cells by specifically binding to epitopes of the δ1 and δ4 TCR; or iv) one or more agents that selectively expand δ1, δ3, δ4 and δ5 T cells by specifically binding to epitopes of δ1, δ3, δ4 and δ5 TCRs γδ T cell populations can be selectively expanded from isolated complex samples or mixed cell populations cultured in vitro by contacting the mixed cell population with one or more agents to produce an enriched γδ T cell population. In some cases, the one or more agents specifically bind to the δ1J1, δ1J2, or δ1J3 TCR, or two of them, or all of them. In some embodiments, γδ cells in a total PBMC population can be activated and expanded without prior depletion of specific cell populations, such as monocytes, αβ T cells, B cells, and NK cells, resulting in an enriched γδ T cell population. In some aspects, the activation and expansion of γδ T cells is performed without the presence of natural or engineered APCs. In some embodiments, activation and expansion of γδ T cells from a tumor sample may be performed using immobilized γδ T cell mitogens, including antibodies specific for activating epitopes unique to the δ1 TCR; the δ1, δ3, δ4 and δ5 TCR, the δ1 and δ4 TCR; or the δ2 ​​TCR, and other activating agents, including lectins that bind to activating epitopes unique to the δ1 TCR; the δ1, δ3, δ4 and δ5 TCR, the δ1 and δ4 TCR; or the δ2 ​​TCR.

[0126] In certain embodiments, the isolated mixed cell population is contacted with one or more agents that selectively expand δ1, δ1 and δ4, δ2, or δ1 and δ2 T cells for about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or any range therebetween. For example, the isolated mixed cell population is contacted with one or more agents that selectively expand δ1 or δ2 T cells for about 1 to about 3 days, about 1 to about 4 days, about 1 to about 5 days, about 2 to about 3 days, about 2 to about 4 days, about 2 to about 5 days, about 3 to about 4 days, about 3 to about 5 days, about 4 to about 5 days, about 5 to about 15 days, or about 5 to about 7 days to produce a first enriched γδ T cell population. In some embodiments, the selectively expanded δ1, δ1 and δ3, δ1 and δ4, δ2, or δ1 and δ2 T cells are further expanded in a second expansion step as described herein.

[0127] In certain embodiments, the isolated starting mixed cell population, e.g., a peripheral blood sample, comprises T lymphocytes in the range of about 20-80%. In certain embodiments, the percentage of residual αβ T cells and NK cells in the enriched γδ T cell population(s) of the invention is about 2.5% or less and about 1% or less, respectively. The percentage of residual αβ T cells or NK cells in the enriched γδ T cell population(s) of the invention is about 1% or less, about 0.5% or less, about 0.4% or less, about 0.2% or less, about 0.1% or less, or about 0.01% or less. In certain embodiments, the percentage of residual αβ T cells in the enriched γδ T cell population(s) of the invention (e.g., after a step of positive selection of γδ T cells or subtypes thereof, or after depletion of αβ T cells) is about 0.4% or less, about 0.2% or less, about 0.1% or less, or about 0.01% or less. In some embodiments, αβ T cells are depleted, but NK cells are not, before or after the first and / or second γδ T cell expansion. In certain aspects, the isolated mixed cell population is derived from a single donor. In other aspects, the isolated mixed cell population is derived from more than one donor or multiple donors (e.g., 2, 3, 4, 5, or 2-5, 2-10, or 5-10, or more donors).

[0128] Thus, in some embodiments, the methods of the present invention provide a clinically relevant number (10 8 Super, 10 9 Super, 10 10 Super, 10 11 Over or 10 12 Over or about 10 8 ~about 10 12 ) of expanded γδ T cells can be obtained from just one donor. In some cases, the methods of the invention can produce clinically relevant numbers (10 8 Super, 10 9 Super, 10 10 Super, 10 11 Over or 10 12 Over or about 10 8 ~about 10 12) of expanded γδ T cells can be obtained in less than 19 days or less than 21 days from the time the donor sample is obtained.

[0129] Following specific activation and expansion of specific γδ T cell subsets in a first enrichment step using activating agents that bind to activation epitopes unique to the δ1, δ1 and δ3 TCR, δ1 and δ4 TCR, or δ2 TCR, the first enriched γδ T cell population(s) of the invention may be further enriched or purified in a second, third, fourth, fifth, etc. enrichment step using techniques known in the art to obtain second or further enriched γδ T cell population(s). For example, αβ T cells, B cells, and NK cells may be removed from the first enriched γδ T cell population(s). γδ T cells or populations of γδ T cell(s) expressing similar cell surface markers can be directly isolated from the first enriched γδ T cell population(s) using positive and / or negative selection of cell surface markers expressed on the collected γδ T cell(s). For example, γδ T cells can be isolated from the first enriched γδ T cell population based on the positive or negative expression of markers such as CD2, CD3, CD4, CD8, CD24, CD25, CD44, Kit, TCRα, TCRβ, TCRγ (or one or more subtypes thereof), TCRδ (or one or more subtypes thereof), NKG2D, CD70, CD27, CD28, CD30, CD16, OX40, CD46, CD161, CCR7, CCR4, DNAM-1, JAML, and other suitable cell surface markers.

[0130] In some embodiments, after the first γδ T cell expansion, first enrichment step, second γδ T cell expansion and / or second enrichment step of the invention, the enriched γδ T cell population is enriched at clinically relevant levels, e.g., in a culture volume of less than 10 mL, less than 25 mL, less than 50 mL, less than 100 mL, less than 150 mL, less than 200 mL, less than 500 mL, less than 750 mL, less than 1 L, less than 2 L, less than 3 L, less than 4 L, less than 5 L, less than 10 L, less than 20 L or less than 25 L. 8For example, the method of the present invention can be used to administer a γδ T cell subset containing more than 10 to 100 mL, 25 to 100 mL, 50 to 100 mL, 75 to 100 mL, 10 to 150 mL, 25 to 150 mL, 50 to 150 mL, 75 to 150 mL, 100 to 150 mL, 10 to 200 mL, 25 to 200 mL, 50 to 200 mL, 75 to 200 mL, 100 to 200 mL, 10 to 250 mL, 25 to 250 mL, 50 to 250 mL, 75 to 250 mL, Clinically relevant levels in growth cultures with volumes of 100-250 mL, 150-250 mL, 5-1,000 mL, 10-1,000 mL, or 100-1,000 mL, 150-1,000 mL, 200-1,000 mL, 250-1,000 mL, 400 mL to 1 L, 1 L to 2 L, 2 L to 5 L, 2 L to 10 L, 4 L to 10 L, 4 L to 15 L, 4 L to 20 L, or 4 L to 25 L, 8 In other embodiments, the enriched γδ T cell population after the second, third, fourth, fifth, etc. enrichment steps of the present invention can result in a clinically relevant level of γδ T cell subsets greater than 10 8 It contains more than one γδ T cell subset.

[0131] In some embodiments, the γδ T cell(s) are capable of rapid proliferation in response to contact with one or more antigens. + γδ T cell(s) rapidly proliferate in vitro in response to contact with several antigens, such as prenyl pyrophosphate, alkylamines, and metabolic products in tissue culture or microbial extracts. Furthermore, some wild-type γδ T cell(s), e.g., Vγ2Vδ2 +γδ T cell(s) rapidly proliferate in vivo in humans in response to certain types of vaccination(s). Stimulated γδ T cells can exhibit a variety of antigen-presenting, costimulatory, and adhesion molecules that can facilitate isolation of γδ T cell(s) from a complex sample. γδ T cell(s) in a complex sample can be stimulated in vitro with at least one antigen for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, about 5-15 days, 5-10 days, or 5-7 days, or another suitable period, before or after expansion with a selective γδ T cell expansion agent described herein, such as an antibody or immobilized antibody. Stimulation of γδ T cells with the appropriate antigen can expand the γδ T cell population in vitro.

[0132] Non-limiting examples of antigens that can be used to stimulate in vitro proliferation of γδ T cell(s) from a complex sample include prenyl pyrophosphates, such as isopentenyl pyrophosphate (IPP), alkylamines, metabolites of human microbial pathogens, metabolites of commensal bacteria, 1-methyl-3-butenyl-1-pyrophosphate (2M3B1PP), (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), ethyl pyrophosphate (EPP), farnesyl pyrophosphate (FPP), dimethylallyl phosphate (DMAP), dimethylallyl pyrophosphate (DMAPP), ethyl-adenosine triphosphate (EPPPA), geranyl pyrophosphate (GPP), geranylgeranyl pyrophosphate (GGPP), and the like. ), isopentenyl-adenosine triphosphate (IPPPA), monoethyl phosphate (MEP), monoethyl pyrophosphate (MEPP), 3-formyl-1-butyl-pyrophosphate (TUBAg1), X-pyrophosphate (TUBAg2), 3-formyl-1-butyl-uridine triphosphate (TUBAg3), 3-formyl-1-butyl-deoxythymidine triphosphate (TUBAg4), monoethyl alkylamines, allyl pyrophosphate, clotyl pyrophosphate, dimethylallyl-γ-uridine triphosphate, clotyl-γ-uridine triphosphate, allyl-γ-uridine triphosphate, ethylamine, isobutylamine, sec-butylamine, isoamylamine, and nitrogen-containing bisphosphonates.

[0133] Activation and expansion of γδ T cells can be performed using the activating and costimulatory agents described herein to induce specific γδ T cell proliferation and persistence populations. In some embodiments, activation and expansion of γδ T cells from different cultures can achieve distinct clonal or mixed polyclonal population subsets. In some embodiments, various agonists can be used to identify agents that provide specific γδ activating signals. In one aspect, agents that provide specific γδ activating signals can be various monoclonal antibodies (MAbs) directed against the γδ TCR.

[0134] In one embodiment, the MAbs can bind to different epitopes on the constant or variable regions of the γ and / or δ TCR. In one embodiment, the MAbs can comprise γδ TCR pan-MAbs. In one embodiment, the γδ TCR pan-MAbs can recognize domains common to various γ and δ TCRs on either the γ or δ chain or both, including the δ1 and δ2 cell populations. In one embodiment, the antibodies can be 5A6.E9 (Thermo scientific), B1 (Biolegend), IMMU510 and / or 11F2 (11F2) (Beckman Coulter). In one embodiment, MAbs can be directed to specific domains unique to the variable region of the gamma chain (7A5 MAb (Thermo Scientific #TCR1720) directed against the Vγ9 TCR analog), or to domains on the Vδ1 variable region (Mab TS8.2 (Thermo Scientific #TCR1730); MAb TS-1 (ThermoFisher #TCR1055), MAb R9.12 (Beckman Coulter #IM1761)), or to the Vδ2 chain (MAb 15D (Thermo Scientific #TCR1732 or Life technologies #TCR2732), B6 ​​(Biolegend #331402), one of the δ1-# antibodies described in Figures 33-34, or one of the δ2-# antibodies described in Figures 35-36).

[0135] In some embodiments, antibodies against different domains of the γδ TCR (pan-antibodies and antibodies recognizing unique variable region epitopes on subset populations) can be combined to assess their ability to enhance γδ T cell activation. In some embodiments, γδ T cell activators can include γδ TCR binding agents, such as MICA, agonist antibodies against NKG2D, fusion proteins of MICA with, for example, an Fc tag, ULBP1, or ULBP3 (R&D systems Minneapolis, MN), ULBP2, or ULBP6 (Sino Biological Beijing, China). In some embodiments, companion costimulators can be identified that help induce specific γδ T cell proliferation without inducing cellular anergy and apoptosis. These costimulatory agents can include ligands for receptors expressed on γδ cells, such as ligand(s) for one or more of the following: NKG2D, CD161, CD70, JAML, DNAX, CD81 accessory molecule-1 (DNAM-1), ICOS, CD27, CD196, CD137, CD30, HVEM, SLAM, CD122, DAP, and CD28. In some embodiments, costimulatory agents can be specific antibodies directed against unique epitopes on CD2 and CD3 molecules. CD2 and CD3 can have different conformational structures when expressed on αβ or γδ T cell(s), and in some cases, specific antibodies against CD3 and CD2 can result in selective activation of γδ T cells.

[0136] The population of γδ T cell(s) may be expanded in vitro prior to manipulation of the γδ T cell(s). Non-limiting examples of reagents that can be used to promote in vitro expansion of γδ T cell populations include anti-CD3 or anti-CD2, anti-CD27, anti-CD30, anti-CD70, anti-OX40 antibodies, IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-18, IL-19, IL-21, IL23, IL-33, IFNγ, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), CD70 (CD27 ligand), concavalin A (ConA), pokeweed (PWM), peanut agglutinin protein (PNA), soybean agglutinin (SBA), Les Culinaris agglutinin (LCA), Pisum Sativum agglutinin (PSA), Helix pomatia agglutinin (HPA), Vicia graminea lectin (VGA), Phaseolus Suitable mitogens include Phaseolus vulgaris erythroagglutinin (PHA-E), Phaseolus vulgaris leucoagglutinin (PHA-L), Sambucus nigra lectin (SNA, EBL), Maackia amurensis lectin II (MAL II), Sophora japonica agglutinin (SJA), Dolichos biflorus agglutinin (DBA), Lens culinaris agglutinin (LCA), Wisteria floribunda lectin (WFA, WFL), or another suitable mitogen capable of stimulating T cell proliferation.

[0137] Genetic engineering of γδ T-cell(s) may include stably integrating into the genome of the isolated γδ T-cell(s) a construct expressing a tumor recognition moiety such as an αβ TCR, a γδ TCR, or a CAR encoding an antibody, antigen-binding fragment thereof, or lymphocyte activation domain, and may include a cytokine (e.g., IL-15, IL-12, IL-2, IL-7, IL-21, IL-18, IL-19, IL-33, IL-4, IL-9, IL-23, or IL1β) that enhances T-cell proliferation, survival, and function in vitro and in vivo. Genetic engineering of isolated γδ T-cells may further include deleting or disrupting gene expression of one or more endogenous genes in the genome of the isolated γδ T-cell, for example, gene expression of an MHC locus(s).

[0138] Ex vivo expansion of γδ T cells In another aspect, the present disclosure provides methods for expanding populations of non-engineered and engineered γδ T cells in vitro for adoptive transfer therapy. The non-engineered or engineered γδ T cells of the present disclosure can be expanded in vitro. The non-engineered or engineered γδ T cells of the present disclosure can be expanded in vitro without activation with APCs or without co-culture with APCs and / or aminophosphonates. Additionally or alternatively, the non-engineered or engineered γδ T cells of the present disclosure can be expanded in vitro with at least one expansion step that includes activation with or co-culture with APCs and / or one or more aminophosphonates.

[0139] In some embodiments, non-engineered or engineered γδ T-cells of the disclosure can be expanded in vitro without activation by APCs in a first γδ T-cell expansion, and then expanded in vitro with activation by APCs in a second γδ T-cell expansion. In some cases, the first γδ T-cell expansion comprises contacting γδ T-cells with one or more agents that (a) expand γδ T-cells, or (b) selectively expand δ1 T-cells; δ2 T-cells; δ1 T-cells and δ3 T-cells; δ1 T-cells and δ4 T-cells; or δ1, δ3, δ4 and δ5 T-cells by binding to activation epitopes unique to the δ1 TCR; δ2 TCR; δ1 and δ4 TCR; or δ1, δ3, δ4 and δ5 TCR, respectively.

[0140] In some cases, the second γδ T cell expansion is performed in culture medium that does not contain one or more agents used in the first γδ T cell expansion. In some cases, the second γδ T cell expansion is performed in culture medium that contains one or more second agents that (a) expand T cells, (b) expand γδ T cells, or (c) selectively expand δ1 T cells; δ2 T cells; δ1 T cells and δ3 T cells; δ1 T cells and δ4 T cells; or δ1, δ3, δ4 and δ5 T cells by binding to an activating epitope unique to the δ1 TCR; δ2 TCR; δ1 and δ4 TCR; or δ1, δ3, δ4 and δ5 TCR, respectively.

[0141] In some cases, the second agent is different (e.g., has a different primary amino acid sequence and / or binds to a structurally different γδ TCR epitope) from the agent used in the first γδ T cell expansion. In some cases, the second agent binds to an overlapping or the same γδ TCR epitope as the agent used in the first γδ T cell expansion, or can compete with the agent for binding to the γδ TCR. In some cases, the second agent is expressed on the cell surface of the APC. In some cases, the second agent is bound to the surface of the APC, for example, by a binding interaction between a constant region of the second agent and an Fc receptor on the surface of the APC. In some cases, the second agent is soluble. In some cases, the second γδ T cell expansion is performed in culture medium containing a soluble second agent and APCs, optionally expressing on or having bound to their cell surface an agent that expands or selectively expands the γδ T cell population.

[0142] In some cases, the first γδ T cell expansion is performed without APCs and the second γδ T cell expansion is performed with APCs. In some cases, the second γδ T cell expansion is performed using APCs and one or more second agents that (a) expand T cells, (b) expand γδ T cells, or (c) selectively expand δ1 T cells; δ2 T cells; δ1 T cells and δ3 T cells; δ1 T cells and δ4 T cells; or δ1, δ3, δ4 and δ5 T cells by binding to an activating epitope unique to the δ1 TCR; δ2 TCR; δ1 and δ4 TCR; or δ1, δ3, δ4 and δ5 TCR, respectively.

[0143] Those skilled in the art will appreciate that in certain embodiments, the methods for the second expansion step described herein may be performed as the first expansion step, and the methods for the first step described herein may be performed as the second expansion step. By way of example and not limitation, in some embodiments, a mixed population of cells (e.g. PBMCs) may be expanded in a first step by contacting them with APCs, and then expanded in the absence of APCs, for example by contacting the expanded population from the first expansion step with an immobilized agent that selectively expands δ1 T cells; δ2 T cells; δ1 T cells and δ3 T cells; δ1 T cells and δ4 T cells; or δ1, δ3, δ4 and δ5 T cells by binding to activating epitopes unique to δ1 TCR; δ2 TCR; δ1 and δ4 TCR; or δ1, δ3, δ4 and δ5 TCR, respectively.

[0144] The methods of the present invention include various γδ T cell(s) populations, e.g., Vγ1 + , Vγ2 + or Vγ3 + In some instances, the methods of the invention can expand a γδ T cell population. + T cell population; Vδ1 + and Vδ3 + T cell population; Vδ1 + and Vδ4 + T cell population; Vδ1 + and Vδ2 + T cell population; or Vδ1 + , Vδ3 + , Vδ4 + and Vδ5 + The T cell population can be expanded.

[0145] In some cases, the γδ T cell population can be expanded in vitro for less than 36 days, less than 35 days, less than 34 days, less than 33 days, less than 32 days, less than 31 days, less than 30 days, less than 29 days, less than 28 days, less than 27 days, less than 26 days, less than 25 days, less than 24 days, less than 23 days, less than 22 days, less than 21 days, less than 20 days, less than 19 days, less than 18 days, less than 17 days, less than 16 days, less than 15 days, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days or less than 3 days.

[0146] In some aspects, methods are provided for selectively expanding various γδ T cells, including engineered and non-engineered γδ T cells, by contacting γδ T cells from a mixed cell population with an activating agent. In some cases, the activation or activating agent binds to a unique epitope on a cell surface receptor of the γδ T cells. The activating agent can be an antibody, e.g., a monoclonal antibody. The activating agent can specifically activate the growth of one or more types of γδ T cells, e.g., δ1, δ2, δ1 and δ3, or δ1 and δ4 cell populations. In some embodiments, the activating agent specifically activates the growth of a δ1 cell population, resulting in an enriched δ1 T cell population. In other cases, the activating agent specifically activates the growth of a δ2 cell population, resulting in an enriched δ2 T cell population.

[0147] Activating agents can stimulate the proliferation of engineered and non-engineered γδ T cells at a fast growth rate. For example, an agent can stimulate one cell division in less than 30 hours, one cell division in less than 29 hours, one cell division in less than 28 hours, one cell division in less than 27 hours, one cell division in less than 26 hours, one cell division in less than 25 hours, one cell division in less than 24 hours, one cell division in less than 23 hours, one cell division in less than 22 hours, one cell division in less than 21 hours, one cell division in less than 20 hours, one cell division in less than 19 hours, one cell division in less than 18 hours, one cell division in less than 17 hours, one cell division in less than 16 hours, or one cell division in less than 18 hours. stimulates proliferation of γδ T cell populations at an average rate of cell division, 1 cell division in less than 15 hours, 1 cell division in less than 14 hours, 1 cell division in less than 13 hours, 1 cell division in less than 12 hours, 1 cell division in less than 11 hours, 1 cell division in less than 10 hours, 1 cell division in less than 9 hours, 1 cell division in less than 8 hours, 1 cell division in less than 7 hours, 1 cell division in less than 6 hours, 1 cell division in less than 5 hours, 1 cell division in less than 4 hours, 1 cell division in less than 3 hours, and 1 cell division in less than 2 hours.

[0148] In some cases, the activator may be activated at an average rate of about 1 division every 4 hours, an average rate of about 1 division every 5 hours, an average rate of about 1 division every 6 hours, an average rate of about 1 division every 7 hours, an average rate of about 1 division every 8 hours, an average rate of about 1 division every 9 hours, an average rate of about 1 division every 10 hours, an average rate of about 1 division every 11 hours, an average rate of about 1 division every 12 hours, an average rate of about 1 division every 13 hours, an average rate of about 1 division every 14 hours, an average rate of about 1 division every 15 hours, an average rate of about 1 division every 16 hours, an average rate of about 1 division every 17 hours, an average rate of about 1 division every 18 hours, an average rate of about 1 division every 19 hours, an average rate of about 1 division every 20 hours, an average rate of about 1 division every 21 hours, an average rate of about 1 division every 22 hours, an average rate of about 1 division every 23 hours, an average rate of about 1 division every 24 hours, an average rate of about 1 division every 25 hours, an average rate of about 1 division every 26 hours, an average rate of about 1 division every 27 hours, a rate of about 1 division every 28 hours, a rate of about 1 division every 29 hours, an average rate of about 1 division every 30 hours, an average rate of about 1 division every 31 hours, an average rate of about 1 division every 32 hours, an average rate of about 1 division every 33 hours, a rate of about 1 division every 34 hours, a rate of about 1 division every 35 hours, an average rate of about 1 division every 36 hours.

[0149] In some cases, the activating agent may stimulate rapid proliferation of engineered and / or non-engineered γδ T cells in a γδ T cell expansion culture, wherein the rapid proliferation is maintained at any one of the above average rates of cell division for about 1 to about 19 consecutive days, about 1 to about 14 consecutive days, about 1 to about 7 consecutive days, about 1 to about 5 consecutive days, about 2 to about 19 consecutive days, about 2 to about 14 consecutive days, about 2 to about 7 consecutive days, about 2 to about 5 consecutive days, about 4 to about 19 consecutive days, about 4 to about 14 consecutive days, about 4 to about 7 consecutive days, or about 4 to about 5 consecutive days.

[0150] In some cases, the activator may induce an average rate of about 1 division every 12 hours (e.g., 10-12 hours), an average rate of about 1 division every 13 hours (e.g., 10-13 hours), an average rate of about 1 division every 14 hours (e.g., 10-14 hours), an average rate of about 1 division every 15 hours (e.g., 10-15 hours), an average rate of about 1 division every 16 hours (e.g., 10-16 hours), an average rate of about 1 division every 17 hours (e.g., 10-17 hours or 12-17 hours), an average rate of about 1 division every 18 hours (e.g., 10-18 hours or 12-18 hours), an average rate of about 1 division every 19 hours (e.g., 20-21 hours), or an average rate of about 20-21 hours in γδ T cell expansion cultures maintained for about 2 consecutive days to about 7 consecutive days, or about 2 to about 5 consecutive days. the proliferation of engineered and / or non-engineered γδ T cells may be stimulated at an average rate of about one division every 10-19 hours or 12-19 hours), about one division every 20 hours (e.g., 12-20 hours, 16-20 hours, or 18-20 hours), about one division every 21 hours (e.g., 12-21 hours, 16-21 hours, or 18-21 hours), about one division every 22 hours (e.g., 12-22 hours, 16-22 hours, or 18-22 hours), about one division every 23 hours or less (e.g., 12-23 hours, 16-23 hours, or 18-23 hours), or about one division every 24 hours (e.g., 12-24 hours, 16-24 hours, or 18-24 hours).

[0151] In some cases, the activating agent induces an average rate of about 1 division every 25 hours (e.g., 12-25 hours, 16-25 hours, 18-25 hours, or 20-25 hours), an average rate of about 1 division every 26 hours (e.g., 12-26 hours, 16-26 hours, 18-26 hours, or 20-26 hours), an average rate of about 1 division every 27 hours (e.g., 12-27 hours, 16-26 hours, 18-26 hours, or 20-26 hours), or an average rate of about 1 division every 28 hours (e.g., 12-28 hours, 16-26 hours, 18-26 hours, or 20-26 hours) in γδ T cell expansion cultures maintained for about 2 consecutive days to about 7 consecutive days, or about 2 to about 5 consecutive days. an average rate of about one division every 6-27 hours (e.g., 18-27 hours, 18-27 hours, or 20-27 hours), a rate of about one division every 28 hours (e.g., 12-28 hours, 16-28 hours, 18-28 hours, 20-28 hours, or 22-28 hours), a rate of about one division every 29 hours (e.g., 16-29 hours, 18-29 hours, 20-29 hours, or 22-29 hours), a rate of about one division every 30 hours (e.g., 16-30 hours, 18-30 hours, 20-30 hours, or 22-30 hours), an average rate of approximately one division every 31 hours (e.g., 16-31 hours, 18-31 hours, 20-31 hours, 22-31 hours, or 24-31 hours); an average rate of approximately one division every 32 hours (e.g., 18-32 hours, 20-32 hours, 22-32 hours, or 24-32 hours); an average rate of approximately one division every 33 hours (e.g., 18-33 hours, 20-33 hours, 22-33 hours, or 24-33 hours). The engineered and / or non-engineered γδ T cells may be stimulated to proliferate at an average rate of about one division every 34 hours (e.g., 18-34 hours, 20-34 hours, 22-34 hours, or 24-34 hours), about one division every 35 hours (e.g., 18-35 hours, 20-35 hours, 22-35 hours, or 24-35 hours), or about one division every 36 hours (e.g., 18-36 hours, 20-36 hours, 22-36 hours, or 24-36 hours).

[0152] In some cases, the activator induces an average rate of about 1 division every 12 hours (e.g., 10-12 hours), an average rate of about 1 division every 13 hours (e.g., 10-13 hours), an average rate of about 1 division every 14 hours (e.g., 10-14 hours), an average rate of about 1 division every 15 hours (e.g., 10-15 hours), an average rate of about 1 division every 16 hours (e.g., 10-16 hours), an average rate of about 1 division every 17 hours (e.g., 10-17 hours or 12-17 hours), an average rate of about 1 division every 18 hours (e.g., 10-18 hours or 12-18 hours), an average rate of about 1 division every 19 hours (e.g., 10-1 The engineered and / or non-engineered γδ T cells may be stimulated to proliferate at an average rate of about one division every 9 hours or 12-19 hours), about one division every 20 hours (e.g., 12-20 hours, 16-20 hours, or 18-20 hours), about one division every 21 hours (e.g., 12-21 hours, 16-21 hours, or 18-21 hours), about one division every 22 hours (e.g., 12-22 hours, 16-22 hours, or 18-22 hours), about one division every 23 hours or less (e.g., 12-23 hours, 16-23 hours, or 18-23 hours), or about one division every 24 hours (e.g., 12-24 hours, 16-24 hours, or 18-24 hours).

[0153] In some cases, the activator induces an average rate of about one division every 25 hours (e.g., 12-25 hours, 16-25 hours, 18-25 hours, or 20-25 hours), an average rate of about one division every 26 hours (e.g., 12-26 hours, 16-26 hours, 18-26 hours, or 20-26 hours), an average rate of about one division every 27 hours (e.g., 12-27 hours, 16-27 hours, 18-27 hours, or 20-26 hours) in γδ T cell expansion cultures maintained for at least 14 consecutive days. an average rate of approximately one division every 27 hours or 20-27 hours), a rate of approximately one division every 28 hours (e.g., 12-28 hours, 16-28 hours, 18-28 hours, 20-28 hours, or 22-28 hours), a rate of approximately one division every 29 hours (e.g., 16-29 hours, 18-29 hours, 20-29 hours, or 22-29 hours), and a rate of approximately one division every 30 hours (e.g., 16-30 hours, 18-30 hours, 20-30 hours, or 22-30 hours). an average rate of about one division, an average rate of about one division every 31 hours (e.g., 16-31 hours, 18-31 hours, 20-31 hours, 22-31 hours, or 24-31 hours); an average rate of about one division every 32 hours (e.g., 18-32 hours, 20-32 hours, 22-32 hours, or 24-32 hours); an average rate of about one division every 33 hours (e.g., 18-33 hours, 20-33 hours, 22-33 hours, or 24-33 hours); The proliferation of engineered and / or non-engineered γδ T cells may be stimulated at an average rate of about one division every 4 hours (e.g., 18-34 hours, 20-34 hours, 22-34 hours, or 24-34 hours), about one division every 35 hours (e.g., 18-35 hours, 20-35 hours, 22-35 hours, or 24-35 hours), or about one division every 36 hours (e.g., 18-36 hours, 20-36 hours, 22-36 hours, or 24-36 hours).

[0154] In some cases, the activator induces an average rate of about 1 division every 12 hours (e.g., 10-12 hours), an average rate of about 1 division every 13 hours (e.g., 10-13 hours), an average rate of about 1 division every 14 hours (e.g., 10-14 hours), an average rate of about 1 division every 15 hours (e.g., 10-15 hours), an average rate of about 1 division every 16 hours (e.g., 10-16 hours), an average rate of about 1 division every 17 hours (e.g., 10-17 hours or 12-17 hours), an average rate of about 1 division every 18 hours (e.g., 10-18 hours or 12-18 hours), an average rate of about 1 division every 19 hours (e.g., 10-1 The engineered and / or non-engineered γδ T cells may be stimulated to proliferate at an average rate of about one division every 9 hours or 12-19 hours), about one division every 20 hours (e.g., 12-20 hours, 16-20 hours, or 18-20 hours), about one division every 21 hours (e.g., 12-21 hours, 16-21 hours, or 18-21 hours), about one division every 22 hours (e.g., 12-22 hours, 16-22 hours, or 18-22 hours), about one division every 23 hours or less (e.g., 12-23 hours, 16-23 hours, or 18-23 hours), or about one division every 24 hours (e.g., 12-24 hours, 16-24 hours, or 18-24 hours).

[0155] In some cases, the activator induces an average rate of about one division every 25 hours (e.g., 12-25 hours, 16-25 hours, 18-25 hours, or 20-25 hours), an average rate of about one division every 26 hours (e.g., 12-26 hours, 16-26 hours, 18-26 hours, or 20-26 hours), an average rate of about one division every 27 hours (e.g., 12-27 hours, 16-27 hours, 18-27 hours, or 20-26 hours) in γδ T cell expansion cultures maintained for at least 19 consecutive days. an average rate of approximately one division every 27 hours or 20-27 hours), a rate of approximately one division every 28 hours (e.g., 12-28 hours, 16-28 hours, 18-28 hours, 20-28 hours, or 22-28 hours), a rate of approximately one division every 29 hours (e.g., 16-29 hours, 18-29 hours, 20-29 hours, or 22-29 hours), and a rate of approximately one division every 30 hours (e.g., 16-30 hours, 18-30 hours, 20-30 hours, or 22-30 hours). an average rate of about one division, an average rate of about one division every 31 hours (e.g., 16-31 hours, 18-31 hours, 20-31 hours, 22-31 hours, or 24-31 hours); an average rate of about one division every 32 hours (e.g., 18-32 hours, 20-32 hours, 22-32 hours, or 24-32 hours); an average rate of about one division every 33 hours (e.g., 18-33 hours, 20-33 hours, 22-33 hours, or 24-33 hours); The proliferation of engineered and / or non-engineered γδ T cells may be stimulated at an average rate of about one division every 4 hours (e.g., 18-34 hours, 20-34 hours, 22-34 hours, or 24-34 hours), about one division every 35 hours (e.g., 18-35 hours, 20-35 hours, 22-35 hours, or 24-35 hours), or about one division every 36 hours (e.g., 18-36 hours, 20-36 hours, 22-36 hours, or 24-36 hours).

[0156] An activating agent can stimulate the proliferation of a subpopulation of engineered or non-engineered γδ T cells at various growth rates. For example, the agent can stimulate the growth of a δ1 cell population over a period of 1 day to 90 days of culture (e.g., from about 1 day to about 19, 21, or 23 days of culture) to be greater than another γδ T cell population, e.g., a δ2 or δ3 population; greater than the starting number of γδ T cells before expansion; greater than the starting number of γδ1 T cells before expansion; or greater than about 10-fold, greater than about 100-fold, or greater than about 200-fold greater than the αβ T cell population in culture. The stimulation may be at a faster rate such that it results in greater than about 300-fold, greater than about 400-fold, greater than about 500-fold, greater than about 600-fold, greater than about 700-fold, greater than about 800-fold, greater than about 900-fold, greater than about 1,000-fold, greater than about 10,000-fold, greater than about 20,000-fold, greater than about 30,000-fold, greater than about 50,000-fold, greater than about 70,000-fold, greater than about 100,000-fold, or greater than about 1,000,000-fold greater expansion.

[0157] In other cases, the agent may increase the growth of the δ1 and δ4 populations over a period of 1 day to 90 days in culture (e.g., from about 1 day to about 19, 21, or 23 days in culture) relative to the δ2 ​​T cell population; relative to another γδ T cell subpopulation; relative to the starting number of γδ T cells before expansion; relative to the starting number of γδ1 T cells before expansion; relative to the starting number of γδ1 and γδ3 T cells before expansion; or relative to the starting number of αβ T cells in culture. The cells may be stimulated at a faster rate such that the cell population is expanded by more than 10-fold, more than 100-fold, more than 200-fold, more than 300-fold, more than 400-fold, more than 500-fold, more than 600-fold, more than 700-fold, more than 800-fold, more than 900-fold, more than 1,000-fold, more than 10,000-fold, more than 20,000-fold, more than 30,000-fold, more than 50,000-fold, more than 70,000-fold, more than 100,000-fold or more than 1,000,000-fold.

[0158] In other cases, the agent may increase the growth of the δ1 and δ4 populations over a period of 1 day to 90 days in culture (e.g., from about 1 day to about 19, 21, or 23 days in culture) relative to the δ2 ​​T cell population; relative to another γδ T cell subpopulation; relative to the starting number of γδ T cells before expansion; relative to the starting number of γδ1 T cells before expansion; relative to the starting number of γδ1 and γδ4 T cells before expansion; or relative to the starting number of αβ T cells in culture. The cells may be stimulated at a faster rate such that the cell population is expanded by more than 10-fold, more than 100-fold, more than 200-fold, more than 300-fold, more than 400-fold, more than 500-fold, more than 600-fold, more than 700-fold, more than 800-fold, more than 900-fold, more than 1,000-fold, more than 10,000-fold, more than 20,000-fold, more than 30,000-fold, more than 50,000-fold, more than 70,000-fold, more than 100,000-fold or more than 1,000,000-fold.

[0159] In other cases, the agent may increase the growth of the δ1, δ3, δ4, and δ5 populations over a period of 1 day to 90 days of culture (e.g., from about 1 day to about 19, 21, or 23 days of culture) relative to the δ2 ​​T cell population; relative to another γδ T cell subpopulation; relative to the starting number of γδ T cells before expansion; relative to the starting number of γδ1 T cells before expansion; relative to the starting number of γδ1 and γδ3 T cells before expansion; relative to the starting number of γδ1, γδ3, γδ4, and γδ5 T cells before expansion. or at a faster rate such that the number of αβ T cells stimulated is greater than 10-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1,000-fold, 10,000-fold, 20,000-fold, 30,000-fold, 50,000-fold, 70,000-fold, 100,000-fold, or 1,000,000-fold greater than the αβ T cell population in culture.

[0160] In other cases, the agent may stimulate growth of the δ2 ​​population at a faster rate such that proliferation over a period of 1 day to 90 days of culture (e.g., from about 1 day to about 19, 21 or 23 days of culture) results in greater than 10-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1,000-fold, 10,000-fold, 20,000-fold, 30,000-fold, 50,000-fold, 70,000-fold, 100,000-fold or 1,000,000-fold greater expansion over a period of 1 day to 90 days of culture (e.g., from about 1 day to about 19, 21 or 23 days of culture) than the δ1 T cell population; than the δ3 T cell population; than another γδ T cell subpopulation; than the starting number of γδ T cells before expansion; than the starting number of γδ T cells before expansion; or than αβ T cells.

[0161] In some aspects, the disclosure provides engineered or non-engineered γδ T cell populations that have been contacted with an agent that stimulates proliferation of the γδ T cell population at a rapid rate, e.g., about one cell division every 30 hours or faster. In some cases, the agent selectively stimulates proliferation of either δ1; δ2; δ1 and δ4; or δ1, δ3, δ4, and δ5 T cells. The γδ T cell population can include an amount of non-engineered γδ T cells and an amount of engineered γδ T cells. In some cases, the γδ T cell population includes different percentages of δ1, δ2, δ3, and δ4 T cells. An engineered or non-engineered γδ T cell population may comprise, for example, less than 90% δ1 T cells, less than 80% δ1 T cells, less than 70% δ1 T cells, less than 60% δ1 T cells, less than 50% δ1 T cells, less than 40% δ1 T cells, less than 30% δ1 T cells, less than 20% δ1 T cells, less than 10% δ1 T cells or less than 5% δ1 T cells. Alternatively, an engineered or non-engineered γδ T cell population may comprise more than 5% δ1 T cells, more than 10% δ1 T cells, more than 20% δ1 T cells, more than 30% δ1 T cells, more than 40% δ1 T cells, more than 50% δ1 T cells, more than 60% δ1 T cells, more than 70% δ1 T cells, more than 80% δ1 T cells or more than 90% δ1 T cells. In some cases, the agent is one of the selective proliferation agents described herein. In some cases, the agent is immobilized on a surface, such as a cell culture surface or the surface of an APC (e.g., expressed on the surface of the APC or bound to an Fc receptor expressed on the surface of the APC).

[0162] An engineered or non-engineered γδ T cell population may, for example, comprise less than 90% δ2T cells, less than 80% δ2T cells, less than 70% δ2T cells, less than 60% δ2T cells, less than 50% δ2T cells, less than 40% δ2T cells, less than 30% δ2T cells, less than 20% δ2T cells, less than 10% δ2T cells or less than 5% δ2T cells. Alternatively, an engineered or non-engineered γδ T cell population may comprise more than 5% δ2T cells, more than 10% δ2T cells, more than 20% δ2T cells, more than 30% δ2T cells, more than 40% δ2T cells, more than 50% δ2T cells, more than 60% δ2T cells, more than 70% δ2T cells, more than 80% δ2T cells or more than 90% δ2T cells.

[0163] An engineered or non-engineered γδ T cell population may for example comprise less than 90% δ1 and δ4 T cells, less than 80% δ1 and δ4 T cells, less than 70% δ1 and δ4 T cells, less than 60% δ1 and δ4 T cells, less than 50% δ1 and δ4 T cells, less than 40% δ1 and δ4 T cells, less than 30% δ1 and δ4 T cells, less than 20% δ1 and δ4 T cells, less than 10% δ1 and δ4 T cells, or less than 5% δ1 and δ4 T cells. Alternatively, an engineered or non-engineered γδ T cell population may comprise more than 5% δ1 and δ4 T cells, more than 10% δ1 and δ4 T cells, more than 20% δ1 and δ4 T cells, more than 30% δ1 and δ4 T cells, more than 40% δ1 and δ4 T cells, more than 50% δ1 and δ4 T cells, more than 60% δ1 and δ4 T cells, more than 70% δ1 and δ4 T cells, more than 80% δ1 and δ4 T cells, or more than 90% δ1 and δ4 T cells.

[0164] An engineered or non-engineered γδ T cell population may comprise, for example, less than 90% δ4 T cells, less than 80% δ4 T cells, less than 70% δ4 T cells, less than 60% δ4 T cells, less than 50% δ4 T cells, less than 40% δ4 T cells, less than 30% δ4 T cells, less than 20% δ4 T cells, less than 10% δ4 T cells or less than 5% δ4 T cells. Alternatively, an engineered or non-engineered γδ T cell population may comprise more than 5% δ1 and δ4 T cells, more than 10% δ1 and δ4 T cells, more than 20% δ1 and δ4 T cells, more than 30% δ1 and δ4 T cells, more than 40% δ1 and δ4 T cells, more than 50% δ1 and δ4 T cells, more than 60% δ1 and δ4 T cells, more than 70% δ1 and δ4 T cells, more than 80% δ1 and δ4 T cells, or more than 90% δ1 and δ4 T cells. An engineered or non-engineered γδ T cell population may for example comprise less than 90% δ1 and δ4 T cells, less than 80% δ1 and δ4 T cells, less than 70% δ1 and δ4 T cells, less than 60% δ1 and δ4 T cells, less than 50% δ1 and δ4 T cells, less than 40% δ1 and δ4 T cells, less than 30% δ1 and δ4 T cells, less than 20% δ1 and δ4 T cells, less than 10% δ1 and δ4 T cells, or less than 5% δ1 and δ4 T cells.

[0165] In certain embodiments, the invention provides a mixture of expanded γδ T cell populations comprising 10-90% δ1 T cells and 90-10% δ2 T cells. In certain embodiments, the invention provides a mixture of expanded γδ T cell populations comprising 10-90% δ1 and δ3 T cells and 90-10% δ2 T cells. In certain embodiments, the invention provides a mixture of expanded γδ T cell populations comprising 10-90% δ1 and δ4 T cells and 90-10% δ2 T cells. In certain embodiments, the invention provides a mixture of expanded γδ T cell populations comprising 10-90% δ1, δ3, δ4, and δ5 T cells and 90-10% δ2 T cells.

[0166] One or more activators may contact a γδ T cell (e.g., an activator γδ T cell natural receptor), after which a costimulatory molecule may contact the γδ T cell to provide further stimulation and proliferate the γδ T cell. In some embodiments, the activators and / or costimulatory agents may be plant and non-plant derived lectins, monoclonal antibodies that activate γδ T cells, and other non-lectin / non-antibody agents. In other cases, the plant lectin may be concanavalin A (ConA), although other plant lectins, etc., may also be used. Other examples of lectins include peanut agglutinin protein (PNA), soybean agglutinin (SBA), Les culinaris agglutinin (LCA), Pisum sativum agglutinin (PSA), Helix pomatia agglutinin (HPA), Vicia graminea lectin (VGA), Phaseolus vulgaris erythroagglutinin (PHA-E), Phaseolus vulgaris leucoagglutinin (PHA-L), Sambucus nigra lectin (SNA, EBL), Maackia amurensis lectin II (MAL II), Sophora japonica agglutinin (SJA), Dolichos biflorus agglutinin (DBA), Lens culinaris agglutinin (LCA), and Wisteria floribunda lectin (WFA, WFL).

[0167] Non-limiting examples of activators and costimulatory molecules include any one or more antibodies selective for the delta or gamma chain or subtypes thereof described herein, such as antibodies 5A6.E9, B1, TS8.2, 15D, B6, B3, TS-1, gamma3.20, 7A5, IMMU510, R9.12, 11F2, or combinations thereof. Other examples of activators and costimulatory molecules include zoledronate, phorbol 12-myristate-13-acetate (TPA), mezerein, staphylococcal enterotoxin A (SEA), streptococcal protein A, or combinations thereof.

[0168] In other cases, the activating agent and / or costimulatory agent can be an antibody or ligand to αTCR, βTCR, γTCR, δTCR, CD277, CD28, CD46, CD81, CTLA4, ICOS, PD-1, CD30, NKG2D, NKG2A, HVEM, 4-1BB (CD137), OX40 (CD134), CD70, CD80, CD86, DAP, CD122, GITR, FcεRIγ, CD1, CD16, CD161, DNAX, accessory molecule-1 (DNAM-1), one or more NCRs (e.g., NKp30, NKp44, NKp46), SLAM, coxsackievirus and adenovirus receptors, or combinations thereof.

[0169] Manipulated γδT cells Engineered γδ T cells (see, e.g., Figure 1 ) can be generated by various methods known in the art. Engineered γδ T cells can be engineered to stably express a specific tumor recognition moiety. A polynucleotide encoding an expression cassette containing a tumor recognition moiety or another type of recognition moiety can be stably introduced into γδ T cells by a transposon / transposase system or a viral-based gene transfer system, such as a lentivirus or retrovirus system, or another suitable method, such as a viral delivery method including transfection, electroporation, transduction, lipofection, calcium phosphate (CaPO), nanoengineered materials such as Ormosil, adenovirus, retrovirus, lentivirus, adeno-associated virus, or another suitable method. An antigen-specific TCR, either αβ or γδ, can be introduced into an engineered γδ T cell by stably inserting a polynucleotide containing the genetic code for the antigen-specific TCR into the genome of the γδ T cell. A polynucleotide encoding a CAR having a tumor recognition moiety can be introduced into an engineered γδ T-cell by stably inserting the polynucleotide into the genome of the γδ T-cell. In some cases, the engineered tumor recognition moiety is an engineered T cell receptor, and the expression cassette integrated into the genome of the engineered γδ T-cell comprises a polynucleotide encoding an engineered TCR α (TCR alpha) gene, an engineered TCR β (TCR beta) gene, a TCR δ (TCR delta) gene, or an engineered TCR γ (TCR gamma) gene. In some cases, the expression cassette integrated into the genome of the engineered γδ T-cell comprises a polynucleotide encoding an antibody fragment or antigen-binding portion thereof. In some cases, the antibody fragment or antigen-binding fragment thereof is a polynucleotide encoding a whole antibody, an antibody fragment, a single-chain variable fragment (scFv), a single-domain antibody (sdAb), a Fab, a F(ab)2, an Fc, a light or heavy chain on an antibody, a variable or constant region of an antibody, or any combination thereof, that binds to a cell surface tumor antigen as part of a chimeric antigen receptor (CAR) construct, or a bispecific construct comprising a CAR and a T-cell receptor (TCR) or a CAR with an antibody directed against a different antigen.In some cases, the polynucleotide is derived from a human or another species. Antibody fragment or antigen-binding fragment polynucleotides derived from non-human species can be modified to improve their similarity to antibody variants naturally occurring in humans, and the antibody fragment or antigen-binding fragment can be partially or fully humanized. Antibody fragment or antigen-binding fragment polynucleotides can also be chimeric, such as mouse-human antibody chimeras. Engineered γδ T cells expressing a CAR can also be engineered to express a ligand for the antigen recognized by the tumor recognition moiety.

[0170] Various techniques known in the art can be used to introduce cloned or synthetically engineered nucleic acids containing the genetic code for tumor recognition moieties into specific locations within the genome of engineered γδ T cells. RNA-guided Cas9 nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) system, as described in WO201409370, WO2003087341, WO2014134412, and WO2011090804 (each of which is incorporated by reference in its entirety), can be used to achieve efficient genome engineering in γδ T cell(s). The techniques described herein can also be used to insert expression cassettes into genomic locations that simultaneously knock out one gene and knock in another. For example, a polynucleotide containing an expression cassette of the present disclosure can be inserted into a genomic region encoding an MHC gene. Such manipulation can result in the simultaneous knock-in of one or more genes, for example genes contained within an expression cassette, and the knock-out of another gene, for example the MHC locus.

[0171] In one case, a Sleeping Beauty transposon containing a nucleic acid encoding a tumor recognition moiety is introduced intracellularly into the γδ T-cells to be engineered. A mutant Sleeping Beauty transposase that provides enhanced integration compared to wild-type Sleeping Beauty, such as the transposase described in US 7,985,739 (incorporated herein by reference in its entirety), may be used to introduce the polynucleotide into the engineered γδ T-cells.

[0172] In some cases, viral methods are used to introduce a polynucleotide comprising a tumor recognition moiety into the genome of engineered γδ T cells. Many viral methods have been used for human gene therapy, such as those described in WO1993020221 (incorporated herein by reference in its entirety). Non-limiting examples of viral methods that can be used to engineer γδ T cells include retroviral, adenoviral, lentiviral, herpes simplex virus, vaccinia virus, poxvirus, or adenovirus-associated virus methods.

[0173] The polynucleotide containing the genetic code for the tumor recognition moiety can include a mutation or other transgene that affects the growth, proliferation, or activation state of the engineered γδ T cell, or a tumor cell-specific antigen, such as a testis-specific cancer antigen. The γδ T cells of the present disclosure can be engineered to express a polynucleotide containing an activation domain linked to the antigen recognition moiety, such as a molecule in the TCR-CD3 complex or a costimulatory factor. The engineered γδ T cell can express an intracellular signaling domain that is a T lymphocyte activation domain. The γδ T cell can be engineered to express an intracellular activation domain gene or an intracellular signaling domain. The intracellular signaling domain gene can be, for example, CD3ζ, CD28, CD2, ICOS, JAML, CD27, CD30, OX40, NKG2D, CD4, OX40 / CD134, 4-1BB / CD137, FcεRIγ, IL-2RB / CD122, IL-2RG / CD132, a DAP molecule, CD70, a cytokine receptor, CD40, or any combination thereof. In some cases, the engineered γδ T cells are further engineered to express cytokines, antigens, cell receptors, or other immunomodulatory molecules.

[0174] An appropriate tumor recognition moiety to be expressed on the engineered γδ T cells can be selected based on the disease to be treated. For example, in some cases, the tumor recognition moiety is a TCR. In some cases, the tumor recognition moiety is a receptor for a ligand expressed on cancer cells. Non-limiting examples of suitable receptors include NKG2D, NKG2A, NKG2C, NKG2F, LLT1, AICL, CD26, NKRP1, CD244 (2B4), DNAM-1, NKp30, NKp44, NKp46, and NKp80. In some cases, the tumor recognition moiety can comprise a ligand, e.g., an IL-13 ligand, or a ligand mimic for a tumor antigen, e.g., an IL-13 mimetic for IL13R.

[0175] γδ T cells can be engineered to express chimeric tumor-recognition moieties comprising ligand-binding domains derived from NKG2D, NKG2A, NKG2C, NKG2F, LLT1, AICL, CD26, NKRP1, CD244 (2B4), DNAM-1, or anti-tumor antibodies such as anti-Her2neu or anti-EGFR, and signaling domains obtained from CD3-zeta, Dap10, Dap12, CD28, 41BB, and CD40L. In some examples, the chimeric receptor is selected from the group consisting of MICA, MICB, Her2neu, EGFR, EGFRvIII, mesothelin, CD38, CD20, CD19, BCMA, PSA, RON, CD30, CD22, CD37, CD38, CD56, CD33, CD138, CD123, CD79b, CD70, CD75, CA6, GD2, alpha fetoprotein (AFP), CS1, carcinoembryonic antigen (CEA), CEACAM5, CA-125, MUC-16, 5T4, NaPi2b, ROR1, ROR2, PLIF, Her2 / Neu, EGFRvIII, GPMNB, LIV-1, glycolipid F77, fibroblast activation protein (FAP), PSMA, STEAP-1, STEAP-2, c-M The antibody binds to one or more antigens of et, CSPG4, CD44v6, PVRL-4, VEGFR2, C4.4a, PSCA, folate binding protein / receptor, SLC44A4, Cripto, CTAG1B, AXL, IL-13Rα2, IL-3R, EPHA3, SLTRK6, gp100, MART1, tyrosinase, SSX2, SSX4, NYESO-1, epithelial tumor antigen (ETA), MAGEA family genes (e.g., MAGEA3, MAGEA4), KKLC1, mutant ras (H, N, K), BRaf, p53, β-catenin, EGFRT790, MHC class I chain-related molecule A (MICA) or MHC class I chain-related molecule B (MICB), or HPV, CMV, or EBV.

[0176] In some cases, the tumor recognition moiety targets an MHC class I molecule (HLA-A, HLA-B, or HLA-C) complexed with a tumor-associated peptide. Methods and compositions for producing and using tumor recognition moieties that target tumor-associated peptides complexed with MHC class I molecules include those described, for example, in Weidanz et al., Int. Rev. Immunol. 30:328-40, 2011; Scheinberg et al., Oncotarget. 4(5):647-8, 2013; Cheever et al., Clin. Cancer Res. 15(17):5323-37, 2009; Dohan & Reiter Expert Rev Mol Med. 14:e6, 2012; Dao et al., Sci Transl Med. 2013 Mar 13;5(176):176ra33; US 9,540,448; and WO 2017 / 011804. In some embodiments, the targeted tumor-associated peptide of the peptide-MHC complex is a peptide of Wilms tumor protein 1 (WT1), human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 (CYP1B), KRAS, or BRAF.

[0177] Two or more tumor recognition moieties may be expressed in a γδ T-cell from genetically distinct, substantially distinct, or substantially identical αβ TCR polynucleotides stably expressed by the engineered γδ T-cell, or from genetically distinct αβ TCR polynucleotides stably integrated into the engineered γδ T-cell. In the case of genetically distinct αβ TCR(s), αβ TCR(s) recognizing different antigens associated with the same condition may be utilized. In one preferred embodiment, a γδ T-cell is engineered to express different TCRs of human or mouse origin from one or more expression cassettes that recognize the same antigen in the context of different MHC haplotypes. In another preferred embodiment, a γδ T-cell is engineered to express a TCR and two or more antibodies directed against the same or different peptides from a given antigen complexed with different MHC haplotypes. In some cases, expression of a single TCR by the engineered γδ T-cell promotes proper TCR pairing. Engineered γδ T cells expressing different TCRs can provide universal allogeneic engineered γδ T cells. In another preferred embodiment, γδ T cells are engineered to express one or more different antibodies directed against peptide-MHC complexes, each antibody directed against the same or different peptides complexed with the same or different MHC haplotypes. In some cases, the tumor recognition moiety can be an antibody that binds to the peptide-MHC complex.

[0178] γδ T cells can be engineered to express TCRs from one or more expression cassettes that recognize the same antigen in the context of different MHC haplotypes. In some cases, engineered γδ T cells are designed to express a single TCR or a TCR in combination with a CAR to minimize the possibility of TCR mispairing within the engineered cells. The tumor recognition moieties expressed from two or more expression cassettes preferably have different polynucleotide sequences, e.g., encode tumor recognition moieties that recognize different epitopes of the same target in the context of different HLA haplotypes. Engineered γδ T cells expressing such different TCRs or CARs can provide universal allogeneic engineered γδ T cells.

[0179] In some cases, γδ T cells are engineered to express one or more tumor recognition moieties. Two or more tumor recognition moieties may be expressed from genetically identical or substantially identical antigen-specific chimeric (CAR) polynucleotides engineered in the γδ T cells. Two or more tumor recognition moieties may be expressed from genetically distinct CAR polynucleotides engineered in the γδ T cells. The genetically distinct CAR(s) may be engineered to recognize different antigens associated with the same condition.

[0180] γδ T-cells may alternatively be bispecific. Bispecific engineered γδ T-cells can express two or more tumor recognition moieties. Bispecific engineered γδ T-cells can express both a TCR tumor recognition moiety and a CAR tumor recognition moiety. Bispecific engineered γδ T-cells can be designed to recognize different antigens associated with the same condition. Engineered γδ T-cells can express two or more CAR / TCR(s) bispecific polynucleotides that recognize the same or substantially the same antigen. Engineered γδ T-cells can express two or more CAR / TCR(s) bispecific constructs that recognize distinct antigens. In some cases, the bispecific constructs of the present disclosure bind to the activation and inactivation domains of target cells, thereby providing improved target specificity. A γδ T-cell can be engineered to express at least 1 tumor recognition moiety, at least 2 tumor recognition moieties, at least 3 tumor recognition moieties, at least 4 tumor recognition moieties, at least 5 tumor recognition moieties, at least 6 tumor recognition moieties, at least 7 tumor recognition moieties, at least 8 tumor recognition moieties, at least 9 tumor recognition moieties, at least 10 tumor recognition moieties, at least 11 tumor recognition moieties, at least 12 tumor recognition moieties, or another suitable number of tumor recognition moieties.

[0181] Proper TCR function can be enhanced by two functional zeta proteins containing ITAM motifs. Proper TCR function can also be enhanced by αβ or γδ activation domains, such as CD3ζ, CD28, CD2, CTLA4, ICOS, JAML, PD-1, CD27, CD30, 41-BB, OX40, NKG2D, HVEM, CD46, CD4, FcεRIγ, IL-2RB / CD122, IL-2RG / CD132, DAP molecules, and CD70. The expressed polynucleotide can contain genetic codes for a tumor recognition moiety, a linker moiety, and an activation domain. Translation of the polynucleotide by engineered γδ T cells can provide the tumor recognition moiety and activation domain connected by a protein linker. Typically, the linker contains amino acids that do not interfere with folding of the tumor recognition moiety and activation domain. The linker molecule can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some cases, at least 50%, at least 70%, or at least 90% of the amino acids in the linker are serine or glycine.

[0182] In some cases, the activation domain may contain one or more mutations. Suitable mutations may, for example, be mutations that make the activation domain constitutively active. Changing the identity of one or more nucleic acids changes the amino acid sequence of the translated amino acid. Nucleic acid mutations can be introduced to change the encoded amino acid to a polar, non-polar, basic, or acidic amino acid. Nucleic acid mutations can be introduced to optimize the tumor recognition moiety to recognize an epitope from a tumor. The engineered tumor recognition moiety, engineered activation domain, or another engineered component of a γδ T cell may have more than 1 amino acid mutation, 2 amino acid mutations, 3 amino acid mutations, 4 amino acid mutations, 5 amino acid mutations, 6 amino acid mutations, 7 amino acid mutations, 8 amino acid mutations, 9 amino acid mutations, 10 amino acid mutations, 11 amino acid mutations, 12 amino acid mutations, 13 amino acid mutations, 14 amino acid mutations, 15 amino acid mutations, 16 amino acid mutations, 17 amino acid mutations, 18 amino acid mutations, 19 amino acid mutations, 20 amino acid mutations, 21 amino acid mutations, 22 amino acid mutations, 23 amino acid mutations, 24 amino acid mutations, The amino acid sequence may include a 25 amino acid mutation, a 26 amino acid mutation, a 27 amino acid mutation, a 28 amino acid mutation, a 29 amino acid mutation, a 30 amino acid mutation, a 31 amino acid mutation, a 32 amino acid mutation, a 33 amino acid mutation, a 34 amino acid mutation, a 35 amino acid mutation, a 36 amino acid mutation, a 37 amino acid mutation, a 38 amino acid mutation, a 39 amino acid mutation, a 40 amino acid mutation, a 41 amino acid mutation, a 42 amino acid mutation, a 43 amino acid mutation, a 44 amino acid mutation, a 45 amino acid mutation, a 46 amino acid mutation, a 47 amino acid mutation, a 48 amino acid mutation, a 49 amino acid mutation, or a 50 amino acid mutation.

[0183] In some cases, the γδ T cells of the present disclosure do not express one or more MHC molecules. Deletion of one or more MHC loci in engineered γδ T cells can reduce the likelihood of the engineered γδ T cells being recognized by the host immune system. Human major histocompatibility complex (MHC) loci, known as the human leukocyte antigen (HLA) system, constitute a large family of genes expressed in antigen-presenting cells, including γδ T cells. HLA-A, HLA-B, and HLA-C molecules function to present intracellular peptides as antigens to antigen-presenting cells. HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR molecules function to present extracellular peptides as antigens to antigen-presenting cells. Some alleles of HLA genes have been associated with GVHD, autoimmune disorders, and cancer. The engineered γδ T cells described herein can be further engineered to lack or disrupt gene expression of one or more HLA genes. The engineered γδ T-cells described herein can be further engineered to lack or disrupt gene expression of one or more components of the MHC complex, for example, to completely delete one or more of the MHC genes, to delete specific exons, or to delete β2 microglobulin (B2m). Genetic ablation or disruption of at least one HLA gene can result in clinically therapeutic γδ T-cells that can be administered to subjects with any HLA haplotype without causing host-versus-graft disease. The engineered γδ T-cells described herein can serve as universal donors for human subjects with any HLA haplotype.

[0184] γδ T cells can be engineered to lack one or various HLA loci(s). The engineered γδ T cells can lack an HLA-A allele, an HLA-B allele, an HLA-C allele, an HLA-DR allele, an HLA-DQ allele, or an HLA-DP allele. In some cases, the HLA allele is associated with a human condition, such as an autoimmune condition. For example, the HLA-B27 allele is associated with arthritis and uveitis, the HLA-DR2 allele is associated with systemic lupus erythematosus and multiple sclerosis, the HLA-DR3 allele is associated with 21-hydroxylase deficiency, and HLA-DR4 is associated with rheumatoid arthritis and type 1 diabetes. For example, engineered γδ T cells lacking the HLA-B27 allele can be administered to a subject suffering from arthritis without being readily recognized by the subject's immune system. In some cases, deletion of one or more HLA loci results in engineered γδ T cells that are universal donors for any subject with any HLA haplotype.

[0185] In some cases, engineering a γδ T cell requires the deletion of a portion of the γδ T cell genome. In some cases, the deleted portion of the genome includes a portion of the MHC locus(s). In some cases, the engineered γδ T cell is derived from a wild-type human γδ T cell, and the MHC locus is an HLA locus. In some cases, the deleted portion of the genome includes a portion of a gene corresponding to a protein in the MHC complex. In some cases, the deleted portion of the genome includes the β2-microglobulin gene. In some cases, the deleted portion of the genome includes an immune checkpoint gene, such as PD-1, CTLA-4, LAG3, ICOS, BTLA, KIR, TIM3, A2aR, B7-H3, B7-H4, and CECAM-1. In some cases, the engineered γδ T cell can be engineered to express an activation domain that enhances T cell activation and cytotoxicity. Non-limiting examples of activation domains that can be expressed in engineered γδ T cells include CD2, ICOS, 4-1BB (CD137), OX40 (CD134), CD27, CD70, CD80, CD86, DAP molecules, CD122, GITR, and FcεRIγ.

[0186] Any portion of the genome of an engineered γδ T cell can be deleted to disrupt expression of endogenous γδ T cell genes. Non-limiting examples of genomic regions that can be deleted or disrupted in the genome of a γδ T cell include promoters, activators, enhancers, exons, introns, non-coding RNAs, microRNAs, small nuclear RNAs, variable number tandem repeats (VNTRs), short tandem repeats (STRs), SNP patterns, hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, or simple sequence repeats. In some cases, the deleted portion of the genome ranges from 1 to about 10 nucleic acids, 1 to about 100 nucleic acids, 1 to about 1,000 nucleic acids, 1 to about 10,000 nucleic acids, 1 to about 100,000 nucleic acids, 1 to about 1,000,000 nucleic acids, or other suitable ranges.

[0187] HLA gene expression in engineered γδ T cells can also be disrupted using various techniques known in the art. In some cases, locus-wide gene editing techniques are used to excise genes from the engineered γδ T cell genome or disrupt gene expression of at least one HLA locus in the engineered γδ T cell. Non-limiting examples of gene editing techniques that can be used to edit desired loci on the genome of engineered γδ T cells include clustered regularly interspaced short palindromic repeats (CRISPR)-Cas, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and meganuclease technologies, as described in WO201409370, WO2003087341, WO2014134412, and WO2011090804, each of which is incorporated by reference in its entirety.

[0188] γδ T-cells may be engineered from isolated, non-engineered γδ T-cells that already express a tumor recognition moiety. The engineered γδ T-cells can retain the tumor cell recognition moiety endogenously expressed in isolated wild-type γδ T-cells, for example, isolated from tumor-infiltrating lymphocytes of a tumor sample. In some cases, the wild-type γδ TCR is replaced with the tumor cell recognition moiety of the engineered γδ T-cell.

[0189] γδ T cells can be engineered to express one or more homing molecules, e.g., lymphocyte homing molecules. Homing molecules can be, for example, lymphocyte homing receptors or cell adhesion molecules. Homing molecules can help engineered γδ T cells migrate and infiltrate solid tumors, including targeted solid tumors, when administered to a subject. Non-limiting examples of homing receptors include members of the CCR family, e.g., CCR2, CCR4, CCR7, CCR8, CCR9, CCR10, CLA, CD44, CD103, CD62L, E-selectin, P-selectin, L-selectin, and integrins, e.g., VLA-4 and LFA-1. Non-limiting examples of cell adhesion molecules include ICAM, N-CAM, VCAM, PE-CAM, L1-CAM, nectins (PVRL1, PVRL2, PVRL3), LFA-1, integrin alphaXbeta2, alphavbeta7, macrophage-1 antigen, CLA-4, glycoprotein IIb / IIIa. Further examples of cell adhesion molecules include calcium-dependent molecules such as T-cadherin and antibodies against matrix metalloproteinases (MMPs), such as MMP9 or MMP2.

[0190] Steps involved in T cell maturation, activation, proliferation, and function can be regulated by costimulatory and inhibitory signals via immune checkpoint proteins. Immune checkpoints are innate costimulatory and inhibitory elements of the immune system. They help maintain self-tolerance and regulate the duration and magnitude of physiological immune responses to prevent tissue damage when the immune system responds to disease conditions, such as cellular transformation or infection. The balance between costimulatory and inhibitory signals used to control immune responses from either γδ T cells or αβ T cells can be regulated by immune checkpoint proteins. Immune checkpoint proteins, such as PD1 and CTLA4, are present on the surface of T cells and can be used to switch the immune response "on" or "off." Tumors can dysregulate checkpoint protein function as a mechanism of immune tolerance, particularly against T cells specific for tumor antigens. The engineered γδ T-cells of the present disclosure can be further engineered to lack one or more immune checkpoint locus(s), for example, PD-1, CTLA-4, LAG3, ICOS, BTLA, KIR, TIM3, A2aR, CEACAM1, B7-H3, and B7-H4. Alternatively, expression of endogenous immune checkpoint genes in the engineered γδ T-cells of the present disclosure can be disrupted by gene editing techniques.

[0191] The immunological checkpoint can be a molecule that regulates an inhibitory signaling pathway (e.g., CTLA4, PD1, and LAG3) or a molecule that regulates a stimulatory signaling pathway (e.g., ICOS) in the engineered γδ T cells of the present disclosure. Several proteins within the broad immunoglobulin superfamily can be ligands for immunological checkpoints. Non-limiting examples of immune checkpoint ligand proteins include B7-H4, ICOSL, PD-L1, PD-L2, MegaCD40L, MegaOX40L, and CD137L. In some cases, the immune checkpoint protein is an antigen expressed on tumors. In some cases, the immune checkpoint gene is the CTLA-4 gene. In some cases, the immune checkpoint gene is the PD-1 gene.

[0192] PD1 is an inhibitory receptor belonging to the CD28 / CTLA4 family and is expressed on activated T lymphocytes, B cells, monocytes, DCs, and T-regs. There are two known ligands for PD1, PD-L1 and PD-L2, which are expressed on T cells, APCs, and malignant cells and function to suppress autoreactive lymphocytes and the effector function of TAA-specific cytotoxic T lymphocytes (CTLs). Thus, engineered γδ T cells lacking PD1 can retain their cytotoxic activity regardless of the expression of PD-L1 and PD-L2 by tumor cells. In some cases, the engineered γδ T cells disclosed herein lack the PD-1 gene locus. In some cases, expression of the PD-1 gene in the engineered γδ T cells is disrupted by gene editing technology.

[0193] CTLA4 (cytotoxic T lymphocyte antigen 4) is also known as CD152 (differentiation antigen 152). Although CTLA4 shares sequence homology and ligands (CD80 / B7-1 and CD86 / B7-2) with the costimulatory molecule CD28, it differs in that it delivers inhibitory signals to T cells that express CTLA4 as a receptor. CTLA4 has a much higher overall affinity for both ligands and can outcompete CD28 for binding when ligand density is limited. CTLA4 binds to CD8 + It is often expressed on the surface of effector T cells and plays a functional role in the early activation stages of both naive and memory T cells. CTLA4 counteracts the activity of CD28 through its increased affinity for CD80 and CD86 during the early stages of T cell activation. The primary functions of CTLA4 include downregulating helper T cells and enhancing the immunosuppressive activity of regulatory T cells. In some cases, the engineered γδ T cells of the present disclosure lack the CTLA4 gene. In some cases, expression of the CTLA4 gene in the engineered γδ T cells is disrupted by gene editing techniques.

[0194] LAG3 (lymphocyte activation gene 3) is expressed on activated antigen-specific cytotoxic T cells and can enhance regulatory T cell function, independently of CD8 + It can suppress effector T cell activity. LAG3 is a CD4-like negative regulatory protein with high binding affinity to MHC class II proteins and is upregulated in some epithelial cancers, resulting in T cell proliferation and homeostatic tolerance. Mitigation of LAG-3 / class II interactions using a LAG-3-IG fusion protein can enhance anti-tumor immune responses. In some cases, the engineered γδ T cells of the present disclosure lack the LAG3 gene locus. In some cases, expression of the LAG3 gene in the engineered γδ T cells is disrupted by gene editing technology.

[0195] Phenotypes of unengineered and engineered γδ T cells Engineered γδ T cells can home to specific bodily locations in a subject's body. The migration and homing of engineered γδ T cells can depend on the combined expression and action of specific chemokines and / or adhesion molecules. The homing of engineered γδ T cells can be controlled by the interaction of chemokines with their receptors. For example, cytokines can influence the homing of engineered γδ T cells, including, but not limited to, CXCR3 (whose ligands are IP-10 / CXCL10 and 6Ckine / SLC / CCL21), CCR4+ CXCR5+ (receptors for RANTES, MIP-1α, and MIP-1β), CCR6+, and CCR7. In some cases, engineered γδ T cells can home to sites of inflammation and injury and diseased cells to exert repair functions. In some cases, engineered γδ T cells can home to cancer. In some cases, the engineered γδ T-cells can home to the thymus, bone marrow, skin, larynx, trachea, pleura, lungs, esophagus, abdomen, stomach, small intestine, large intestine, liver, pancreas, kidneys, urethra, bladder, testes, prostate, vas deferens, ovaries, uretus, mammary gland, parathyroid gland, spleen, or another site in a subject's body. The engineered γδ T-cells can express one or more homing moieties, e.g., specific TCR alleles and / or lymphocyte homing molecules.

[0196] The engineered γδ T-cells can have a particular phenotype, which can be expressed in terms of cell surface marker expression. Various types of γδ T-cells can be engineered as described herein. In a preferred embodiment, the engineered γδ T-cells are derived from humans, although the engineered γδ T-cells may also be derived from another source, such as mammalian or synthetic cells.

[0197] The immunophenotype of the expanded cell population can be determined using markers including, but not limited to, CD27, CD45RA, CD45RO, CCR7, and CD62L (Klebanoff et al., Immunol Rev. 211:214 2006). CD45RA is expressed on naive T lymphocytes and is replaced by CD45RO upon antigen encounter, but is re-expressed on subsequent effector cells (Michie et al., Nature 360, 264-265 (1992)). CD62L is a cell adhesion molecule that acts as a homing molecule for entry into secondary lymphoid tissues and is lost after T cell activation when T cells acquire effector function (Sallusto et al., Nature. 401:708 (1999)). CD27 is a costimulatory marker that is lost during T cell differentiation (Appay et al., Nat Med. 8:379 (2002); Klebanoff et al., Immunol Rev. 211:214 2006).

[0198] antigen The invention disclosed herein provides engineered γδ T cells expressing an antigen recognition moiety, where the antigen recognition moiety recognizes a disease-specific epitope. An antigen can be a molecule that elicits an immune response. This immune response can involve either antibody production or activation of specific cells capable of immune response, or both. An antigen can be, for example, a peptide, protein, hapten, lipid, carbohydrate, bacterium, pathogen, or virus. An antigen can be a tumor antigen. A tumor epitope can be presented on the surface of tumor cells by MHC1 or MHCII complexes. An epitope can be a portion of an antigen that is expressed on the cell surface and recognized by the tumor recognition moiety.

[0199] Non-limiting examples of antigens recognized by engineered γδ T cells include CD19, CD20, CD30, CD22, CD37, CD38, CD56, CD33, CD138, CD123, CD79b, CD70, CD75, CA6, GD2, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), RON, CEACAM5, CA-125, MUC-16, 5T4, NaPi2b, ROR1, ROR2, PLIF, Her2 / Neu, EGFRvIII, GPMNB, LIV-1, glycolipid F77, fibroblast activation protein (FAP), PSMA, STEAP-1, STEAP-2, mesothelin, c-Met, CSPG4, and PVRL-4. , VEGFR2, PSCA, CLEC12a, L1CAM, GPC2, GPC3, folate binding protein / receptor, SLC44A4, Cripto, CTAG1B, AXL, IL-13R, IL-3Rα2, SLTRK6, gp100, MART1, tyrosinase, SSX2, SSX4, NYESO-1, WT-1, PRAME, epithelial tumor antigen (ETA), MAGEA family genes (e.g., MAGEA3, MAGEA4), KKLC1, mutant ras, □□af, p53, MHC class I chain-related molecule A (MICA) or MHC class I chain-related molecule B (MICB), or one or more antigens of HPV, CMV, or EBV.

[0200] The antigen can be expressed in the intracellular or extracellular compartment of a cell, and the engineered γδ T cell can recognize an intracellular or extracellular tumor antigen. In some cases, the αβ TCR of the engineered γδ T cell recognizes a peptide derived from either an intracellular or extracellular tumor antigen. For example, the antigen can be a protein produced intracellularly or extracellularly by a cell infected with a virus, such as an HIV, EBV, CMV, or HPV protein. The antigen can also be a protein expressed intracellularly or extracellularly in a cancer cell.

[0201] The antigen recognition moiety can recognize antigens from cells under stress, such as cancer cells or cells infected with viruses. For example, the human MHC class I chain-related genes (MICA and MICB) are located within the HLA class I region of chromosome 6. The MICA and MICB proteins are considered markers of "stress" in human epithelia and act as ligands for cells expressing the general natural killer cell receptor (NKG2D). MICA and MICB can be highly expressed by cancer cells as stress markers. Engineered γδ T cells can recognize the MICA or MICB tumor epitope.

[0202] Tumor recognition moieties can be engineered to recognize antigens with a particular avidity. For example, tumor recognition moieties encoded by TCR or CAR constructs can be at least at least 10 fM, at least 100 fM, at least 1 picomolar (pM), at least 10 pM, at least 20 pM, at least 30 pM, at least 40 pM, at least 50 pM, at least 60 pM, at least 7 pM, at least 80 pM, at least 90 pM, at least 100 pM, at least 200 pM, at least 300 pM, at least 400 pM, at least 500 pM, at least 600 pM, at least 700 pM, at least 800 pM, at least 900 pM, at least 1 nanomolar (nM), at least 2 nM, at least 3 nM, at least 4 nM, at least 5 nM, at least 6 nM, at least 7 nM, at least 8 nM, at least 9 nM, at least 10 nM, at least 2 and / or at least 1 μM, at least 2 μM, at least 3 μM, at least 4 μM, at least 5 μM, at least 6 μM, at least 7 μM, at least 8 μM, at least 9 μM, at least 10 μM, at least 20 μM, at least 30 μM, at least 40 nM, at least 50 nM, at least 60 nM, at least 70 nM, at least 80 nM, at least 90 nM, at least 100 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, at least 1 μM, at least 2 μM, at least 3 μM, at least 4 μM, at least 5 μM, at least 6 μM, at least 7 μM, at least 8 μM, at least 9 μM, at least 10 μM, at least 20 μM, at least 30 μM, at least 40 μM, at least 50 μM, at least 60 μM, at least 70 μM, at least 80 μM, at least 90 μM or at least 100 μM.

[0203] In some cases, the tumor recognition moiety is 10 fM or less, 100 fM or less, 1 picomolar (pM) or less, 10 pM or less, 20 pM or less, 30 pM or less, 40 pM or less, 50 pM or less, 60 pM or less, 7 pM or less, 80 pM or less, 90 pM or less, 100 pM or less, 200 pM or less, 300 pM or less, 400 pM or less, 500 pM or less, 600 pM or less, 700 pM or less, 800 pM or less, 900 pM or less, 1 nanomolar (nM) or less, 2 nM or less, 3 nM or less, 4 nM or less, 5 nM or less, 6 nM or less, 7 nM or less, 8 nM or less, 9 nM or less, 10 nM or less, 20 nM or less, 30 nM or less, 4 The antibody may be engineered to recognize an antigen with a dissociation constant of 0 nM or less, 50 nM or less, 60 nM or less, 70 nM or less, 80 nM or less, 90 nM or less, 100 nM or less, 200 nM or less, 300 nM or less, 400 nM or less, 500 nM or less, 600 nM or less, 700 nM or less, 800 nM or less, 900 nM or less, 1 μM or less, 2 μM or less, 3 μM or less, 4 μM or less, 5 μM or less, 6 μM or less, 7 μM or less, 8 μM or less, 9 μM or less, 10 μM or less, 20 μM or less, 30 μM or less, 40 μM or less, 50 μM or less, 60 μM or less, 70 μM or less, 80 μM or less, 90 μM or less, or 100 μM or less.

[0204] New activator The present inventors have identified activating agents that bind to specific subtypes of γδ TCRs and thus activate specific populations of γδ T cells. In one aspect, the present invention provides novel activating agents that bind to novel activating epitopes identified and described in Examples 14 and 39 and Figures 23-30 and 33-36 herein. Activating agents include, but are not limited to, MAbs TS8.2, TS-1, 15D, B6, R9.12, δ1-05, δ1-08, δ1-18, δ1-22, δ1-23, δ1-26, δ1-35, δ1-37, δ1-39, δ1-113, δ1-143, δ1-149, δ1-155, δ1-182, δ1-183, δ1-191, δ1-192, δ1-19 5, δ1-197, δ1-199, δ1-201, δ1-203, δ1-239, δ1-253, δ1-257, δ1-278, δ1-282, δ1-285, δ2-14, δ2-17, δ2-22, δ2-30, δ2-31, δ2-32, δ2-33, δ2-35, δ2-36 and δ2-37.

[0205] These activators further include, but are not limited to, TS8.2, TS-1, 15D, B6, R9.12, δ1-05, δ1-08, δ1-18, δ1-22, δ1-23, δ1-26, δ1-35, δ1-37, δ1-39, δ1-113, δ1-143, δ1-149, δ1-155, δ1-182, δ1-183, δ1-191, δ1-192, δ1-195, δ1-197, δ1-199, Included are activators that bind to the same epitope as or compete with one or more MAbs selected from the group consisting of δ1-201, δ1-203, δ1-239, δ1-253, δ1-257, δ1-278, δ1-282, δ1-285, δ2-14, δ2-17, δ2-22, δ2-30, δ2-31, δ2-32, δ2-33, δ2-35, δ2-36 and δ2-37.

[0206] These activators further include, but are not limited to, TS8.2, TS-1, 15D, B6, R9.12, δ1-05, δ1-08, δ1-18, δ1-22, δ1-23, δ1-26, δ1-35, δ1-37, δ1-39, δ1-113, δ1-143, δ1-149, δ1-155, δ1-182, δ1-183, δ1-191, δ1-192, δ1-195, δ1-197, δ1- 199, δ1-201, δ1-203, δ1-239, δ1-253, δ1-257, δ1-278, δ1-282, δ1-285, δ2-14, δ2-17, δ2-22, δ2-30, δ2-31, δ2-32, δ2-33, δ2-35, δ2-36 and δ2-37.

[0207] The present invention further provides (i) TS8.2, TS-1, 15D, B6, R9.12, δ1-05, δ1-08, δ1-18, δ1-22, δ1-23, δ1-26, δ1-35, δ1-37, δ1-39, δ1-113, δ1-1 43, δ1-149, δ1-155, δ1-182, δ1-183, δ1-191, δ1-192, δ1-195, δ1-197, δ1-199, δ1-201, δ1-203, δ1-239, δ1-253, δ1-257, Nucleic acids are provided that encode an activator that contains the complementarity-determining regions (CDRs) and / or variable regions of a MAb selected from the group consisting of δ1-278, δ1-282, δ1-285, δ2-14, δ2-17, δ2-22, δ2-30, δ2-31, δ2-32, δ2-33, δ2-35, δ2-36, and δ2-37; (ii) binds to the same epitope as or competes with the MAb; or (iii) is the MAb. In some cases, the nucleic acid is in a host cell (e.g., a heterologous host cell). In some cases, the nucleic acid is operably linked to a heterologous promoter or to a nucleic acid encoding a heterologous polypeptide. As used herein, the term "heterologous" refers to two components that do not naturally occur together.

[0208] In certain embodiments, the activating agent (e.g., an antibody) expands or activates one or more γδ T cell populations (e.g., δ1 T cells or δ2 T cells). In certain embodiments, the activating agent selectively activates δ1 and δ3 T cells. In certain embodiments, the activating agent selectively activates δ1 and δ4 T cells. In certain embodiments, the activating agent selectively activates δ1 T cells. In certain embodiments, the activating agent selectively activates δ1, δ3, δ5, and δ5 T cells. In certain embodiments, the activating agent selectively activates δ2 T cells.

[0209] The present invention further provides methods for producing one or more of the above activators, for example, by culturing a host cell containing nucleic acid encoding the activator to produce one or more of the above activators.

[0210] APC Further described herein are APCs for the expansion of engineered or non-engineered γδ T cells, e.g., one or more subpopulations of γδ T cells. In some embodiments, described herein are APCs that contain heterologous nucleic acid encoding one or more of the above-mentioned activating agents. In some embodiments, described herein are APCs that express one or more of the above-mentioned activating agents on their cell surface. In some embodiments, described herein are APCs that express one or more Fc receptors on their cell surface, wherein the Fc receptor(s) are in contact with and / or bind to one or more of the above-mentioned activating agents.

[0211] In some cases, APCs (e.g., APCs expressing one or more of the above-described activating agents on their cell surface or binding to Fc receptors expressed on their cell surface) do not express or exhibit reduced expression of HLA class I, HLA class I invariant chain, and / or HLA-DM. In some cases, APCs express adhesion molecules, such as adhesion molecule-1, CD11a, CD18, CD54, and / or leukocyte function-associated antigen-3. In some cases, APCs express Fc receptors, e.g., Fc receptors specific for the isotype of an activating agent used in the γδ T cell expansion methods described herein. In some cases, APCs express one or more Fc receptors selected from the group consisting of CD64, CD32A, CD32B, CD32C, CD16A, CD16B, FcRn, TRIM21, or CD307, or engineered variants thereof with higher affinity or altered specificity.

[0212] Further described herein are cultures comprising one or more of the above-described APCs. The cultures may further comprise expanded or unexpanded, engineered or non-engineered γδ T cells. The cultures may additionally or alternatively comprise a selective or non-selective γδ T cell activator, including any one of the γδ T cell activators described herein. In some cases, the cultures do not contain IL-21. In some cases, the cultures do not contain IL-4, IL-2, or IL-15, or a combination thereof. In some cases, the cultures do not contain cytokines that selectively expand a subpopulation of γδ T cells.

[0213] Epitope identification The inventors of the present invention have demonstrated that the γδ TCR activating MAbs TS8.2, TS-1, 15D, B6, R9.12, δ1-05, δ1-08, δ1-18, δ1-22, δ1-23, δ1-26, δ1-35, δ1-37, δ1-39, δ1-113, δ1-143, δ1-149, δ1-155, δ1-182, δ1-183, δ1-191, δ1-192, δ1-195, δ1- 197, δ1-199, δ1-201, δ1-203, δ1-239, δ1-253, δ1-257, δ1-278, δ1-282, δ1-285, δ2-14, δ2-17, δ2-22, δ2-30, δ2-31, δ2-32, δ2-33, δ2-35, δ2-36 and δ2-37 epitopes were identified. Exemplary epitopes include, but are not limited to, γδ TCR activating MAbs TS8.2, TS-1, 15D, B6, R9.12, δ1-05, δ1-08, δ1-18, δ1-22, δ1-23, δ1-26, δ1-35, δ1-37 , δ1-39, δ1-113, δ1-143, δ1-149, δ1-155, δ1-182, δ1-183, δ1-191, δ1-192, δ1-195, δ In some embodiments, the epitope is a γδ TCR activating MAb TS8.2, TS-1, 15D, B6, R9.12, δ1-05, δ1-08, δ1-22, δ1-26, δ1-35, δ1-37, δ1-39, δ1 -113, δ1-143, δ1-149, δ1-155, δ1-182, δ1-183, δ1-191, δ1-192, δ1-195, δ1-197, An epitope that specifically binds to one or more of δ1-199, δ1-201, δ1-203, δ1-253, δ1-257, δ1-278, δ1-282, δ1-285, δ2-14, δ2-17, δ2-30, δ2-31, δ2-32, δ2-33, δ2-35, δ2-36 and δ2-37.

[0214] In one aspect, the present disclosure provides a method for identifying epitopes of agents that stimulate the proliferation of engineered and non-engineered γδ T cells at a rapid growth rate. The epitope may comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in a unique spatial conformation. Epitopes can be formed both from contiguous amino acids or from noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents.

[0215] Epitope localization can be performed to identify the linear or nonlinear, discontinuous amino acid sequence(s), i.e., epitope, that is (e.g., specifically) recognized by an activator of interest, such as the TS8.2, 15D, B6, TS-1, and R9.12 antibodies. A general approach to epitope localization may involve expressing the full-length polypeptide sequence recognized by the antibody or ligand of interest, as well as various fragments or truncated forms of the polypeptide sequence, often in a heterologous expression system. These various recombinant polypeptide sequences or fragments thereof (e.g., fused to an N-terminal protein (e.g., GFP)) can then be used to determine whether the antibody or ligand of interest can bind to one or more truncated forms of the polypeptide sequence.

[0216] In some embodiments, the recombinant polypeptide sequence is a chimera containing a fragment formed by joining two or more homologous parent polypeptides, where at least one parent polypeptide binds to an activator of interest and at least one parent polypeptide does not bind to the activator of interest. For example, a segment of a human δ1 chain gene can be joined with a segment of a homologous dolphin δ chain gene and tested for its ability to generate a chimeric TCR in a recombinant expression system. The resulting chimeric TCR gene, as indicated, for example, by detection on the cell surface with a pan-γδ TCR antibody, can then be tested for its ability to bind to an activator of interest. In another example, a segment of a human δ2 chain gene can be joined with a segment of a homologous macaque δ chain gene and tested for its ability to generate a chimeric TCR in a recombinant expression system. The resulting chimeric TCR gene, as indicated, for example, by detection on the cell surface with a pan-γδ TCR antibody, can then be tested for its ability to bind to an activator of interest.

[0217] By repeatedly truncating and creating recombinant polypeptide sequences with overlapping amino acid regions, it is possible to identify the region of a polypeptide sequence recognized by an antibody of interest (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996)). The method relies on the ability of an agent, such as an antibody of interest, to bind to sequences regenerated from an epitope library, e.g., an epitope library derived from a synthetic peptide array on a membrane support or a combinatorial phage display peptide library. The epitope library thus offers a variety of possibilities for screening antibodies. Furthermore, site-directed mutagenesis or random Ala scanning targeting one or more residues of the epitope can be performed to confirm the identity of the epitope.

[0218] Various possible modifications of the γδ T cell receptor (γδ TCR) can be synthetically designed as cDNA constructs and expressed in an appropriate system to generate a library of epitopes. For example, multiple Vδ1 gene segments with different Jδ regions, such as Jδ1, Jδ2, and Jδ3 gene segments, can be synthetically designed. Alternatively, Vδ2Jδ1 and Vδ3Jδ1 chains can be ordered as synthetic genes and cloned into an appropriate vector. Multiple synthetically cloned δ TCR chains, such as Vδ1Jδ1, Vδ1Jδ2, Vδ1Jδ3, Vδ1Jδ4, Vδ2, and Vδ3 chains, can be co-transfected into a host system together with synthetically cloned γ TCR chains, such as Vγ2, Vγ3, Vγ4, Vγ5, Vγ8, Vγ9, and Vγ10 synthetically designed gene segments. In other cases, delta TCR chains, such as V51J51, V51J52, V51J53, V51J54, V52 and V53 chains, can be amplified from total RNA extracted from human PBMCs or γδ T cells isolated from human normal and malignant tissues.

[0219] The host system can be any suitable expression system, such as 293 cells, insect cells, or a suitable in vitro translation system. The wide variety of possible recombinations of synthetically designed γδ T-cell segments transfected into the host system can result in more than 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 possible pairing combinations of γδ TCRs. Binding of an agent to one of the epitopes in the aforementioned library can be detected by contacting a labeled antibody, such as TS8.2, 15D, B6, TS-1, and R9.12, with the epitope in the library and detecting a signal from the label.

[0220] Computational algorithms have also been developed for epitope localization that have been shown to locate conformationally discontinuous epitopes. Conformational epitopes can be identified by determining the spatial conformation of amino acids using methods including, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. Some epitope localization methods, such as X-ray analysis of crystals of antigen:antibody complexes, can provide atomic resolution of epitopes. In other cases, computational combinatorial methods for epitope localization can be employed to model potential epitopes based on the sequence of an antibody, such as the TS-1 or TS8.2 antibody. In such cases, the antigen-binding portion of the antibody is sequenced, and a computational model is used to reconstruct and predict potential binding sites of the antibody.

[0221] In some cases, the present disclosure provides a method for determining epitopes of a γδ T cell receptor, the method comprising: (a) preparing a library of epitopes from the γδ T cell receptor; (b) contacting the library of epitopes with an antibody; and (b) identifying an amino acid sequence of at least one epitope in the library of epitopes that binds to the antibody. In some cases, the antibody is selected from the group consisting of TS8.2, 15D, B6, TS-1, and R9.12 antibodies. In some cases, the antibody is attached to a solid support. The library of epitopes can include sequences corresponding to continuous or discontinuous epitopes of a T cell receptor, such as a γTCR or a δTCR. In some cases, the library of epitopes includes fragments from the γδ T cell receptor ranging in length from about 10 amino acids to about 30 amino acids, from about 10 amino acids to about 20 amino acids, or from about 5 amino acids to about 12 amino acids. In some cases, the antibody is labeled, and the label is a radioactive molecule, a luminescent molecule, a fluorescent molecule, an enzyme, or biotin.

[0222] δ1 epitope bin In some embodiments, the epitope recognized (e.g., specifically) by an activating agent of interest is an epitope consisting of amino acids 47-70 (SKEMIFLIRQGSDEQNA) and J1 (TDKLIFGKGTRVTVEP) or J2 (LTAQLFFGKGTQLIVEP) of human V51, and the activating agent does not bind to epitopes containing a K120T mutation in J1 or J2. This epitope is referred to herein as the bin1 δ1 epitope. Examples of activators that bind to the bin1 δ1 epitope include, but are not limited to, TS-1 and δ1-18. The human V51 J region can vary depending on the (D)(J) rearrangement; an example of a V51 J region of the δ1 chain of a γδ TCR that has a bin1 δ1 epitope is: SKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP and another example of a Vδ1J region bin 1δ1 epitope of the δ1 chain of the γδ TCR is SKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGEAPSAWGKHLTAQLFFGKGTQLIVEP is.

[0223] In some cases, an activator that binds to the bin1 delta1 epitope is AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP It binds to δ1 of γδTCR having the sequence AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDSWDTRQMFFGTGIKLFVEP It does not bind to δ1 of γδTCR having the sequence

[0224] Activators that bind to the bin1δ1 epitope are AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGEAPSAWGKHLTAQLFFGKGTQLIVEP It can also bind to δ1 of γδTCR having the sequence

[0225] In some embodiments, the epitope recognized (e.g., specifically) by an activating agent of interest is an epitope consisting of amino acids 47-70 of human V51 (SKEMIFLIRQGSDEQNA) and J1 (TDKLIFGKGTRVTVEP), and the activating agent does not bind to an epitope containing a K120T mutation in J1. This epitope is referred to herein as the bin1bδ1 epitope. Examples of activators that bind to the bin1bδ1 epitope include, but are not limited to, δ1-37. An example of the V51J region of the δ1 chain of a γδ TCR that has the bin1bδ1 epitope is: SKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP is.

[0226] In some cases, an activator that binds to the bin 1b δ1 epitope is AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP It binds to the δ1 chain of the γδ TCR, which has the sequence AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDSWDTRQMFFGTGIKLFVEP It does not bind to the δ1 chain of γδTCR, which has the sequence

[0227] Activators that bind to the bin1bδ1 epitope are AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKS.GRYSVNFKKAAKSVALTISALQLEDSAKYFCALGEAPSAWGKHLTAQLFFGKGTQLIVEP It also does not bind to the δ1 chain of the γδTCR, which has the sequence

[0228] In some embodiments, the epitope recognized (e.g., specifically) by an activating agent of interest is an epitope consisting of amino acids 11-21 of human Vδ1 (VSMPVRKAVTL). This epitope is referred to herein as the bin2δ1 epitope. Examples of activators that bind to the bin2δ1 epitope include, but are not limited to, δ1-285.

[0229] In some cases, an activator that binds to the bin2 delta 1 epitope is AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP It binds to the δ1 chain of the γδ TCR, which has the sequence AQKVTQVQRAMSSQLGEAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEPRSQPHHTKPSVFVMKNGTNVACLVKEF It does not bind to the δ1 chain of γδTCR, which has the sequence

[0230] In some embodiments, the epitope recognized (e.g., specifically) by an activating agent of interest is an epitope consisting of amino acids 11-21 of human V51 (VSMPVRKAVTL), and the activating agent does not bind to an epitope containing an R16 mutation in V51, e.g., an R16N mutation. This epitope is referred to herein as the bin2b51 epitope. An example of an activating agent that binds to the bin2b51 epitope includes, but is not limited to, R9.12.

[0231] In some cases, an activator that binds to the bin 2b δ1 epitope is AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP It binds to the δ1 chain of the γδ TCR, which has the sequence AQKVTQAQSSVSMPVNKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP and / or does not bind to the δ1 chain of a γδ TCR having the sequence AQKVTQVQRAMSSQLGEAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEPRSQPHHTKPSVFVMKNGTNVACLVKEF It does not bind to the δ1 chain of γδTCR, which has the sequence

[0232] In some embodiments, the epitope recognized (e.g., specifically) by an activating agent of interest is an epitope consisting of amino acids 11-21 of human V51 (VSMPVRKAVTL), and activating agents that bind to this epitope also bind (cross-react) with 53, 54, and 55γδ TCRs. This epitope is referred to herein as the bin2cδ1 epitope. Examples of activating agents that bind to the bin2cδ1 epitope include, but are not limited to, 51-39.

[0233] In some cases, an activator that binds to the bin2cδ1 epitope is AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP It binds to the δ1 chain of the γδ TCR, which has the sequence AQKVTQVQRAMSSQLGEAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEPRSQPHHTKPSVFVMKNGTNVACLVKEF It does not bind to the δ1 chain of γδTCR, which has the sequence

[0234] In some embodiments, the epitope recognized (e.g., specifically) by an activating agent of interest is an epitope consisting of amino acids 80-95 of human V51 (FKKAAKSVALTISALQ) or amino acids 70-95 of human V51 (AKSGRYSVNFKKAAKSVALTISALQ). This epitope is referred to herein as the bin3δ1 epitope. Examples of activators that bind to the bin3δ1 epitope include, but are not limited to, δ1-08 and δ1-23.

[0235] In some cases, an activator that binds to the bin3 delta 1 epitope is AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP It binds to the δ1 chain of the γδ TCR, which has the sequence AQKVTQVQRAMSSQLGEAVTLSCQYETSLSWYDIFWYKQLPSGEMTFLIHQISSDQNAKNGRYSVNFQERHKFISLTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEPRSQPHHTKPSVFVMKNGTNVACLVKEFYPKD It does not bind to the δ1 chain of γδTCR, which has the sequence

[0236] In some embodiments, the epitope recognized (e.g., specifically) by an activator of interest is an epitope consisting of amino acids 1-11 (AQKVTQAQSSV) and J1 or J2 of human V51. This epitope is referred to herein as the bin4 51 epitope. In some cases, activators that bind to the bin4 51 epitope do not bind to epitopes containing a K120T mutation in J1 or J2. Examples of activators that bind to bin4 51 epitopes include, but are not limited to, 51-35 and 51-203.

[0237] In some cases, an activator that binds to the bin 4 delta 1 epitope is AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP It binds to the δ1 chain of the γδ TCR, which has the sequence AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDSWDTRQMFFGTGIKLFVEP It does not bind to the δ1 chain of γδTCR, which has the sequence

[0238] In some cases, an activator that binds to the bin 4 delta 1 epitope is AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGEAPSAWGKHLTAQLFFGKGTQLIVEP It binds to the δ1 chain of the γδTCR, which has the sequence:

[0239] In some embodiments, the epitope recognized (e.g., specifically) by an activating agent of interest is an epitope consisting of amino acids 28-47 (SWWSYYIFWYKQLPS) and J1 of human V51. This epitope is referred to herein as the bin5 δ1 epitope. Examples of activators that bind to the bin5 δ1 epitope include, but are not limited to, δ1-113, δ1-155, δ1-183, δ1-191, δ1-278, and δ1-282. An example of the V51J1 region of the δ1 chain of a γδ TCR that has a bin5 δ1 epitope is: SWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP is.

[0240] In some cases, an activator that binds to the bin5 delta 1 epitope is AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP It binds to the δ1 chain of the γδ TCR, which has the sequence AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGEAPSAWGKHLTAQLFFGKGTQLIVEP It does not bind to the δ1 chain of γδTCR, which has the sequence

[0241] In some embodiments, the epitope recognized (e.g., specifically) by an activating agent of interest is an epitope consisting of amino acids 21-28 (LNCLYETS) and J1 of human V51. This epitope is referred to herein as the bin651 epitope. Examples of activators that bind to the bin651 epitope include, but are not limited to, TS8.2 and 51-143. An example of the V51J1 region of the 51 chain of a γ5 TCR that has the bin651 epitope is: LNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP is.

[0242] In some cases, an activator that binds to the bin6 delta 1 epitope is AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP It binds to the δ1 chain of the γδ TCR, which has the sequence AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDSWDTRQMFFGTGIKLFVEP It does not bind to the δ1 chain of γδTCR, which has the sequence

[0243] In some embodiments, the epitope recognized (e.g., specifically) by an activating agent of interest is an epitope consisting of amino acids 47-70 (SKEMIFLIRQGSDEQNA) and J1 or J2 of human V51. This epitope is referred to herein as the bin7δ1 epitope. Examples of activators that bind to the bin7δ1 epitope include, but are not limited to, δ1-149, δ1-253, and δ1-257. An example of the V51J region of the δ1 chain of a γδ TCR that has a bin7δ1 epitope is: SKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP, and another example of a Vδ1J region bin 7 δ1 epitope of the δ1 chain of the γδ TCR is SKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGEAPSAWGKHLTAQLFFGKGTQLIVEP is.

[0244] In some cases, an activator that binds to the bin 7 delta 1 epitope is AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP It binds to the δ1 chain of the γδ TCR, which has the sequence AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDSWDTRQMFFGTGIKLFVEP It does not bind to the δ1 chain of γδTCR, which has the sequence

[0245] In some embodiments, the epitope recognized (e.g., specifically) by an activating agent of interest is an epitope consisting of amino acids 70-80 (AKSGRYSVNF) and J1 or J2 of human V51. This epitope is referred to herein as the bin8δ1 epitope. Examples of activators that bind to the bin8δ1 epitope include, but are not limited to, δ1-192. An example of the V51J region of the δ1 chain of a γδ TCR that has the bin8δ1 epitope is: AKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP and another example of a Vδ1J region bin 8 δ1 epitope of the δ1 chain of the γδ TCR is AKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGEAPSAWGKHLTAQLFFGKGTQLIVEP is.

[0246] In some cases, an activator that binds to the bin8 delta 1 epitope is AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP It binds to the δ1 chain of the γδ TCR, which has the sequence AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDSWDTRQMFFGTGIKLFVEP It does not bind to the δ1 chain of γδTCR, which has the sequence

[0247] In some embodiments, the epitope recognized (e.g., specifically) by an activating agent of interest is an epitope consisting of amino acids 80-95 of human Vδ1 (FKKAAKSVALTISALQ). This epitope is referred to herein as the bin9δ1 epitope. Examples of activators that bind to the bin9δ1 epitope include, but are not limited to, δ1-201.

[0248] In some cases, an activator that binds to the bin9 delta 1 epitope is AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP It binds to the δ1 chain of the γδ TCR, which has the sequence AQKVTQVQRAMSSQLGEAVTLSCQYETSLSWYDIFWYKQLPSGEMTFLIHQISSDQNAKNGRYSVNFQERHKFISLTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP It does not bind to the δ1 chain of γδTCR with the sequence

[0249] The δ1-specific antibodies described herein selectively bind to δ1-containing γδ TCRs over δ2-containing γδ TCRs. Thus, the δ1-specific antibodies may be used, for example, in AIELVPEHQTVPVSIGVPATLRCSMKGEAIGNYYINWYRKTQGNTMTFIYREKDIYGPGFKDNFQGDIDIAKNLAVLKILAPSERDEGSYYCACDPLGGPPDKLIFGKGTRVTVEP and / or γδTCRs containing said sequences.

[0250] δ2 epitope bin In some embodiments, the epitope recognized (e.g., specifically) by an activator of interest is an epitope consisting of amino acids 83-94 of human V52 (AKNLAVLKILAP). This epitope is referred to herein as the bin1 52 epitope. In some cases, activators that bind to the bin1 52 epitope do not bind to epitopes containing a K90 mutation in V52, such as a K90N mutation. Examples of activators that bind to the bin1 52 epitope include, but are not limited to, 52-17 and B6.

[0251] In some instances, an activator that binds to the bin 1 δ2 epitope is AIELVPEHQTVPVSIGVPATLRCSMKGEAIGNYYINWYRKTQGNTMTFIYREKDIYGPGFKDNFQGDIDIAKNLAVLKILAPSERDEGSYYCACDPLGGPPDKLIFGKGTRVTVEP It binds to the δ2 ​​chain of the γδ TCR, which has the sequence AIELVPEHQTVPVSIGVPATLRCSMKGEAIGNYYINWYRKTQGNTMTFIYREKDIYGPGFKDNFQGDIDFLNNQAVLNILEASERDEGSYYCACDPLGGPPDKLIFGKGTRVTVEP It does not bind to the δ2 ​​chain of γδTCR, which has the sequence

[0252] In some embodiments, the epitope recognized (e.g., specifically) by an activator of interest is an epitope consisting of amino acids 28-38 (EAIGNYY) of human V52. This epitope is referred to herein as the bin2 52 epitope. In some cases, an activator that binds to the bin2 52 epitope does not bind to an epitope containing a G35 mutation in V52, e.g., a G35S mutation. Examples of activators that bind to the bin2 52 epitope include, but are not limited to, 15D.

[0253] In some cases, an activator that binds to the bin 2 δ2 epitope is AIELVPEHQTVPVSIGVPATLRCSMKGEAIGNYYINWYRKTQGNTMTFIYREKDIYGPGFK.DNFQGDIDIAKNLAVLKILAPSERDEGSYYCACDPLGGPPDKLIFGKGTRVTVEP It binds to the δ2 ​​chain of the γδ TCR, which has the sequence AIELVPEHQTVPVSIGVPATLRCSMKGDSISNYYTFWYRRTPGNTMTLIYREGGTYGPGFEDNLQGEIDFLNNQAVLNILEASERDEGSYYCACDPLGGPPDKLIFGKGTRVTVEP It does not bind to the δ2 ​​chain of γδTCR, which has the sequence

[0254] In some embodiments, the epitope recognized (e.g., specifically) by an activating agent of interest is an epitope consisting of amino acids 72-83 (KDNFQGDIDIA) of human V52. This epitope is referred to herein as the bin3 52 epitope. Examples of activators that bind to the bin3 52 epitope include, but are not limited to, 52-32.

[0255] In some cases, an activator that binds to the bin3 delta 2 epitope is AIELVPEHQTVPVSIGVPATLRCSMKGEAIGNYYINWYRKTQGNTMTFIYREKDIYGPGFK.DNFQGDIDIAKNLAVLKILAPSERDEGSYYCACDPLGGPPDKLIFGKGTRVTVEP It binds to the δ2 ​​chain of the γδ TCR, which has the sequence AIELVPEHQTVPVSIGVPATLRCSMKGEAIGNYYINWYRKTQGNTMTFIYREKDIYGPGFEDNLQGEIDFLNNQAVLNILEASERDEGSYYCACDPLGGPPDKLIFGKGTRVTVEP It does not bind to the δ2 ​​chain of γδTCR, which has the sequence

[0256] In some embodiments, the epitope recognized (e.g., specifically) by an activating agent of interest is an epitope consisting of amino acids 1-27 of human V52 (AIELVPEHQTVPVSIGVPATLRCSMKG). This epitope is referred to herein as the bin4 52 epitope. Examples of activators that bind to the bin4 52 epitope include, but are not limited to, 52-14, 52-22, 52-30, 52-31, 52-36, and 52-37.

[0257] In some cases, an activator that binds to the bin 4 δ2 epitope is AIELVPEHQTVPVSIGVPATLRCSMKGEAIGNYYINWYRKTQGNTMTFIYREKDIYGPGFK.DNFQGDIDIAKNLAVLKILAPSERDEGSYYCACDPLGGPPDKLIFGKGTRVTVEP It binds to the δ2 ​​chain of the γδ TCR, which has the sequence AVTLVPQNQARSVSVGESVTLRCSMKGDSISNYYTFWYRRTPGNTMTLIYREGGTYGPGFEDNLQGEIDFLNNQAVLNILEASERDEGSYYCACDPLGGPPDKLIFGKGTRVTVEP It does not bind to the δ2 ​​chain of γδTCR, which has the sequence

[0258] The δ2-specific antibodies described herein bind preferentially to δ2-containing γδ TCRs over δ1-containing γδ TCRs. Thus, the δ2-specific antibodies may be used, for example, in AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP and / or γδTCRs containing said sequences.

[0259] In general, the δ1- and δ2-specific antibodies described herein recognize conformational epitopes associated with γδ TCRs. In some cases, the δ1- and δ2-specific antibodies described herein are specific for one or more pairs of γδ1- or γδ2-TCRs, respectively. For example, in some cases, the δ1-specific antibodies described herein are specific for γ8δ1 TCRs. In some cases, the δ1-specific antibodies described herein bind to γ8δ1 TCRs but not γ9δ1 TCRs.

[0260] Treatment method The pharmaceutical compositions described herein containing non-manipulated enriched γδ T cell populations, manipulated enriched γδ T cell populations, and / or mixtures thereof can be administered for prophylactic and / or therapeutic treatments. In therapeutic applications, the compositions can be administered to a subject already suffering from a disease or condition in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition. Non-manipulated enriched γδ T cell populations, manipulated enriched γδ T cell populations, and / or mixtures thereof can also be administered to reduce the likelihood of onset, contraction, or worsening of the condition. The effective amount of non-manipulated enriched γδ T cell populations, manipulated enriched γδ T cell populations, and / or mixtures thereof used for treatment can vary based on the severity and course of the disease or condition, previous therapies, the subject's health, weight, and / or response to drugs, and / or the judgment of the treating physician.

[0261] The non-manipulated enriched γδ T cell populations, manipulated enriched γδ T cell populations, and / or mixtures thereof of the present disclosure can be used to treat a subject in need of treatment for a condition. Examples of conditions include cancer, infectious diseases, autoimmune disorders, and sepsis. The subject can be a human, a non-human primate, such as chimpanzees and other apes and monkey species; a livestock animal, such as a cow, horse, sheep, goat, or pig; a domestic animal, such as a rabbit, dog, or cat; or a laboratory animal, such as a rodent, such as a rat, mouse, or guinea pig. The subject can be of any age. The subject can be, for example, an elderly adult, an adult, an adolescent, a juvenile, a child, a young child, or an infant.

[0262] A method for treating a condition (e.g., a disease) in a subject with an enriched γδ T cell population of the present invention may include administering to the subject a therapeutically effective amount of a non-manipulated enriched γδ T cell population, an engineered enriched γδ T cell population, and / or a mixture thereof. The enriched γδ T cell population and / or a mixture thereof of the present disclosure may be administered in various dosing regimens (e.g., timing, concentration, dosage, treatment interval, and / or dosage form). The subject may also be pretreated with, for example, chemotherapy, radiation, or a combination of both, before receiving the enriched γδ T cell population and / or a mixture thereof of the present disclosure. The non-manipulated enriched γδ T cell population, the engineered enriched γδ T cell population, and / or a mixture thereof may be administered to the subject in a first dosing regimen as part of the treatment, and the subject may be followed up to determine whether the treatment in the first regimen meets a given level of therapeutic efficacy. In some cases, the engineered γδ T cells or another engineered γδ T cell may be administered to the subject in a second dosing regimen. Figure 2 schematically illustrates a method for treating a subject. In a first operation 201, at least one engineered γδ T-cell is administered to a subject having or suspected of having a given condition (e.g., cancer). The engineered γδ T-cells may be administered in a first dosing regimen. In a second operation 202, the subject may be followed up by, for example, a healthcare provider (e.g., a treating physician or nurse). In some examples, the subject is followed up to determine or measure the effectiveness of the engineered γδ T-cells in treating the subject's condition. In some situations, the subject may be followed up to determine the in vivo expansion of a γδ T-cell population in the subject. Then, in a third operation 203, at least one other engineered γδ T-cell is administered to the subject in a second dosing regimen. The second dosing regimen may be the same as the first dosing regimen or may be different from the first dosing regimen. In some situations, the third operation 203 is not performed, for example, if the administration of the engineered γδ T-cells in the first operation 201 is found to be effective (e.g., a first administration may be sufficient to treat the condition). Due to the allogeneic and universal donor characteristics, a population of engineered γδ T-cells can be administered to a variety of subjects with different MHC haplotypes. The engineered γδ T-cells can be frozen or cryopreserved before being administered to a subject.

[0263] Enriched populations of γδ T cells (i.e., engineered or non-engineered) and / or mixtures thereof may be frozen or cryopreserved before administration to a subject. In certain embodiments, a population of engineered enriched γδ T cells may contain two or more cells expressing the same tumor recognition moiety, different tumor recognition moieties, or a combination of the same and different tumor recognition moieties. For example, a population of engineered enriched γδ T cells may contain several distinct engineered γδ T cells designed to recognize different antigens or different epitopes of the same antigen. For example, human cells suffering from melanoma may express the NY-ESO-1 oncogene. Infected cells within a human may process the NY-ESO-1 oncoprotein into smaller fragments and present various portions of the NY-ESO-1 protein for antigen recognition. A population of engineered enriched γδ T cells may contain various engineered γδ T cells expressing different tumor recognition moieties designed to recognize different portions of the NY-ESO-1 protein. FIG. 3 schematically illustrates a method of treating a subject with a population of engineered γδ T cells that recognize different epitopes of the melanoma antigen NY-ESO-1. In a first step 301, a population of engineered γδ T cells that recognize different epitopes of the same antigen is selected. For example, the population of engineered γδ T cells can include two or more cells expressing different tumor recognition moieties that recognize different portions of the NY-ESO-1 protein. In a second step 302, the population of engineered γδ T cells can be administered in a first dosing regimen. In a second step 303, the subject can be followed and evaluated, for example, by a healthcare provider (e.g., a treating physician or nurse).

[0264] The enriched γδ T cell populations of the present disclosure, i.e., non-engineered or engineered, and / or mixtures thereof, can be used to treat a variety of conditions. In some cases, the non-engineered enriched γδ T cell populations of the present disclosure, engineered enriched γδ T cell populations, and / or mixtures thereof can be used to treat cancer, including solid tumors and hematological malignancies.Non-limiting examples of cancer include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, neuroblastoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors, e.g., cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, optic tract hypothalamic glioma, breast cancer, bronchial adenoma, Burkitt's lymphoma, carcinoma of unknown primary, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, disease, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liposarcoma, liver cancer, lung cancer, including non-small cell and small cell lung cancer, lymphoma, leukemia, Macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma of bone, medulloblastoma, melanoma, mesothelioma, metastatic squamous neck cancer of unknown primary, oral cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myeloid leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, epithelial ovarian cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic islet cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal These include somatic germinoma, pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasm, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, cutaneous Merkel cell carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T-cell lymphoma, throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (gestational), cancer of unknown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0265] In some cases, the non-engineered enriched γδ T cell population, the engineered enriched γδ T cell population, and / or a mixture thereof of the present disclosure can be used to treat infectious diseases. Infectious diseases can be caused, for example, by pathogenic bacteria or viruses. Various pathogenic proteins, nucleic acids, lipids, or fragments thereof can be expressed in diseased cells. Antigen-presenting cells can internalize such pathogenic molecules, for example, by phagocytosis or receptor-mediated endocytosis, and present antigen fragments bound to appropriate MHC molecules. For example, various 9-mer fragments of pathogenic proteins can be presented by APCs. The engineered enriched γδ T cells of the present disclosure can recognize various antigens and antigen fragments of pathogenic bacteria or viruses. Non-limiting examples of pathogenic bacteria include a) the genus Bordetella, such as the species Bordetella pertussis; b) the genus Borrelia, such as the species Borrelia burgdorferi; c) the genus Brucelia, such as the species Brucella abortus, Brucella canis, Brucella meliterisis, and / or Brucella suis; d) the genus Campylobacter, such as the species Campylobacter jejuni; e) the genera Chlamydia and Chlamydophila, such as the species Chlamydia pneumonia, Chlamydia trachomatis, and / or Chlamydophila psittaci; f) the genus Clostridium, such as the species Clostridium botulinum, Clostridium difficile, Clostridium perfringens, and Clostridium tetani; g) the genus Corynebacterium, such as the species Corynebacterium diphtheria species; h) Enterococcus species, such as Enterococcus faecalis and / or Enterococcus faecium species; i) Escherichia species, such as Escherichia coli species; j) Francisella species, such as Francisella tularensis species; k) Haemophilus species, such as Haemophilus influenza species;l) the genus Helicobacter, for example the species Helicobacter pylori; m) the genus Legionella, for example the species Legionella pneumophila; n) the genus Leptospira, for example the species Leptospira interrogans; o) the genus Listeria, for example the species Listeria monocytogenes; p) the genus Mycobacterium, for example the species Mycobacterium leprae, mycobacterium tuberculosis, and / or mycobacterium ulcerans; q) the genus Mycoplasma, for example the species Mycoplasma pneumonia; r) the genus Neisseria, for example the species Neisseria gonorrhoeae and / or Neisseria meningitidia; s) the genus Pseudomonas, for example the species Pseudomonas aeruginosa; t) the genus Rickettsia, for example the species Rickettsia rickettsii; u) the genus Salmonella, for example Salmonella typhi and / or Salmonella Typhimurium species; v) Shigella genus, for example Shigella sonnei species; w) Staphylococcus genus, for example Staphylococcus aureus, Staphylococcus epidermidis, and / or Staphylococcus saprophyticus species; x) Streptpcoccus genus, for example Streptococcus agalactiae, Streptococcus pneumonia, and / or Streptococcus pyogenes species; y) Treponema genus, for example Treponema pallidum species; z) Vibrio genus, for example Vibrio cholera; and / or aa) Yersinia genus, for example Yersinia pestis species;

[0266] In some cases, the non-manipulated enriched γδ T cell populations of the present disclosure, the manipulated enriched γδ T cell populations, and / or mixtures thereof may be used to treat an infectious disease, which may be caused by a virus.Non-limiting examples of viruses can be found in the following viral families, with representative species: a) Adenoviridae, e.g., Adenovirus species; b) Herpesviridae, e.g., Herpes simplex type 1, Herpes simplex type 2, Varicella-zoster virus, Epstein-Barr virus, Human cytomegalovirus, Human herpesvirus type 8 species; c) Papillomaviridae, e.g., Human papillomavirus species; d) Polyomaviridae, e.g., BK virus , JC virus species; e) Poxviridae family, for example, smallpox species; f) Hepadnaviridae family, for example, hepatitis B virus species; g) Parvoviridae family, for example, human bocavirus, parvovirus B19 species; h) Astroviridae family, for example, human astrovirus species; i) Caliciviridae family, for example, Norwalk virus species; j) Flaviviridae family, for example, hepatitis C virus (HCV), yellow fever virus, dengue virus, West Nile virus species; k) l) the family Hepeviridae, for example the Hepatitis E virus species; m) the family Retroviridae, for example the Human Immunodeficiency Virus (HIV) species; n) the family Orthomyxoviridae, for example the Influenza virus species; o) the family Arenaviridae, for example the Guanarito virus, Junin virus, Lassa virus, Machupo virus, and / or Sabia virus species; p) the family Bunyaviridae, for example the Crimean-Congo hemorrhagic fever virus spp.; q) the family Filoviridae, such as Ebola virus and / or Marburg virus species; the family Paramyxoviridae, such as measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus and / or Nipah virus species; r) the genus Rhabdoviridae, such as rabies virus species; s) the family Reoviridae, such as rotavirus, orbivirus, coltivirus and / or bannavirus species. In some examples, the virus cannot be assigned to a viral family, such as hepatitis D.

[0267] In some cases, the non-engineered enriched γδ T cell population, the engineered enriched γδ T cell population, and / or a mixture thereof of the present disclosure may be used to treat an immune disease, such as an autoimmune disease. Inflammatory diseases, including autoimmune diseases, are also a class of diseases associated with B cell disorders. Examples of immune diseases or conditions, including autoimmune conditions, include rheumatoid arthritis, rheumatic fever, multiple sclerosis, experimental autoimmune encephalomyelitis, psoriasis, uveitis, diabetes mellitus, systemic lupus erythematosus (SLE), lupus nephritis, eczema, scleroderma, polymyositis / scleroderma, polymyositis / dermatomyositis, ulcerative proctitis, and the like. protitis), severe combined immunodeficiency (SCID), DiGeorge syndrome, ataxia-telangiectasia, seasonal allergies, perennial allergies, food allergies, anaphylaxis, mastocytosis, allergic rhinitis, atopic dermatitis, Parkinson's disease, Alzheimer's disease, hypersplenism, leukocyte adhesion deficiency, X-linked lymphoproliferative disease, X-linked agammaglobulinemia, selective immunoglobulin A deficiency, hyper-IgM syndrome, HIV, autoimmune lymphoproliferative syndrome, Wiskott-Aldrich syndrome, chronic granulomatous disease, common variable immunodeficiency (CVID), hyperimmunoglobulin E syndrome, Hashimoto's thyroid disease adenitis, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, polyglandular syndrome, bullous pemphigoid, Henoch-Schönlein purpura, poststreptococcal nephritis, erythema nodosum, erythema multiforme, gamma-associated nephropathy, Takayasu's arteritis, Addison's disease, sarcoidosis, ulcerative colitis, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangitis obliterans, Sjögren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, chronic active hepatitis, polychondritis, pemphigus vulgaris vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis / polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, fibrosing alveolitis, and cancer.

[0268] Treatment with the γδ T cell populations and / or mixtures thereof of the present disclosure can be provided to a subject before, during, or after the clinical onset of symptoms. Treatment can be provided to a subject 1 day, 1 week, 6 months, 12 months, or 2 years after the clinical onset of disease. Treatment can be provided to a subject 1 day, 1 week, 1 month, 6 months, 12 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more after the clinical onset of disease. Treatment can be provided to a subject less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after the clinical onset of disease. Treatment can also include treating humans in clinical trials. Treatment can include administering to a subject a non-manipulated enriched γδ T cell population, an engineered enriched γδ T cell population, and / or mixtures thereof of the present disclosure.

[0269] In some cases, administration of the non-engineered enriched γδ T cell population, the engineered enriched γδ T cell population, and / or a mixture thereof of the present disclosure to a subject modulates the activity of endogenous lymphocytes in the subject. In some cases, administration of the non-engineered enriched γδ T cell population, the engineered enriched γδ T cell population, and / or a mixture thereof to a subject can provide antigen to endogenous T cells and enhance the immune response. In some cases, memory T cells are CD4 + In some cases, memory T cells are CD8 + In some cases, administration of the un-engineered enriched γδ T cell population, the engineered enriched γδ T cell population, and / or a mixture thereof to a subject activates the cytotoxicity of another immune cell. In some cases, the other immune cell is a CD8 +In some cases, the other immune cells are T cells. In some cases, the other immune cells are natural killer T cells. In some cases, administration of the non-engineered enriched γδ T cell population, the engineered enriched γδ T cell population, and / or a mixture thereof to a subject suppresses regulatory T cells. In some cases, the regulatory T cells are Fox3+ Treg cells. In some cases, the regulatory T cells are Fox3- Treg cells. Non-limiting examples of cells whose activity can be modulated by the non-engineered enriched γδ T cell population, the engineered enriched γδ T cell population, and / or a mixture thereof of the present disclosure include hematopoietic stem cells, B cells, CD4, CD8, red blood cells, white blood cells, dendritic cells, e.g., dendritic antigen-presenting cells, leukocytes, macrophages, memory B cells, memory T cells, monocytes, natural killer cells, neutrophil granulocytes, T helper cells, and T killer cells.

[0270] During most bone marrow transplants, a combination of cyclophosphamide and total body irradiation is routinely employed to prevent rejection of the hematopoietic stem cells (HSCs) in the transplant by the subject's immune system. In some cases, ex vivo incubation of donor bone marrow with interleukin-2 (IL-2) is performed to enhance the production of killer lymphocytes in the bone marrow. Interleukin-2 (IL-2) is a cytokine required for the growth, proliferation, and differentiation of wild-type lymphocytes. Current studies of adoptively transferring γδ T cells into humans may require coadministration of γδ T cells with IL-2. However, both low- and high-dose IL-2 can have toxic side effects. IL-2 toxicity can manifest in multiple organs / systems, most notably the heart, lungs, kidneys, and central nervous system. In some cases, the present disclosure provides methods of administering to a subject a non-engineered enriched γδ T cell population, an engineered enriched γδ T cell population, and / or a mixture thereof without co-administration of a cytokine, e.g., IL-2, IL-15, IL-12, or IL-21. In some cases, the non-engineered enriched γδ T cell population, an engineered enriched γδ T cell population, and / or a mixture thereof can be administered to a subject without co-administration of IL-2. In some cases, the non-engineered enriched γδ T cell population, an engineered enriched γδ T cell population, and / or a mixture thereof is administered to a subject without co-administration of IL-2 during a procedure such as a bone marrow transplant.

[0271] Administration method One or multiple non-manipulated enriched γδ T cell populations, manipulated enriched γδ T cell populations, and / or mixtures thereof of the present invention can be administered to a subject in any order or simultaneously. If simultaneously, multiple non-manipulated enriched γδ T cell populations, manipulated enriched γδ T cell populations, and / or mixtures thereof of the present invention can be provided in the form of a single, unified dose, such as an intravenous injection, or in multiple doses, for example, multiple intravenous infusions, subcutaneous injections, or tablets. The non-manipulated enriched γδ T cell populations, manipulated enriched γδ T cell populations, and / or mixtures thereof of the present invention can be packaged together or separately in a single package or multiple packages. One or all of the non-manipulated enriched γδ T cell populations, manipulated enriched γδ T cell populations, and / or mixtures thereof of the present invention can be given in multiple doses. If not simultaneously, the timing between the multiple doses can vary from about one week, one month, two months, three months, four months, five months, six months, or even about one year. In some cases, the non-manipulated enriched γδ T cell population, the manipulated enriched γδ T cell population, and / or a mixture thereof of the present invention can be expanded in vivo in the subject's body after administration to the subject. The non-manipulated enriched γδ T cell population, the manipulated enriched γδ T cell population, and / or a mixture thereof can be frozen to provide cells for multiple treatments with the same cell preparation. The non-manipulated enriched γδ T cell population, the manipulated enriched γδ T cell population, and / or a mixture thereof of the present disclosure, and pharmaceutical compositions comprising the same, can be packaged as a kit. The kit may include instructions (e.g., instructions for use) for using the non-manipulated enriched γδ T cell population, the manipulated enriched γδ T cell population, and / or a mixture thereof, and pharmaceutical compositions comprising the same.

[0272] In some instances, a method for treating cancer comprises administering a therapeutically effective amount of a non-engineered enriched γδ T cell population, an engineered enriched γδ T cell population, and / or a mixture thereof to a subject, whereby the cancer is treated. In some embodiments, the therapeutically effective amount of the non-engineered enriched γδ T cell population, the engineered enriched γδ T cell population, and / or a mixture thereof is administered for at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In some embodiments, the therapeutically effective amount of the non-engineered enriched γδ T cell population, the engineered enriched γδ T cell population, and / or a mixture thereof is administered for at least 1 week. In some embodiments, the therapeutically effective amount of the non-manipulated enriched γδ T cell population, the manipulated enriched γδ T cell population and / or a mixture thereof is administered for at least two weeks.

[0273] The non-engineered enriched γδ T cell population, the engineered enriched γδ T cell population, and / or a mixture thereof described herein can be administered before, during, or after the onset of a disease or symptom, and the timing of administering a pharmaceutical composition containing the non-engineered enriched γδ T cell population, the engineered enriched γδ T cell population, and / or a mixture thereof can vary. For example, the non-engineered enriched γδ T cell population, the engineered enriched γδ T cell population, and / or a mixture thereof can be used as a prophylactic and can be administered continuously to a subject with a symptom or a tendency toward a disease to reduce the likelihood of the disease or symptom occurring. The non-engineered enriched γδ T cell population, the engineered enriched γδ T cell population, and / or a mixture thereof can be administered to a subject during the onset of symptoms or as soon as possible thereafter. Administration of the non-manipulated enriched γδ T cell population, the manipulated enriched γδ T cell population, and / or a mixture thereof can begin as soon as possible, such as until the onset of symptoms, within the first 3 hours after onset of symptoms, within the first 6 hours after onset of symptoms, within the first 24 hours after onset of symptoms, within 48 hours after onset of symptoms, or any time from onset of symptoms. The initial administration can be by any practical route, e.g., by any route described herein using any dosage form described herein. In some cases, the administration of the non-manipulated enriched γδ T cell population, the manipulated enriched γδ T cell population, and / or a mixture thereof of the present disclosure is intravenous. Single or multiple dosages of unmanipulated enriched γδ T cell populations, manipulated enriched γδ T cell populations, and / or mixtures thereof can be administered as soon as practicable after the onset of cancer, infection, immune disease, sepsis, or in conjunction with a bone marrow transplant, and for the length of time necessary to treat the immune disease, e.g., from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 1 month, or from about 1 month to about 3 months. For the treatment of cancer, single or multiple dosages of unmanipulated enriched γδ T cell populations, manipulated enriched γδ T cell populations, and / or mixtures thereof can be administered several years after the onset of cancer, before or after other treatments.In some examples, the non-engineered enriched γδ T cell population, the engineered enriched γδ T cell population, and / or mixtures thereof can be administered for at least about 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years. The length of treatment can vary from subject to subject.

[0274] Dosage The non-manipulated enriched γδ T cell populations, manipulated enriched γδ T cell populations, and / or mixtures thereof disclosed herein can be formulated as unit dosage forms suitable for single administration of precise dosages. In some cases, the unit dosage form includes additional lymphocytes. In unit dosage forms, the formulation is divided into unit doses containing appropriate amounts of one or more compounds. The unit dosages can be in the form of packages containing individual quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be in single-dose non-resealable containers. Multi-dose resealable containers can be used, for example, with or without preservatives. In some examples, the pharmaceutical composition does not contain preservatives. Formulations for parenteral injection can be provided in unit dosage forms, for example, ampoules, or in multi-dose containers with preservatives.

[0275] The non-engineered enriched γδ T cell populations, engineered enriched γδ T cell populations and / or mixtures thereof described herein may be cultured in a medium containing at least 5 cells, at least 10 cells, at least 20 cells, at least 30 cells, at least 40 cells, at least 50 cells, at least 60 cells, at least 70 cells, at least 80 cells, at least 90 cells, at least 100 cells, at least 200 cells, at least 300 cells, at least 400 cells, at least 500 cells, at least 600 cells, at least 700 cells, at least 800 cells, at least 900 cells, at least 1 × 10 3 Cells, at least 2 x 10 3 Cells, at least 3 x 10 3 Cells, at least 4 x 10 3 Cells, at least 5 x 10 3 Cells, at least 6 x 10 3 Cells, at least 7 x 10 3 Cells, at least 8 x 10 3 cells, at least 9 x 10 3 cells, at least 1 x 10 4 Cells, at least 2 x 10 4 Cells, at least 3 x 10 4 Cells, at least 4 x 10 4 Cells, at least 5 x 10 4 Cells, at least 6 x 10 4 Cells, at least 7 x 10 4 Cells, at least 8 x 10 4 cells, at least 9 x 10 4 cells, at least 1 x 10 5 Cells, at least 2 x 10 5 Cells, at least 3 x 10 5 Cells, at least 4 x 10 5 Cells, at least 5 x 10 5 Cells, at least 6 x 10 5 Cells, at least 7 x 10 5 Cells, at least 8 x 10 5 cells, at least 9 x 10 5 cells, at least 1 x 10 6 Cells, at least 2 x 10 6 Cells, at least 3 x 10 6Cells, at least 4 x 10 6 Cells, at least 5 x 10 6 Cells, at least 6 x 10 6 Cells, at least 7 x 10 6 Cells, at least 8 x 10 6 cells, at least 9 x 10 6 cells, at least 1 x 10 7 Cells, at least 2 x 10 7 Cells, at least 3 x 10 7 Cells, at least 4 x 10 7 Cells, at least 5 x 10 7 Cells, at least 6 x 10 7 Cells, at least 7 x 10 7 Cells, at least 8 x 10 7 cells, at least 9 x 10 7 cells, at least 1 x 10 8 Cells, at least 2 x 10 8 Cells, at least 3 x 10 8 Cells, at least 4 x 10 8 Cells, at least 5 x 10 8 Cells, at least 6 x 10 8 Cells, at least 7 x 10 8 Cells, at least 8 x 10 8 cells, at least 9 x 10 8 cells, at least 1 x 10 9 Cells or larger amounts may be present in the composition.

[0276] The therapeutically effective dose of the non-manipulated enriched γδ T cell population, the manipulated enriched γδ T cell population, and / or a mixture thereof of the present invention is about 1 cell to about 10 cells, about 1 cell to about 100 cells, about 1 cell to about 10 cells, about 1 cell to about 20 cells, about 1 cell to about 30 cells, about 1 cell to about 40 cells, about 1 cell to about 50 cells, about 1 cell to about 60 cells, about 1 cell to about 70 cells, about 1 cell to about 80 cells, about 1 cell to about 90 cells, about 1 cell to about 100 cells, about 1 cell to about 1 × 10 3 Cells, about 1 cell ~ about 2 x 10 3 Cells, about 1 cell ~ about 3 x 10 3 Cells, about 1 cell ~ about 4 x 10 3 Cells, about 1 cell ~ about 5 x 10 3Cells, approximately 1 cell to approximately 6 × 10⁶ 3 Cells, approximately 1 cell to approximately 7 × 10⁻⁶ cells 3 Cells, approximately 1 cell to approximately 8 × 10⁸ cells 3 Cells, approximately 1 cell to approximately 9 × 10⁻⁶ cells 3 Cells, approximately 1 cell to approximately 1 × 10⁻⁶ 4 Cells, approximately 1 cell to approximately 2 × 10⁻⁶ cells 4 Cells, approximately 1 cell to approximately 3 × 10⁻⁶ cells 4 Cells, approximately 1 cell to approximately 4 × 10⁻⁶ cells 4 Cells, approximately 1 cell to approximately 5 × 10⁻⁶ cells 4 Cells, approximately 1 cell to approximately 6 × 10⁶ 4 Cells, approximately 1 cell to approximately 7 × 10⁻⁶ cells 4 Cells, approximately 1 cell to approximately 8 × 10⁸ cells 4 Cells, approximately 1 cell to approximately 9 × 10⁻⁶ cells 4 Cells, approximately 1 cell to approximately 1 × 10⁻⁶ 5 Cells, approximately 1 cell to approximately 2 × 10⁻⁶ cells 5 Cells, approximately 1 cell to approximately 3 × 10⁻⁶ cells 5 Cells, approximately 1 cell to approximately 4 × 10⁻⁶ cells 5 Cells, approximately 1 cell to approximately 5 × 10⁻⁶ cells 5 Cells, approximately 1 cell to approximately 6 × 10⁶ 5 Cells, approximately 1 cell to approximately 7 × 10⁻⁶ cells 5 Cells, approximately 1 cell to approximately 8 × 10⁸ cells 5 Cells, approximately 1 cell to approximately 9 × 10⁻⁶ cells 5 Cells, approximately 1 cell to approximately 1 × 10⁻⁶ 6 Cells, approximately 1 cell to approximately 2 × 10⁻⁶ cells 6 Cells, approximately 1 cell to approximately 3 × 10⁻⁶ cells 6 Cells, approximately 1 cell to approximately 4 × 10⁻⁶ cells 6 Cells, approximately 1 cell to approximately 5 × 10⁻⁶ cells 6 Cells, approximately 1 cell to approximately 6 × 10⁶ 6 Cells, approximately 1 cell to approximately 7 × 10⁻⁶ cells 6 Cells, approximately 1 cell to approximately 8 × 10⁸ cells 6 Cells, approximately 1 cell to approximately 9 × 10⁻⁶ cells 6 Cells, approximately 1 cell to approximately 1 × 10⁻⁶ 7 Cells, approximately 1 cell to approximately 2 × 10⁻⁶ cells 7 Cells, approximately 1 cell to approximately 3 × 10⁻⁶ cells 7 Cells, approximately 1 cell to approximately 4 × 10⁻⁶ cells 7 Cells, approximately 1 cell to approximately 5 × 10⁻⁶ cells 7 Cells, approximately 1 cell to approximately 6 × 10⁶ 7 Cells, approximately 1 cell to approximately 7 × 10⁻⁶ cells 7 Cells, approximately 1 cell to approximately 8 × 10⁸ cells7 Cells, about 1 cell ~ about 9 x 10 7 Cells, about 1 cell ~ about 1 x 10 8 Cells, about 1 cell ~ about 2 x 10 8 Cells, about 1 cell ~ about 3 x 10 8 Cells, about 1 cell ~ about 4 x 10 8 Cells, about 1 cell ~ about 5 x 10 8 Cells, about 1 cell ~ about 6 x 10 8 Cells, about 1 cell ~ about 7 x 10 8 Cells, about 1 cell ~ about 8 x 10 8 Cells, about 1 cell ~ about 9 x 10 8 cells, or approximately 1 cell to approximately 1 x 10 9 It may be a cell.

[0277] In some cases, a therapeutically effective dose of the non-manipulated enriched γδ T cell population, the manipulated enriched γδ T cell population and / or mixtures thereof of the present invention is about 1×10 3 cells ~ approx. 2 x 10 3 cells, approximately 1 x 10 3 cells ~ approx. 3 x 10 3 cells, approximately 1 x 10 3 cells ~ approx. 4 x 10 3 cells, approximately 1 x 10 3 Cells ~ approx. 5 x 10 3 cells, approximately 1 x 10 3 cells ~ approx. 6 x 10 3 cells, approximately 1 x 10 3 cells ~ approx. 7 x 10 3 cells, approximately 1 x 10 3 cells ~ approx. 8 x 10 3 cells, approximately 1 x 10 3 cells ~ approx. 9 x 10 3 cells, approximately 1 x 10 3 cells ~ approx. 1 x 10 4 cells, approximately 1 x 10 3 cells ~ approx. 2 x 10 4 cells, approximately 1 x 10 3 cells ~ approx. 3 x 10 4 cells, approximately 1 x 10 3 cells ~ approx. 4 x 10 4 cells, approximately 1 x 10 3 Cells ~ approx. 5 x 10 4 cells, approximately 1 x 10 3 cells ~ approx. 6 x 10 4 cells, approximately 1 x 103 Cells ~ Approximately 7 × 10 4 Cells, approximately 1 × 10 3 Cells ~ Approximately 8 × 10 4 Cells, approximately 1 × 10 3 Cells ~ approximately 9 × 10 4 Cells, approximately 1 × 10 3 Cells ~ Approximately 1×10 5 Cells, approximately 1 × 10 3 Cells ~ approximately 2 × 10 5 Cells, approximately 1 × 10 3 Cells ~ Approximately 3 × 10 5 Cells, approximately 1 × 10 3 Cells ~ approximately 4 × 10 5 Cells, approximately 1 × 10 3 Cells ~ approximately 5 × 10 5 Cells, approximately 1 × 10 3 Cells ~ Approximately 6 × 10 5 Cells, approximately 1 × 10 3 Cells ~ Approximately 7 × 10 5 Cells, approximately 1 × 10 3 Cells ~ Approximately 8 × 10 5 Cells, approximately 1 × 10 3 Cells ~ approximately 9 × 10 5 Cells, approximately 1 × 10 3 Cells ~ Approximately 1×10 6 Cells, approximately 1 × 10 3 Cells ~ approximately 2 × 10 6 Cells, approximately 1 × 10 3 Cells ~ Approximately 3 × 10 6 Cells, approximately 1 × 10 3 Cells ~ approximately 4 × 10 6 Cells, approximately 1 × 10 3 Cells ~ approximately 5 × 10 6 Cells, approximately 1 × 10 3 Cells ~ Approximately 6 × 10 6 Cells, approximately 1 × 10 3 Cells ~ Approximately 7 × 10 6 Cells, approximately 1 × 10 3 Cells ~ Approximately 8 × 10 6 Cells, approximately 1 × 10 3 Cells ~ approximately 9 × 10 6 Cells, approximately 1 × 10 3 Cells ~ Approximately 1×10 7 Cells, approximately 1 × 10 3 Cells ~ approximately 2 × 10 7 Cells, approximately 1 × 10 3 Cells ~ Approximately 3 × 10 7cells, approximately 1 x 10 3 cells ~ approx. 4 x 10 7 cells, approximately 1 x 10 3 Cells ~ approx. 5 x 10 7 cells, approximately 1 x 10 3 cells ~ approx. 6 x 10 7 cells, approximately 1 x 10 3 cells ~ approx. 7 x 10 7 cells, approximately 1 x 10 3 cells ~ approx. 8 x 10 7 cells, approximately 1 x 10 3 cells ~ approx. 9 x 10 7 cells, approximately 1 x 10 3 cells ~ approx. 1 x 10 8 cells, approximately 1 x 10 3 cells ~ approx. 2 x 10 8 cells, approximately 1 x 10 3 cells ~ approx. 3 x 10 8 cells, approximately 1 x 10 3 cells ~ approx. 4 x 10 8 cells, approximately 1 x 10 3 Cells ~ approx. 5 x 10 8 cells, approximately 1 x 10 3 cells ~ approx. 6 x 10 8 cells, approximately 1 x 10 3 cells ~ approx. 7 x 10 8 cells, approximately 1 x 10 3 cells ~ approx. 8 x 10 8 cells, approximately 1 x 10 3 cells ~ approx. 9 x 10 8 cells, approximately 1 x 10 3 cells ~ approx. 1 x 10 9 It may be a cell.

[0278] In some cases, a therapeutically effective dose of the non-manipulated enriched γδ T cell population, the manipulated enriched γδ T cell population and / or mixtures thereof of the present invention is about 1×10 6 cells ~ approx. 2 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 3 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 4 x 10 6 cells, approximately 1 x 10 6 Cells ~ approx. 5 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 6 x 10 6 cells, approximately 1 x 106 Cells ~ Approximately 7 × 10 6 Cells, approximately 1 × 10 6 Cells ~ Approximately 8 × 10 6 Cells, approximately 1 × 10 6 Cells ~ approximately 9 × 10 6 Cells, approximately 1 × 10 6 Cells ~ Approximately 1×10 7 Cells, approximately 1 × 10 6 Cells ~ approximately 2 × 10 7 Cells, approximately 1 × 10 6 Cells ~ Approximately 3 × 10 7 Cells, approximately 1 × 10 6 Cells ~ approximately 4 × 10 7 Cells, approximately 1 × 10 6 Cells ~ approximately 5 × 10 7 Cells, approximately 1 × 10 6 Cells ~ Approximately 6 × 10 7 Cells, approximately 1 × 10 6 Cells ~ Approximately 7 × 10 7 Cells, approximately 1 × 10 6 Cells ~ Approximately 8 × 10 7 Cells, approximately 1 × 10 6 Cells ~ approximately 9 × 10 7 Cells, approximately 1 × 10 6 Cells ~ Approximately 1×10 8 Cells, approximately 1 × 10 6 Cells ~ approximately 2 × 10 8 Cells, approximately 1 × 10 6 Cells ~ Approximately 3 × 10 8 Cells, approximately 1 × 10 6 Cells ~ approximately 4 × 10 8 Cells, approximately 1 × 10 6 Cells ~ approximately 5 × 10 8 Cells, approximately 1 × 10 6 Cells ~ Approximately 6 × 10 8 Cells, approximately 1 × 10 6 Cells ~ Approximately 7 × 10 8 Cells, approximately 1 × 10 6 Cells ~ Approximately 8 × 10 8 Cells, approximately 1 × 10 6 Cells ~ approximately 9 × 10 8 Cells, approximately 1 × 10 6 Cells ~ Approximately 1×10 9 Cells, approximately 1 × 10 6 Cells ~ approximately 2 × 10 9 Cells, approximately 1 × 10 6 Cells ~ Approximately 3 × 10 9Cells, approximately 1 × 10 6 Cells ~ approximately 4 × 10 9 Cells, approximately 1 × 10 6 Cells ~ approximately 5 × 10 9 Cells, approximately 1 × 10 6 Cells ~ Approximately 6 × 10 9 Cells, approximately 1 × 10 6 Cells ~ Approximately 7 × 10 9 Cells, approximately 1 × 10 6 Cells ~ Approximately 8 × 10 9 Cells, approximately 1 × 10 6 Cells ~ approximately 9 × 10 9 Cells, approximately 1 × 10 7 Cells ~ Approximately 1×10 9 Cells, approximately 1 × 10 7 Cells ~ approximately 2 × 10 9 Cells, approximately 1 × 10 7 Cells ~ Approximately 3 × 10 9 Cells, approximately 1 × 10 7 Cells ~ approximately 4 × 10 9 Cells, approximately 1 × 10 7 Cells ~ approximately 5 × 10 9 Cells, approximately 1 × 10 7 Cells ~ Approximately 6 × 10 9 Cells, approximately 1 × 10 7 Cells ~ Approximately 7 × 10 9 Cells, approximately 1 × 10 7 Cells ~ Approximately 8 × 10 9 Cells, approximately 1 × 10 7 Cells ~ approximately 9 × 10 9 Cells, approximately 1 × 10 8 Cells ~ Approximately 1×10 9 Cells, approximately 1 × 10 8 Cells ~ approximately 2 × 10 9 Cells, approximately 1 × 10 8 Cells ~ Approximately 3 × 10 9 Cells, approximately 1 × 10 8 Cells ~ approximately 4 × 10 9 Cells, approximately 1 × 10 8 Cells ~ approximately 5 × 10 9 Cells, approximately 1 × 10 8 Cells ~ Approximately 6 × 10 9 Cells, approximately 1 × 10 8 Cells ~ Approximately 7 × 10 9 Cells, approximately 1 × 10 8 Cells ~ Approximately 8 × 10 9 Cells, approximately 1 × 10 8 Cells ~ approximately 9 × 109 cells, or approximately 1 x 10 9 cells ~ approx. 1 x 10 10 It may be a cell.

[0279] keep In some embodiments, enriched γδ T cell populations and / or mixtures thereof can be formulated in freezing medium and placed in a cryopreservation unit, such as a liquid nitrogen freezer (−195°C) or an ultra-low temperature freezer (−65°C, −80°C, or −120°C), for long-term storage of at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, or at least 5 years. The freezing medium can contain dimethyl sulfoxide (DMSO) and / or sodium chloride (NaCl) and / or dextrose and / or dextran sulfate and / or hydroxyethyl starch (HES) along with a physiological pH buffer to maintain a pH of about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5, about 7.5 to about 8.0, or about 6.5 to about 7.5. Cryopreserved γδ T cells can be thawed and further treated by stimulation with antibodies, proteins, peptides, and / or cytokines as described herein. Cryopreserved γδ T cells can be thawed and genetically modified by viral vectors (including retroviral and lentiviral vectors) or non-viral means (including RNA, DNA, and proteins) as described herein. Alternatively, non-manipulated γδ T cells can be expanded, genetically modified, and cryopreserved by the methods described herein.

[0280] In this manner, the engineered and / or non-engineered γδ T cells can be further cryopreserved to produce at least about 10 cells per mL of cryosol medium. 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or at least about 10 10Cell banks can be generated in quantities of at least 1, 5, 10, 100, 150, 200, or 500 vials of cells. Cryopreserved cell banks can retain their functionality and can be thawed for further stimulation and expansion. In some embodiments, thawed cells can be stimulated and expanded in a suitable closed vessel, such as a cell culture bag and / or a bioreactor, to generate large quantities of cells as an allogeneic cell product. Cryopreserved γδ T cells can maintain their biological function under frozen storage conditions for at least about 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 15 months, 18 months, 20 months, 24 months, 30 months, 36 months, 40 months, 50 months, or at least about 60 months. In some embodiments, no preservatives are used in the formulation. Cryopreserved γδ T cells can be thawed and infused into a large number of patients as an allogeneic stock cell product.

[0281] All publications and patents mentioned herein are incorporated by reference in their entirety for the purpose of describing and disclosing, for example, the constructs and methodology described therein that might be used in connection with the invention described herein. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the inventors described herein are not entitled to antedate such disclosure by virtue of prior invention or for any other reason.

[0282] The present invention is further illustrated by the following non-limiting examples. [Example]

[0283] Example 1. Isolation, digestion and culture of primary cells Primary human peripheral blood mononuclear cells (PBMCs) are collected from healthy donors using an apheresis machine. PBMCs are purified using a Ficoll-Paque™ PLUS system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) or a similar system. Cells are then resuspended in the appropriate growth medium.

[0284] Alternatively, peripheral blood primary human cells are collected from peripheral blood, umbilical cord blood, bone marrow, normal tissue, or diseased tissue, eg, cancer tissue.

[0285] Tissue from healthy donors Fresh tissue from healthy donors is received from The Cooperative Human Tissue Network (CHTN) and transported to the laboratory in RPMI-1640 medium. The tissue is cut into 1-3 mm pieces with a scalpel. 3 The tissue was then thinly sliced ​​into 100-μm sections. Two to five sections per well were placed in 2 mL of RPMI-1640 supplemented with GlutaMAX, 25 mM HEPES pH 7.2, 100 U / ml penicillin, 100 U / ml streptomycin, 10% human AB serum, and 100 IU / ml rhIL-2 in a 24-well plate (Costar), or digested as described below. The plates were incubated in a humidified incubator at 37°C and 5% CO2 in air. Cultures were examined every other day to monitor lymphocyte proliferation. Half of the medium was replaced in all wells every 7 days after initiation of culture. Lymphocytes were harvested when densely packed lymphocytes surrounded the periphery of the section or when the lymphocyte population from the digested tissue reached a suitable concentration as described below.

[0286] Enzymatic digestion of tissue Fresh tissue samples from healthy donors were received from The Cooperative Human Tissue Network (CHTN) and transported to the laboratory in RPMI-1640 medium. Lymphocytes were isolated by enzymatic digestion using the two-enzyme blend Liberase™ DL Research Grade (Sigma-Aldrich Co., St. Louis, MO) or Liberase™ Research Grade (Sigma-Aldrich Co., St. Louis, MO), or the Miltenyi Tumor Dissociation Kit (130-095-929) equipped with a gentleMACS dissociator.

[0287] Cut the tissue into 2-3 mm pieces. 3 The cells were cut into 1×10 sections and digested for 1 hour at 37°C and 5% CO2. The digested cell suspension was passed through a 40-micron filter, centrifuged, and washed with RPMI-1640 medium. The cells were counted and resuspended in RPMI medium (GIBCO BRL) supplemented with 10% human AB serum (Corning) and 100 IU / ml rhIL-2. The collected cell population was divided into 0.5–1×10 6 Cells were seeded at 1.5 × 10 cells / ml into 24-well tissue culture plates. 6 When the concentration exceeded 100 cells / ml, the cells were split into RPMI-IL2 containing medium.

[0288] Culture of tumor specimens Fresh tumor specimens from patients with primary and metastatic cancers, including those of the colon, breast, ovary, kidney, head and neck, oral cavity, pancreas, and liver, were received from The Cooperative Human Tissue Network (CHTN) and transported to the laboratory in RPMI medium. Tumor specimens were cut with a scalpel into 1-3 mm pieces. 3The cells were thinly sliced ​​into 100-μm sections. Two to five sections per well were placed in 2 mL of RPMI-1640 supplemented with GlutaMAX, 25 mM HEPES pH 7.2, 100 U / ml penicillin, 100 U / ml streptomycin, 10% human AB serum, and 100 IU / ml rhIL-2 in a 24-well plate (Costar). The plates were incubated in a humidified incubator at 37°C and 5% CO2 in air. Cultures were examined every other day to monitor lymphocyte proliferation. Half of the medium was replaced in all wells every 7 days after the initiation of culture. Lymphocytes were harvested when densely packed lymphocytes surrounded the periphery of the section.

[0289] Enzymatic digestion of fresh tumor specimens Fresh tumor specimens from patients with primary and metastatic cancers, including those of the colon, breast, ovary, kidney, head and neck, oral cavity, pancreas, and liver, were received from The Cooperative Human Tissue Network (CHTN) and transported to the laboratory in RPMI medium. Lymphocytes were isolated by enzymatic digestion using the enzyme blends Liberase™ DL Research Grade (Sigma Aldrich Co., St. Louis, MO), Liberase™ Research Grade (Sigma Aldrich Co., St. Louis, MO), or the Miltenyi Tumor Dissociation Kit (130-095-929) equipped with a gentleMACS dissociator. Tissues were cut into 2-3 mm pieces. 3 The cells were cut into 1×10 sections and digested for 1 hour at 37°C and 5% CO2 in air. The digested cell suspension was passed through a 40-micron filter, centrifuged, and washed with RPMI-1640 medium. The cells were counted and resuspended in RPMI medium (GIBCO BRL) supplemented with 10% human AB serum (Corning) and 100 IU / ml rhIL-2. The collected cell population was 0.5–1×10 6 Cells were seeded at 1.5 × 10 cells / ml into 24-well tissue culture plates. 6 When the concentration exceeded 100 cells / ml, the cells were split into RPMI-IL2 containing medium.

[0290] Cultivation of primary cells in typical serum-supplemented media PBMC populations were generated by isolation from buffy coats derived from healthy donors using Ficoll-Paque™ PLUS (GE Healthcare Bio-Sciences PA, USA). 1 × 10 PBMCs were cultured in 24-well tissue culture plates in RPMI-1640 (Corning CellGro) supplemented with 10% fetal bovine serum (Gibco), 2 mmol / L L-glutamine, 100 U / mL penicillin, 100 U / mL streptomycin, and 100 IU rhIL-2 / mL. 6 cells / mL of PBMCs were cultured.

[0291] Similar culture conditions can be used to grow primary human cells harvested from peripheral blood, umbilical cord blood, bone marrow, normal tissue, or diseased tissue, such as the cancer tissues mentioned above.

[0292] Cultivating primary cells in typical serum-free media PBMC populations were generated by isolation from buffy coats derived from healthy donors using Ficoll-Paque™ PLUS (GE Healthcare Bio-Sciences PA, USA). 1 × 10 PBMCs were cultured in 24-well tissue culture plates in CTS serum-free medium with CTS-OpTmizer supplements. 6 cells / mL of PBMCs were cultured.

[0293] Similar culture conditions can be used to grow primary human cells harvested from peripheral blood, umbilical cord blood, bone marrow, normal tissue, or diseased tissue, such as the cancer tissues mentioned above.

[0294] Example 2: Removal of adherent monocytes and macrophages and CD4+ and CD8+ αβ T cells PBMCs are collected by apheresis as described above, and red blood cells are removed by hypotonic treatment or density separation using Ficoll gradient centrifugation. Red blood cell-free PBMCs are incubated in large culture vessels such as 10- or 40-layer Cell Factory (Nunc) or roller bottles (Nunc). The adherent population, which includes macrophages and monocytes, typically remains attached to the surface of the cell culture vessel. Cell populations grown in suspension are enriched for γδ T cells. Approximately 10 8 , 10 9 or 10 10 PBMCs are incubated with iron-containing microbeads (e.g., Miltenyi Biotech microbeads) coated with anti-human CD4 and anti-human CD8. The incubated cell population is then passed through a magnetic field, where the CD4 + and CD8 + T cells are retained. The "flow-through" population is enriched for γδ T cells.

[0295] Example 3: Depletion of monocytes and macrophages PBMCs are collected by apheresis as previously described, and red blood cells are removed by hypotonic treatment or density separation using Ficoll gradient centrifugation. Red blood cell-free PBMCs are incubated in large culture vessels such as 10- or 40-layer Cell Factory (Nunc) or roller bottles (Nunc). Monocytes and macrophages are removed by passing the red blood cell-depleted PBMCs through a packed glass wool column. The "flow-through" cell population is enriched for γδ T cells for further processing.

[0296] Example 4: Enrichment of γδ T cells PBMCs are collected by apheresis as described above, and red blood cells are removed by hypotonic treatment or density separation using Ficoll gradient centrifugation. Red blood cell-free PBMCs are incubated in large culture vessels such as 10- or 40-layer Cell Factory (Nunc) or roller bottles (Nunc). Monocytes and macrophages are removed using either the method described in Example 2 or Example 3. Approximately 108 , 10 9 or 10 10 PBMCs are incubated with iron-containing microbeads (e.g., Miltenyi Biotech microbeads) coated with CD4 and CD8. The incubated cell population is then passed through a magnetic field, through which anti-human CD4 + and anti-human CD8 + T cells are retained. The "flow-through" cell population is enriched for γδ T cells. Unwanted cells, such as NK, γδ T cells, B cells, monocytes, and macrophages, are removed by immunomagnetic bead separation (e.g., Miltenyi Biotech AutoMACS system) using a cocktail of antibodies directed against unwanted cell types.

[0297] Alternatively, unwanted cell types are removed using a mouse anti-human αβ TCR (IP26, BW242 / 412) antibody, or one or more other antibodies directed against surface receptors on NK, αβ T cells, B cells, monocytes, or macrophages and attached to anti-mouse IgG microbeads (Miltenyi 130-048-401), or one or more tetrameric antibody complexes or bispecific antibodies directed against surface receptors on NK, αβ T cells, B cells, monocytes, or macrophages, or combinations thereof.

[0298] Antibody-mediated isolation of γδ T cells from primary cells In one example, natural γδ T cells are isolated from primary cultures by flow cytometry sorting based on positive (i.e., γδ TCR) or negative (i.e., αβ TCR, CD4, CD8, CD56) expression of cell surface markers.

[0299] Example 5. Isolation of γδ T cells from primary tumors γδ T cells were isolated according to the method detailed in Example 1. Briefly, fresh tumor specimens were obtained from the NCI Cooperative Human Tissue Network (CHTN). Colon adenocarcinoma metastasis to the liver (TIL1) and renal tumor (TIL2) were shipped in RPMI-1640 medium. Tumor tissue was cut into 2 mm sized pieces using a flat blade. 3 The tumors were minced into small pieces, followed by digestion with 2 mL of Liberase enzyme cocktail (Sigma Chemical Co., St. Louis, MO) and 3000 units of DNase in RPMI as described. After digestion, the tumors were filtered through a sterile gauze 40-micron nylon mesh and washed twice with RPMI-1640. Cells were counted and seeded into 24-well plates at 1 x 106 cells / mL in RPMI-1640 containing 10% human AB serum supplemented with L-glutamine and 100 U / mL rhIL-2. After 6 days of culture, tumor-infiltrating lymphocytes were harvested. The presence of γδ T lymphocytes was analyzed by flow cytometry using anti-δ1 TCR (FITC-conjugated anti-Vδ1 TS8.2, Thermo Fisher Scientific) and anti-Vδ2 B6 (Biolegend) antibodies. Data were analyzed using FlowJo software.

[0300] Figure 4 depicts a graph showing the growth of γδ1 and γδ2 lymphocytes isolated from a colon adenocarcinoma metastasis to the liver (TIL1) and a renal tumor (TIL2). These lymphocytes were shown to express CCR4 and CCR7 (data not shown). As shown in Figure 4, the Vδ1 subset was the predominant population isolated from both types of tumors.

[0301] Example 6. Stimulation and proliferation of γδ T cells γδ T cells are stimulated and expanded in serum-free media such as Ex-Vivo10 (Lonza 04-380Q), Ex-Vivo15 (Lonza 04-744Q), Ex-Vivo20 (Lonza 04-448Q), AIMV medium (ThermoFisher Scientific 12055091), or Optimizer CTS (ThermoFisher Scientific A1048501), containing cytokines (IL-2, IL-4, IL-7, IL-15, IL-12, IL-21, IL-23, or IL-33), growth factors (insulin and transferrin, insulin-like growth factors), albumin, lipids (cholesterol, lipid solution, lipid precursors), vitamins, copper, iron, selenium, protein hydrolysate, essential amino acids, non-essential amino acids, and shear protectant (Pluronic F-68).

[0302] The serum-free medium described in the Examples herein is capable of sustaining γδ T cell bioactivity while maintaining the biological functionality of γδ T cells in suspension culture (e.g., WAVE bioreactor). 5 ~2×10 7 It can also be supplemented with additives that support γδ T cell growth at high cell densities of cells / mL.

[0303] Additional additives that resulted in robust γδ T cell growth include, for example, calcium chloride, anhydrous, calcium nitrate, copper sulfate, pentahydrate, ferric citrate, ferric nitrate, ferrous sulfate, zinc sulfate, and / or putrescine.

[0304] To provide low levels of elemental components to replace serum, serum-free media was supplemented with trace metals including ammonium paramolybdate, vanadium, manganese, nickel, sodium selenate, sodium metasilicate nonahydrate, tin chloride, aluminum chloride, barium acetate, cadmium chloride, chromium chloride, cobalt, germanium dioxide, potassium bromide, potassium iodide, rubidium chloride, silver nitrate, sodium fluoride, and / or zirconium chloride.

[0305] Other components added to cell culture media that support robust growth of γδ T cells include, for example, adenosine, guanosine, cytidine, uridine, betaine, taurine, folinic acid, ethanolamine, linoleic acid, oleic acid, hydrocortisone, pyruvate, plant hydrolysates, yeast hydrolysates, and / or β-mercaptoethanol.

[0306] Vitamins that may be added to promote robust γδ T cell growth include, for example, biotin (B7), D-calcium pantothenate (B5), choline chloride, cyanocobalamin (B12), folic acid (B9), i-inositol (myo-inositol), niacinamide (B3), pyridoxal, monohydrochloride, pyridoxine, monohydrochloride (B6), riboflavin (B2), thiamine and / or thiamine monohydrochloride (B1).

[0307] Example 7: Characterization of expanded γδ T cells: immunophenotype Expanded T cell populations can be characterized, for example, by FACS staining for cell surface markers that distinguish between different populations. Cells were washed once with HEPES-buffered saline (HBSS) containing 2% fetal bovine serum, incubated with an appropriate amount of MAb for 30 minutes at 4°C, and washed again with HBSS. Briefly, CD2, CD3 (BioLegend, clone OKT3), CD4 (BioLegend clone OKT4), CD7, CD8 (BioLegend, clone RPAT8), CD11a, CD16, CD18, CD19, CD27, CD28, CD38, CD45RA, CD56, CD57, CD69, CD71, CD95, CD107, ICAM-1, MICA / B, NKG2D DR5, CCR1, CCR2, CCR3, CCR4, CCR5 CCR6, CCR7, CCR10, CXCR1, CXCR2, CXCR3, CXCR5, CXCR5, CXCR6, CXCR7, IL-2R, IL-7R, Ki67, L-selectin, VLA-4, JAML, PD1, PDL1, CTLA-4, Ox40, TCR 1 × 10 cells in a volume of 100 μl of FACS staining medium (FSM; HBSS containing 2% fetal bovine serum) containing fluoroisothiocyanate (FITC)- or phycoerythrin (PE)-conjugated MAbs directed against TCR Vδ1 (ThermoFisher Scientific, clone TS8.2) or TCR Vδ2 (BioLegend, clone B6). 6 Stain cells.

[0308] In addition to surface markers, cytokine secretion, intracellular cytokines and / or membrane-bound cytokines, such as TNF-α, IFN-γ, GM-CSF, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-17, or IL-21, may also be assessed according to methods known in the art.

[0309] In a specific embodiment herein, viable cells were determined by the absence or low uptake of zombie violet (BioLegend) amine dye. Fluorescence Minus One (FMO) controls were used to define the boundaries between positive and negative surface expression of each antigen. Stained cells were collected on a Sony SH800 cytometer, and data were analyzed using FlowJo v10.1. Flow cytometry data were visualized as dot plots.

[0310] Example 8. δ2 T cell proliferation in serum-containing and serum-free medium The growth and proliferation rates of various δ2 T cells were evaluated in serum-containing medium (R2: RPMI + 10% FBS) and serum-free medium (AIMV + bovine albumin; CTS serum-free supplement). Figure 5 depicts a graph showing the growth of δ2 T cells. All media used in this experiment contained 100 IU / mL IL-2, 2 mM glutamine, and 1x penicillin / streptomycin. Additionally, cells were stimulated with 1, 5, and 20 μM zoledronic acid on day 0. Every 2-3 days, the medium was replenished without further zoledronic acid. 10 6 The total number of δ2 T cells expanded from PBMCs and the fold expansion for each treatment after a 13-day period are shown in Figure 5. These results suggest that δ2 T cells can be expanded in serum-free medium.

[0311] Example 9. Blocking and competition assays of anti-γδ TCR antibodies Figures 6 and 7 depict graphs showing anti-γδ TCR antibody blocking experiments, and Figures 8 and 9 depict antibody competition assays. Figures 6 and 7 show the results of blocking experiments in which PBMCs were preincubated with various antibodies, namely, 5A6.E9, B1, TS8.2, 15D, B3, B6, TS-1, γ3.20, IMMU510, or 11F2. Cells were then washed and stained with TS8.2-FITC (δ1-specific) or B6-PE (δ2-specific) secondary antibodies. PBMC samples were analyzed by flow cytometry. The degree of blocking was estimated using the decrease in geometric mean fluorescence intensity (gMFI). The levels of inhibition for TS8.2-FITC (Figure 6) and B6-PE (Figure 7) are shown graphically.

[0312] Competition studies of MAbs TS-1 and TS8.2 with antibodies 5A6.E9, B1, IMMU510, R9.12-2, or 11F2 for binding to the γδ1 TCR-expressing cell line BE13 were performed by simultaneously incubating 1 × 10 cells with 1, 2, or 10 μg of unlabeled competing antibody (IgG1, 5A6.E9, B1, TS8.2, TS-1, R9.12, IMMU510, or 11F2) and 0.2 μg of anti-Vδ1 TCR clone TS8.2 (Figure 8) or anti-Vδ1 TCR clone TS-1 (Figure 9) conjugated to FITC. 5 The assay was performed by incubating the antibody with γδ1 cells on ice for 30 minutes. Percent competition was calculated by dividing the change in geometric mean fluorescence by the maximum change in geometric mean fluorescence. As depicted in Figure 9, the TS-1 antibody competed with TS8.2 binding to the cells as effectively as TS8.2 itself. None of the other antibodies tested was able to compete with TS8.2 binding. The TS8.2 antibody competed with TS-1 binding to the cells, although not as effectively as TS-1 itself. Some level of competition with TS-1 binding was also observed with the 11F2 antibody. These results suggest that both the TS-1 and TS8.2 antibodies bind to γδ1 but may not bind to the same epitope.

[0313] Example 10. Enzymatic digestion of tumor specimens and γδ T cell proliferation by antibodies against specific γδ epitopes 24-well plates were coated with 0.5-1 μg of anti-γδ TCR antibody. Cells isolated from digested tumor tissues as described in Example 4 were counted and cultured at 0.5-1 × 10 in RPMI 1640 supplemented with 10% human AB serum and rhIL-2 (100 IU / mL). 6 Cells / ml were seeded onto antibody-coated wells. Cultures were incubated at 37°C, 5% CO for 7-21 days.

[0314] Example 11. Enzymatic digestion of tumor specimens and γδ T cell proliferation by antibodies against specific γδ epitopes Cells isolated from digested tumor tissues as described in the "Enzymatic Digestion of Fresh Tumor Specimens" section of Example 1 were counted and cultured at 0.5-1 × 10 in RPMI 1640 medium supplemented with 10% human AB serum and rhIL-2 (100 IU / mL). 6 Cells / ml were seeded into antibody-coated wells of 3D cell culture plates (Corning® Costar® Ultra-Low Attachment Multiwell Plates). Cultures were incubated at 37°C, 5% CO2 for 7-21 days.

[0315] Example 12. Activation and proliferation of γδ T cells from PBMCs Activators were tested as soluble agents or immobilized on culture wells. 6 Soluble antigens and antibodies were added to human PBMCs cultured in 24-well plates at a cell density of 1000 cells / ml at final concentrations of 0.1–5 μg / ml. Alternatively, the same anti-γδ TCR antibodies were applied to the wells of 24-well tissue culture plates and immobilized. The anti-γδ TCR antibodies were added at concentrations of 0.1–10 μg / ml. The wells were washed twice with PBS, and then the PBMCs were transferred to the plates and cultured in either RPMI-1640, AIM-V, or CTS-OpTmizer medium as described above. The culture medium was supplemented with 100 IU / ml rhIL-2.

[0316] Specifically, on day 0, one million PBMCs / ml from donor B3 were stimulated with various antibodies at 0.5, 1, and 2 μg per well of a 24-well plate. The antibodies tested were mouse IgG1 isotype control clone MG1-45 (Bio Legend), UCHT-1, 5A6.E9, B1, TS8.2, 15D, B6, B3, TS-1, γ3.20, 7A5, and zoledronate. Figures 10 and 11 depict graphs showing the activation and proliferation of δ1 and δ2 T cells, respectively, from PBMCs. Cells were activated and expanded in medium containing RPMI with 10% FBS, 100 IU / ml rhIL-2, glutamine, and 1× penicillin-streptomycin. Seven days after priming, cells were passaged into fresh medium and placed into newly coated 24-well plates with the same antibodies at the same concentrations. The medium in the restimulated cultures was replenished every 2–3 days until day 13 and analyzed by flow cytometry.

[0317] Figure 12 shows the total number of δ1 T cells, and Figure 13 shows the total number of δ2 T cells after 13 days of growth and expansion. The total number of γδ T cells was calculated by multiplying the percentage of δ1 and δ2 T cells (determined by flow cytometry using TS8.2-FITC and B6-PE, respective...

Claims

1. 1. An in vitro method for producing an enriched γδ T cell population from an isolated mixed cell population, comprising directly contacting the mixed cell population with one or more agents that selectively expand δ2γδ T cells by binding to an activating epitope specific to the δ2 ​​TCR to provide an enriched γδ T cell population, wherein the one or more agents are selected from antibodies that specifically bind to an epitope comprising the δ2 ​​variable region of the human δ2 TCR, the antibodies comprising: i) a heavy chain variable region / light chain variable region (HCVR / LCVR) sequence pair, the HCVR / LCVR sequence pair comprising: or ii) the six CDRs of an HCVR / LCVR sequence pair selected from the group consisting of SEQ ID NOs: 207 / 216, 208 / 217, 209 / 219, 210 / 220, 211 / 221, 212 / 222, 213 / 223, 214 / 224, and 215 / 225 according to Kabat numbering.

2. 2. The method of claim 1, wherein the activating epitope is selected from bin 3 δ2 epitopes and bin 4 δ2 epitopes of the human δ2 TCR.

3. (i) the enriched γδ T cell population has not been expanded by antigen-presenting cells or aminophosphates; (ii) one or more agents that selectively expand δ2 T cells are immobilized on the surface; (iii) the isolated mixed cell population is selected from a peripheral blood sample, an umbilical cord blood sample, or a tumor; (iv) the enriched γδ T cell population comprises polyclonal TCR diversity; (v) the enriched γδ T cell population is formulated for administration to a subject; (vi) the enriched γδ T cell population comprises a therapeutically effective amount of γδ T cells; and / or (vii) the enriched γδ T-cell population is engineered to stably express one or more antigen recognition moieties. The method of claim 1.

4. (i) in a first γδ T cell expansion, selectively expanding δ2 T cells by binding to an activating epitope specific for the δ2 ​​TCR, thereby producing a first enriched γδ T cell population; and thereafter (ii) directly contacting at least a portion of said first enriched γδ T cell population with antigen-presenting cells (APCs) in a second γδ T cell expansion, thereby producing a second enriched γδ T cell population. wherein the second enriched γδ T cell population comprises a clinically relevant number of γδ T cells.

5. (i) in a first γδ T cell expansion, directly contacting the mixed cell population with one or more first activating agents that selectively expand δ2 T cells by binding to an activating epitope specific for the δ2 ​​TCR, thereby producing a first enriched γδ T cell population; and thereafter (ii) in a second γδ T cell expansion, at least a portion of said first enriched γδ T cell population is (a) expanded to γδ T cells, or (b) - selectively expand δ1 T cells by binding to activation epitopes specific for the δ1 TCR; - selectively expand δ2 T cells by binding to activation epitopes specific for the δ2 ​​TCR; - selectively expanding δ1 T cells, δ3 T cells, δ4 T cells and δ5 T cells by binding to activation epitopes specific for the δ1 TCR, δ3 TCR, δ4 TCR and δ5 TCR; or - selectively expands δ1 and δ4 T cells by binding to activation epitopes specific to the δ1 and δ4 TCRs directly contacting the cells with one or more second activating agents, thereby producing a second enriched γδ T cell population. The method of claim 1 , comprising:

6. 6. The method of claim 5, wherein (a) one or more of the second activators are structurally distinct from one or more of the first activators of γδ T cell proliferation.

7. (i) in a first γδ T cell expansion, directly contacting the mixed cell population with one or more first activating agents that selectively expand δ2 T cells by binding to an activating epitope specific for the δ2 ​​TCR, thereby producing a first enriched γδ T cell population; and thereafter (ii) directly contacting at least a portion of said first enriched γδ T cell population with one or more agents that deplete αβ T cells, thereby producing a second enriched γδ T cell population. The method of claim 1 , comprising:

8. The method of any one of claims 1 to 7, wherein the percentage of δ2 T cells in the enriched γδ T cell population is higher than 60% or 80%.

9. The method of any one of claims 1 to 8, comprising serum-free culture conditions and / or suspension cell culture conditions.

10. - the onset of primary proliferation, or - a first step of directly contacting said mixed cell population with one or more activating agents; and (c) at least 10 cells expanded from said isolated mixed cell population within less than 90 days, less than 60 days, less than 30 days, less than 21 days, or less than 19 days from the day of the first cell division. ８ The method according to any one of claims 1 to 9, wherein the method achieves γδ T cells.

11. 4. The method of claim 3, wherein the γδ T cell population is engineered to express two or more antigen recognition moieties.

12. The method described in claim 11, wherein the two or more antigen recognition moieties are different, and each of the different antigen recognition moieties is engineered to recognize a different epitope of the same tumor or a different epitope of a different tumor.

13. The method of claim 3 or 11, wherein the antigen recognition moiety recognizes a tumor antigen, an antigen associated with an autoimmune disease, or a pathogen antigen.

14. The antigen recognition portion comprises: (i) cell surface tumor antigens, (ii) a peptide derived from a tumor antigen expressed on the cell surface as a complex with MHC (peptide-MHC complex); (iii) a cell surface tumor antigen associated with an autoimmune disease or pathogen, or (iv) Peptides derived from antigens associated with autoimmune diseases or pathogens that are expressed on the cell surface as a complex with MHC (peptide-MHC complex). TCR, αβTCR, γδTCR, chimeric antigen receptor (CAR), whole antibody or antigen-binding fragment thereof, single-chain variable fragment (scFv), heavy or light chain single domain antibody (sdAb), Fab, F(ab), which bind to ２ 14. The method of claim 13, wherein the compound is selected from the group consisting of:

15. 14. The method of claim 13, wherein the pathogen antigen is a bacterial antigen or a viral antigen.

16. 4. The method of claim 3, wherein the engineered γδ T-cells are further engineered to lack gene expression from at least one HLA locus.

17. 4. The method of claim 3, wherein the engineered γδ T cells are derived from universal donor cells.

18. 18. The method of any one of claims 1 to 17, wherein the isolated mixed cell population comprises a total PBMC population without prior depletion of monocytes, αβ T cells, B cells, and NK cells.

19. The method of any one of claims 1 to 17, further comprising depleting one or more of red blood cells, αβ T cells, B cells, and NK cells.

20. 20. The method of any one of claims 1-19, wherein contacting the mixed cell population with one or more agents that selectively expand δ2 T cells further comprises culture conditions comprising a cytokine selected from IL-2, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL23, and IL-33.

21. 21. The method of claim 20, wherein the cytokine is selected from the group consisting of IL-2, IL-7, and IL-15.

22. i) comprises a heavy chain variable region / light chain variable region (HCVR / LCVR) sequence pair selected from the group consisting of SEQ ID NOs: 207 / 216, 208 / 217, 209 / 219, 210 / 220, 211 / 221, 212 / 222, 213 / 223, 214 / 224, and 215 / 225 according to Kabat numbering; or ii) An antibody comprising six CDRs of HCVR / LCVR sequence pairs selected from the group consisting of 207 / 216, 208 / 217, 209 / 219, 210 / 220, 211 / 221, 212 / 222, 213 / 223, 214 / 224, and 215 / 225 according to the Kabat numbering system.

23. A nucleic acid encoding the antibody of claim 22, wherein the nucleic acid is operably linked to a heterologous promoter.

24. A host cell comprising the nucleic acid of claim 23 and / or the antibody of claim 22.

25. 25. The host cell of claim 24, wherein the nucleic acid encodes a membrane anchor or transmembrane domain fused to the antibody, and the antibody is displayed on the extracellular surface of the host cell.

26. 23. A composition comprising an expanded γδ T cell population and the antibody of claim 22, wherein the antibody binds to an amino acid sequence of the δ2 ​​TCR variable region and selectively expands δ2 γδ T cells.

27. 27. The composition of claim 26, wherein the expanded γδ T cell population comprises a percentage of δ2 T cells greater than 60% or 80%.

28. 27. The composition of claim 26, wherein the antibody is immobilized on a surface.

29. 27. The composition of claim 26, wherein the γδ T cells are derived from γδ T cells isolated from a complex sample of the subject.

30. The composition described in claim 29, wherein the composite sample of the subject is a blood sample, an umbilical cord blood sample, a tumor, a stem cell precursor, a tumor biopsy, tissue, lymph, or an epithelial site of the subject that is in direct contact with the external environment.

31. 27. The composition of claim 26, further comprising a cytokine selected from IL-2, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, and IL-33.

32. 32. The composition of claim 31, wherein the cytokine is selected from the group consisting of IL-2, IL-7, and IL-15.

33. 27. The composition of claim 26, wherein the γδ T-cell is engineered to stably express at least one antigen recognition moiety, wherein the antigen recognition moiety recognizes a tumor antigen, an antigen associated with an autoimmune disease, or a pathogen antigen.

34. The expanded γδ T cell population comprises at least 10 ８ 34. The composition of any one of claims 26 to 33, comprising δ2γδ T cells.

35. 35. Use of the composition of any one of claims 26 to 34 in the manufacture of a medicament for treating cancer, an autoimmune disease or an infectious disease in a subject in need thereof, wherein γδ T-cells have been engineered to express an antigen recognition moiety, wherein the antigen recognition moiety recognizes a tumor antigen, an antigen associated with an autoimmune disease, or a pathogen antigen, respectively.

36. A TCR, αβTCR, γδTCR, chimeric antigen receptor (CAR), whole antibody or antigen-binding fragment thereof, single-chain variable fragment (scFv), heavy or light chain single domain antibody (sdAb), Fab, or F(ab), wherein the antigen recognition portion binds to: (i) a cell surface tumor antigen, (ii) a peptide derived from a tumor antigen expressed on the cell surface as a complex with MHC (peptide-MHC complex), (iii) a cell surface tumor antigen associated with an autoimmune disease or pathogen, or (iv) a peptide derived from an antigen associated with an autoimmune disease or pathogen expressed on the cell surface as a complex with MHC (peptide-MHC complex). ２ 36. The use of claim 35, wherein the compound is selected from the group consisting of:

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