Methods for selective in vivo expansion of gamma delta t cell populations and compositions thereof

Abstract

To provide clinically acceptable in vivo methods for expanding specific γδ T cell subsets.SOLUTION: Disclosed is an in vivo method for activating, expanding and / or maintaining γδ T cell population in a subject, the method comprising: administering to the subject an effective amount of one or more agents that selectively expand δ1 T cells, δ2 T cells, or δ3 T cells or a combination thereof, where the one or more agents that selectively expand δ1 T cells binds to an activating epitope specific to δ1 TCR, the one or more agents that selectively expand δ2 T cells binds to an activating epitope specific to δ2 TCR, and the one or more agents that selectively expand δ3 T cells binds to an activating epitope specific to δ3 TCR, thereby activating, expanding and / or maintaining the γδ T cell population in the subject.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 774,817, filed December 3, 2018, the contents of which are hereby incorporated by reference in their entirety for all purposes. [Background technology]

[0002] Antigen recognition by T lymphocytes can be achieved by a diverse heterodimeric receptor, 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. Moreover, activation of αβ T cells 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 independent 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, a more extensive junctional diversification process during rearrangement of the TCR γ and δ chains renders the potential γδ TCR repertoire broader than that of αβ TCRs (Carding and Egan, Nat Rev Immunol (2002) 2:336).

[0004] Human γδ T cells use three major Vδ (Vδ1, Vδ2, Vδ3) region genes and up to six Vγ region genes to make 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 with various Vγs make up 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 lymphatic system.

[0005] The ability of γδ T cells to directly recognize antigens on diseased cells and exert an innate ability to kill tumor cells makes them an attractive therapeutic tool. The abundant Vγ9Vδ2 subtype of γδ T cells recognizes pyrophosphate compounds, such as the microbial compound (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate. However, the ligands recognized by other γδ T cell subtypes are unknown.

[0006] Adoptive transfer of Vγ9Vδ2 T cells has resulted in limited targeted clinical responses in the treatment of cancers at the research stage (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.

[0007] The ability to selectively expand γδ T cell subset populations with potent antitumor activity at improved purity and clinically relevant levels is highly desirable. Although cocktails of antibodies and cytokines have been used to expand a more diverse set of γδ T cells, activation of specific γδ T cell subsets to sufficient purity and clinically relevant levels has not been achieved (Dokouhaki et al., 2010; Kang et al., 2009; Lopez et al., 2000; Kress, 2006).

[0008] Selective expansion of γδ T cell subtypes has been demonstrated in vitro and in vivo by the use of known ligands for Vγ9Vδ2. For example, Pressey et al., Medicine (Baltimore). 2016 Sep; 95(39):e4909, report the in vivo expansion of Vγ9Vδ2 using intravenous zoledronate, a synthetic pyrophosphate mimetic, and subcutaneous IL-2. Selective expansion of other γδ T cell subtypes has been demonstrated in vitro using immobilized antibodies that selectively bind and crosslink, e.g., δ1, δ2, and δ3 subtypes. See WO2016 / 081518, WO2017 / 197347, and WO2019 / 099744, the contents of which are incorporated in their entirety. Unfortunately, however, in vivo immobilization is typically performed by binding antibodies to cell surface Fc receptors, which would generally be expected to induce antibody-dependent cell-mediated cytotoxicity (ADCC) effects, thereby reducing the population of γδ T cells recognized by the antibody. Similarly, methods that reduce the interaction between Fc receptors and antibodies would also be expected to reduce immobilization and therefore not achieve robust in vivo expansion. Thus, there is a great need for clinically relevant methods of expanding specific γδ T cell subsets in vivo, and the cells produced thereby. Summary of the Invention

[0009] The inventors have identified i) activators that selectively activate and expand 51 T cells by binding to an activation epitope specific for the 51 TCR, ii) activators that selectively activate and expand 52 T cells by binding to an activation epitope specific for the 52 TCR, and 53 T cells by binding to an activation epitope specific for the 53 TCR. The inventors have surprisingly found that, in a physiologically relevant environment, such antibodies are able to effectively activate, expand and support the persistence of adoptively transferred chimeric antigen receptor (CAR) γδ T cells and / or endogenous γδ T cells in vivo.

[0010] Described herein are methods and compositions for using these activators, individually or in combination, for the in vivo expansion of γδ T cells. These methods and compositions may be suitable for the selective activation and expansion of one or more γδ T cell subpopulations. The in vivo expansion methods and compositions may be suitable for maintaining and / or expanding endogenous γδ T cells or subpopulations thereof. Additionally or alternatively, the in vivo expansion methods and compositions may be suitable for maintaining and / or expanding γδ T cells or subpopulations thereof administered to a subject.

[0011] In a first aspect, the invention provides an in vivo method for activating, expanding and / or maintaining a T cell population in a subject, the method comprising administering to the subject an effective amount of one or more agents that selectively expand 51 T cells, 52 T cells or 53 T cells, or a combination thereof, wherein the one or more agents that selectively expand 51 T cells bind to an activation epitope unique to the 51 TCR, the one or more agents that selectively expand 52 T cells bind to an activation epitope unique to the 52 TCR, and the one or more agents that selectively expand 53 T cells bind to an activation epitope unique to the 53 TCR, thereby activating, expanding or maintaining a γδ T cell population in the subject.

[0012] In some embodiments, the method comprises administering to the subject an effective amount of one or more agents that selectively expand δ1 T cells. In some embodiments, the agent that selectively expands δ1 T cells is selected from an agent that binds to the same epitope as an antibody selected from TS-1 and TS8.2. In some embodiments, the agent that binds to the same epitope as an antibody selected from TS-1 and TS8.2 comprises the CDRs of TS-1 or TS8.2 and / or is humanized TS-1 or TS8.2. In some embodiments, the agent that selectively expands δ1 T cells is selected from an agent that does not compete with TS-1, TS8.2, or R9.12. In some embodiments, the agent that selectively expands δ1 T cells is selected from an agent that specifically binds to an epitope comprising the δ1 variable region. In some embodiments, the agent that selectively expands δ1 T cells binds to an epitope comprising residues Arg71, Asp72, and Lys120 of the δ1 variable region. In some embodiments, the agent that selectively expands .delta.1 T cells has reduced binding to mutant .delta.1 TCR polypeptides comprising a mutation at K120 of deltaJ1 and deltaJ2.

[0013] In some embodiments, the agent that selectively expands δ1 T cells is an agent that binds to the δ1TCR bin 1 δ1 epitope, bin 1b δ1 epitope, bin 2 δ1 epitope, bin 2b δ1 epitope, bin 2c δ1 epitope, bin 3 δ1 epitope, bin 4 δ1 epitope, bin 5 δ1 epitope, bin 6 δ1 epitope, bin 7 δ1 epitope, bin 8 δ1 epitope or bin 9 δ1 epitope of human δ1 TCR. In some embodiments, the agent that selectively expands δ1 T cells is selected from agents that bind to or compete with the same or essentially the same epitope as 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.

[0014] In some embodiments the agent that selectively expands 51 T cells is an antibody selected from the group consisting of 51-05, 51-08, 51-18, 51-22, 51-23, 51-26, 51-35, 51-37, 51-39, 51-113, 51-143, 51-149, 51-155, 51-182, 51-183, 51-191, 51-192, 51-195, 51-197, 51-199, 51-201, 51-203, 51-239, 51-253, 51-257, 51-278, 51-282, and 51-285. In some embodiments the agent that selectively expands 51 T cells selectively expands 51 T cells and 51-3 T cells. In some embodiments, the agent that selectively expands δ1 T cells selectively expands δ1, δ3, δ4, and δ5 γδ T cells.

[0015] In some embodiments the method comprises administering to the subject an effective amount of one or more agents that selectively expand 52 T cells. In some embodiments the agent that selectively expands 52 T cells is selected from an agent that binds to the same epitope as an antibody selected from 15D and B6. In some embodiments the agent that binds to the same epitope as an antibody selected from 15D and B6 comprises the CDRs of 15D or B6 and / or is humanized 15D and B6. In some embodiments the agent that selectively expands 52 T cells is selected from an agent that binds to a different epitope than antibody 15D and / or B6. In some embodiments the agent that selectively expands 52 T cells is selected from an agent that specifically binds to an epitope comprising the 52 variable region. In some embodiments the agent that selectively expands 52 T cells has reduced binding to a mutant 52 TCR polypeptide comprising a mutation at G35 in the 52 variable region. In some embodiments, the agent that selectively expands 52 T cells binds to a 52TCR bin 1 52 epitope, a bin 2 52 epitope, a bin 3 52 epitope, or a bin 4 52 epitope of a human 52TCR.

[0016] In some embodiments, the agent that selectively expands 52 T cells is an agent that binds to or competes with the same or essentially the same epitope as 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. In some 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.

[0017] In some embodiments the method comprises administering to the subject an effective amount of one or more agents that selectively expand 53 T cells. In some embodiments the agents that selectively expand 53 T cells are selected from agents that bind to or compete with the same or essentially the same epitope as an antibody selected from the group consisting of 53-08, 53-20, 53-23, 53-31, 53-42, 53-47, and 53-58. In some embodiments the agents that selectively expand 53 T cells are selected from agents that bind to or compete with the same or essentially the same epitope as an antibody selected from the group consisting of 53-08, 53-23, 53-31, 53-42, 53-47, and 53-58. In some embodiments, the agent that selectively expands δ3 T cells is selected from agents that bind to or compete with the same or essentially the same epitope as an antibody selected from the group consisting of δ3-23, δ3-31, δ3-42, δ3-47, and δ3-58.

[0018] In some embodiments the agent that selectively expands 53 T cells is an antibody or a fragment thereof selected from the group consisting of 53-08, 53-20, 53-23, 53-31, 53-42, 53-47, and 53-58. In some embodiments the agent that selectively expands 53 T cells is an antibody or a fragment thereof selected from the group consisting of 53-08, 53-23, 53-31, 53-42, 53-47, and 53-58. In some embodiments the agent that selectively expands 53 T cells is an antibody or a fragment thereof selected from the group consisting of 53-23, 53-31, 53-42, 53-47, and 53-58.

[0019] Generally, the agent is a human or humanized agent, such as a human antibody or fragment thereof, or a humanized antibody or fragment thereof. In some cases, the agent that selectively expands δ1 T cells, δ2 T cells, or δ3 T cells, or a combination thereof, is multivalent, preferably the multivalent agent comprises at least two or more antigen binding sites that specifically bind to the same antigen, or the multivalent agent comprises at least two or more antigen binding sites that specifically bind to the same epitope of the same antigen. In some cases, the agent that selectively expands δ1 T cells, δ2 T cells, or δ3 T cells, or a combination thereof, is trivalent, tetravalent, or pentavalent, or is at least trivalent, tetravalent, or pentavalent. In some cases, the agent that selectively expands δ1 T cells, δ2 T cells, or δ3 T cells, or a combination thereof, is trivalent, tetravalent, or pentavalent, or is at least trivalent, tetravalent, or pentavalent and monospecific.

[0020] In some embodiments, the method comprises administering to the patient a population of engineered and / or non-engineered γδ T cells. In some embodiments, the method comprises administering the population of engineered and / or non-engineered γδ T cells before administering one or more agents that selectively expand δ1 T cells, δ2 T cells, or δ3 T cells. In some embodiments, the method comprises administering the population of engineered and / or non-engineered γδ T cells after administering one or more agents that selectively expand δ1 T cells, δ2 T cells, or δ3 T cells. In some embodiments, the method comprises administering to the subject one or more agents that selectively expand δ1 T cells, δ2 T cells, or δ3 T cells, isolating a sample from the subject comprising in vivo expanded γδ T cells, optionally manipulating one or more of the isolated γδ T cells, optionally expanding one or more of the isolated (e.g., and engineered) γδ T cells in vitro, and then administering to the same or a different subject the population of isolated, engineered, and / or non-engineered, and / or ex vivo expanded γδ T cells. In some embodiments, the administered population of engineered and / or non-engineered γδ T-cells is a population of cells that is autologous to the subject.

[0021] In some embodiments, the administration population of engineered and / or non-engineered γδ T cells is a population of cells allogeneic to the subject. In some embodiments, the administration population of engineered and / or non-engineered γδ T cells is a population that comprises at least 60% (e.g., at least 70%, 80%, or 90%, about 60% to about 80%, or about 60% to about 90%) δ1 γδ T cells, and the method comprises administering γδ T cells and sequentially or simultaneously administering one or more agents that selectively expand δ1 T cells.

[0022] In some embodiments, the administered population of engineered and / or non-engineered γδ T cells is a population that comprises at least 60% (e.g., at least 70%, 80%, or 90%, about 60% to about 80%, or about 60% to about 90%) δ2 γδ T cells, and the method comprises administering γδ T cells and sequentially or simultaneously administering one or more agents that selectively expand δ2 T cells.

[0023] In some embodiments, the administered population of engineered and / or non-engineered γδ T cells is a population that comprises at least 10% (e.g., at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, about 10% to about 80%, about 20% to about 40%, about 20% to about 50%, or about 20% to about 60%) δ3 γδ T cells, and the method comprises administering γδ T cells and sequentially or simultaneously administering one or more agents that selectively expand δ3 T cells.

[0024] In some embodiments, the method comprises administering an aminophosphonate (e.g., an aminobisphosphonate) or a prenylphosphonate. In some embodiments, the method comprises administering an aminophosphonate selected from the group consisting of zoledronate, pamidronate, alendronate, risedronic acid, ibandronate, and incadronic acid, or salts and / or hydrates thereof. In some embodiments, the method comprises repeatedly administering one or more agents that selectively expand δ1 T cells, δ2 T cells, and / or δ3 T cells. In some embodiments, the method comprises expanding or maintaining a population of the administered γδ T cells in the subject. In some embodiments, the method comprises expanding or maintaining a population of endogenous γδ T cells in the subject. In some embodiments, the method comprises activating, expanding or maintaining a population of endogenous γδ T cells in a subject, isolating a sample containing endogenous γδ T cells from the subject, manipulating the γδ T cells of the isolated sample (e.g., by purification, ex vivo expansion and / or manipulation), administering the engineered γδ T cells to the same or a different subject, and then activating, expanding or maintaining the population of administered γδ T cells in the same or a different subject by administering one or more selective in vivo activating agents.

[0025] In some embodiments, the methods comprise expanding the population of endogenous and / or administered γδ T cells in the subject by a detectable amount (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 75%, 2-fold, 10-fold, or 50-fold, or about 10% to about 20%, about 10% to about 50%, about 10% to about 75%, about 10% to about 2-fold, about 10% to about 10-fold, about 2-fold to about 10-fold, or about 10-fold to about 50-fold) relative to the amount of γδ T cells in a subject not receiving one or more agents that selectively expand δ1 T cells, δ2 T cells, and / or δ3 T cells. In some embodiments, the methods comprise maintaining a larger population of endogenous and / or administered γδ T cells in the subject in a detectable amount (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 75%, 2-fold, 10-fold, or 50-fold, or about 10% to about 20%, about 10% to about 50%, about 10% to about 75%, about 10% to about 2-fold, about 10% to about 10-fold, about 2-fold to about 10-fold, or about 10-fold to about 50-fold) relative to the number of γδ T cells in a subject not receiving one or more agents that selectively expand δ1 T cells, δ2 T cells, and / or δ3 T cells.

[0026] In some embodiments, the agent that selectively expands δ1 T cells, δ2 T cells, or δ3 T cells, or a combination thereof, is multivalent, preferably the multivalent agent comprises at least two antigen binding sites that specifically bind to the same antigen, or the multivalent agent comprises at least two antigen binding sites that specifically bind to the same epitope of the same antigen. In some embodiments, the agent that selectively expands δ1 T cells, δ2 T cells, or δ3 T cells, or a combination thereof, is multivalent, preferably the multivalent agent comprises at least three antigen binding sites that specifically bind to the same antigen, or the multivalent agent comprises at least three antigen binding sites that specifically bind to the same epitope of the same antigen. In some cases, the agent that selectively expands δ1 T cells, δ2 T cells, or δ3 T cells, or a combination thereof, is bivalent, trivalent, tetravalent, or pentavalent, or is at least bivalent, trivalent, tetravalent, or pentavalent. In some cases, the agent that selectively expands 51 T cells, 52 T cells, or 53 T cells, or a combination thereof, is trivalent, tetravalent, or pentavalent, or at least trivalent, tetravalent, or pentavalent. In some cases, the agent that selectively expands 51 T cells, 52 T cells, or 53 T cells, or a combination thereof, is tetravalent, or at least tetravalent.

[0027] In some embodiments, the method comprises administering a cytokine to the subject, either simultaneously or sequentially. In some embodiments, the method comprises administering an engineered γδ T-cell to the subject, the engineered γδ T-cell comprising a transgene encoding a secreted cytokine. In some embodiments, the cytokine is a general gamma chain cytokine or a cytokine selected from the group consisting of IL-2, IL-15, and IL-4, preferably the cytokine is IL-2, IL-15, and / or IL-4. In some embodiments, the method comprises administering a lymphodepletion protocol to the subject prior to administering the γδ T-cells.

[0028] In a second aspect, the present invention provides a method of treating cancer, an infectious disease, an inflammatory disease or an autoimmune disease in a subject in need thereof, the method comprising performing any one of the in vivo expansion methods described above and / or using any one or more of the γδ T cell activators described herein for the in vivo expansion of γδ T cells in a subject in need thereof.

[0029] In a third aspect, the invention provides the use of an agent that selectively expands δ1 T cells, δ2 T cells or δ3 T cells in the manufacture of a medicament for the in vivo expansion of γδ T cells in a subject in need thereof. In some embodiments, the in vivo expansion of γδ T cells in a subject comprises treating cancer, an infectious disease, an inflammatory disease or an autoimmune disease in the subject.

[0030] 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.

[0031] 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 description of the drawings]

[0032] [Figure 1] 1 depicts the amino acid sequences of the framework and complementarity determining regions of the heavy chain of a δ1-specific MAb. [Diagram 2] 2 depicts the amino acid sequences of the framework and complementarity determining regions of the light chain of the .delta.1-specific MAb described in FIG. [Diagram 3] 1 depicts the amino acid sequences of the framework and complementarity determining regions of the heavy chain of a δ2-specific MAb. [Figure 4] FIG. 4 depicts the amino acid sequences of the framework and complementarity determining regions of the light chains of the .delta.2-specific MAbs described in FIG. [Diagram 5] Variable region sequences of δ3-specific anti-γδ TCR antibodies are shown. Sequence above the heavy chain variable region. Sequence below the light chain variable region. [Figure 6] Figure 1 shows the in vivo effect of γδ T cell subtype-specific activators on the number of total human CD45+ cells detected in mouse blood, bone marrow, lung, and spleen after adoptive transfer of human γδ T cells into mice. Untreated mice were not treated with activators. Treated mice were treated with 3 or 10 μg of activators as indicated. D1-35 refers to the δ1 γδ T cell-specific antibody δ1-35 described herein. Some animals also received an intraperitoneal injection of a nonspecific mouse IgG fraction 4-5 h prior to administration of cells to saturate unoccupied Fc receptors. [Figure 7A] By 5 days after treatment with activator, there is proliferation of adoptively transferred human γδ T cells in blood, bone marrow, and spleen, as evidenced by a left shift of the CellTrace Violet trace towards reduced MFI due to dye dilution in the cell progeny at both the 3 and 10 μg dose levels. [Figure 7B] By 5 days after treatment with activator, there is proliferation of adoptively transferred human γδ T cells in blood, bone marrow, and spleen, as evidenced by a left shift of the CellTrace Violet trace towards reduced MFI due to dye dilution in the cell progeny at both the 3 and 10 μg dose levels. [Figure 8] Figure 1 shows the in vivo activating effect of the indicated activating antibodies D1-35 and D1-08 (δ1-08) as detected by CellTrace Violet. Row 1, mice were not treated with activator. Row 2, mice were treated with activator D1-35. Row 3, mice were treated with activator and non-specific mouse IgG. Row 4, mice were treated with hIgG4 isotype version of D1-35 activator. Row 5, mice were treated with activator D1-08. [Figure 9A] Figure 1 shows the in vivo expansion of V52 and V53 cells detected in bone marrow and spleen of animals treated with D2-37 (52-37) and D3-23 (53-23) activating antibodies, respectively, as detected by CellTrace Violet dye dilution. [Figure 9B] Figure 1 shows the in vivo expansion of V52 and V53 cells detected in bone marrow and spleen of animals treated with D2-37 (52-37) and D3-23 (53-23) activating antibodies, respectively, as detected by CellTrace Violet dye dilution. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] While various embodiments of the present invention have been shown and described herein, it will be apparent 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.

[0034] 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 definition set forth herein conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0035] The term "γδ T cells (gamma delta T cells)" as used herein refers to a subset of T cells expressing on their surface a unique T cell receptor (TCR), γδ TCR, consisting of one γ chain and one δ chain. The term "γδ T cells" specifically includes all subsets of γδ T cells and combinations thereof, 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, Vγ11 γδ T cells.

[0036] As used herein, the term "T lymphocyte" or "T cell" refers to an immune cell that expresses CD3 (CD3+) and expresses the T cell receptor (TCR+). T cells play a central role in cell-mediated immunity.

[0037] 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.

[0038] The term "MHC" (major histocompatibility complex) refers to a subset of genes that code for cell surface antigen-presenting proteins. In humans, these genes are called human leukocyte antigen (HLA) genes. In this specification, the abbreviations MHC or HLA are used interchangeably.

[0039] 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%.

[0040] 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 may then be cultured in a test tube. 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 with the present invention. The cell population may be purified to homogeneity, substantial homogeneity, or depletion of one or more cell types (e.g., αβ T cells) by various culture techniques and / or negative or positive selection of specific cell types. The cell population may be a mixed heterogeneous cell population derived, for example, from a peripheral blood sample, an umbilical cord blood sample, a tumor, a stem cell precursor, a tumor biopsy, a tissue, lymph, skin, a sample of or containing tumor-infiltrating lymphocytes, or an epithelial site of a subject in direct contact with the external environment, or derived from stem progenitor cells. Alternatively, the mixed cell population may be derived from in vitro cultures of mammalian cells or from stem progenitor cells established from samples of or containing peripheral blood samples, umbilical cord blood samples, tumors, stem cell precursors, tumor biopsies, tissues, lymph, skin, tumor infiltrating lymphocytes, or from epithelial sites of a subject in direct contact with the external environment.

[0041] An "enriched" cell population or preparation refers to a cell population derived from a starting mixed cell population that contains a higher percentage of a particular cell type than the percentage of that cell type in the starting population. For example, the 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 the cell type in the starting population. As another example, the enriched γδ T cell population can contain a higher percentage of δ1 cells and a higher percentage of δ3 cells than the percentage of each cell type in the starting population. As yet another example, the enriched γδ T cell population can contain a higher percentage of δ1 cells and a higher percentage of δ4 cells than the percentage of each cell type in the starting population. As another example, the enriched γδ T cell population can contain a higher percentage of δ1 cells and a higher percentage of δ5 cells than the percentage of each cell type in the starting population. In yet another example, the enriched γδ T cell population may contain a higher percentage of δ1 T cells, δ3 T cells, δ4 T cells and δ5 T cells than the percentage of each of the respective cell types 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 another embodiment, the enriched γδ T cell population contains a higher percentage of δ3 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 and δ2 cells than the percentage of the respective cell types in the starting population. In yet another embodiment, the enriched γδ T cell population contains a higher percentage of δ1 cells, δ2 cells and δ3 cells than the percentage of the respective cell types in the starting population. In all embodiments, the enriched γδ T cell population contains a lower percentage of the αβ T cell population.

[0042] As used herein, "expanded" means that the number of desired or target cell types (e.g. δ1 and / or δ2 T cells and / or δ3 T cells) in the enriched preparation is greater than in the initial or starting cell population.

[0043] By "selectively expand" it is meant that the target cell type (e.g. δ1, δ2, or δ3 T cells) is preferentially expanded over other non-target cell types, e.g. αβ T cells or NK cells, or untargeted subpopulations of γδ T cells. In certain embodiments, the activating agent of the invention selectively expands, e.g. engineered or non-engineered δ1, δ2, and / or δ3 T cells, without the expansion of αβ T cells or without significant expansion of αβ T cells. In certain embodiments, the activating agent of the invention selectively expands, e.g. engineered or non-engineered δ1 T cells, without the expansion of δ2 T cells or without significant expansion of δ2 T cells. In other embodiments, the activating agent of the invention selectively expands, e.g. engineered or non-engineered δ2 T cells, without the expansion of δ1 T cells or without significant expansion of δ1 T cells. In a particular embodiment, the activating agent of the invention selectively expands, e.g., engineered or non-engineered, δ3 T cells, without proliferation of δ2 T cells or without significant proliferation of δ1 T cells or without significant proliferation of δ1 T cells. In a particular embodiment, the activating agent of the invention selectively expands, e.g., engineered or non-engineered, δ1 and δ3 T cells, without proliferation of δ2 T cells or without significant proliferation of δ2 T cells. In a particular embodiment, the activating agent of the invention selectively expands, e.g., engineered or non-engineered, δ1 and δ4 T cells, without proliferation of δ2 T cells or without significant proliferation of δ2 T cells. In a particular embodiment, the activating agent of the invention selectively expands, e.g., engineered or non-engineered, δ1 and δ5 T cells, without proliferation of δ2 T cells or without significant proliferation of δ2 T cells. In a particular embodiment, the activating agent of the invention selectively expands δ1, δ3, δ4 and δ5 T cells, e.g. engineered or not, without proliferation of δ2 T cells or without significant proliferation of δ2 T cells. In this context, the term "without significant proliferation" 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.

[0044] The term "mixture" as used herein 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 invention is an isolated γδ T cell population. According to certain embodiments, the cell population of the invention is expanded ex vivo and / or provided in vivo and administered to a subject, thereby becoming an in vivo γδ T cell population. According to certain embodiments, the cell population of the invention is expanded in vivo by administering one or more agents that selectively expand the γδ T cell population.

[0045] 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 associated with said cell population in vivo or in vitro.

[0046] In the context of a cell population, the term "contacting" as used herein refers to incubating an isolated cell population with a reagent, such as 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 a bead or plate. In one embodiment, the antibody is immobilized to an Fc-coated well. In a desired embodiment, the contacting occurs in vivo.

[0047] 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 has a much lower affinity (KD>10) with, other polypeptides. -6The 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.

[0048] An "effective amount" in the context of an in vivo method of expanding or maintaining an in vivo population of γδ T cells in a subject refers to a dose that results in a verifiable increase in the expansion or maintenance of an in vivo population of γδ T cells in a subject. As an example, an effective dose may selectively expand a target population of administered γδ T cells by a detectable amount (e.g., at least 1%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 2-fold, or by about 1% to about 10%, or by about 10% to about 2-fold). As another example, an effective dose may selectively expand a target population of endogenous in vivo γδ T cells by a detectable amount (e.g., at least 1%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 2-fold, or by about 1% to about 10%, or by about 10% to about 2-fold). As another example, an effective dose may maintain a greater number of viable target γδ T cells in a subject or in a tissue of a subject (e.g., a tumor tissue) compared to a control subject that does not receive one or more agents that selectively proliferate γδ T cells.

[0049] The term "effective amount" as used herein in the context of an in vivo method of treating cancer, an infectious disease, an inflammatory disease, or an autoimmune disease in a subject in need thereof refers to an amount of a composition containing one or more agents that selectively expand γδ T cells administered to a subject, e.g., a human patient, already suffering from a disease, condition, or disorder, sufficient to cure or at least partially arrest or alleviate to some extent one or more of the symptoms of the disease, disorder, or condition being treated. The effectiveness of such compositions depends on conditions including, but not limited to, the severity and course of the disease, disorder, or condition, previous therapies, 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 can be determined by routine experimentation, for example, but not limited to, a dose escalation trial.

[0050] The term "chimeric antigen receptor (CAR)" as used herein may refer to an artificial T cell receptor, T body, single chain immune receptor, chimeric T cell receptor, or chimeric immune receptor, and may include engineered receptors that fuse artificial specificity to a particular immune effector cell, for example. CARs may be employed to confer the specificity of a monoclonal antibody to T cells, thereby generating a large number of specific T cells, for example, for adoptive cell therapy. In a specific embodiment, the CAR directs the specificity of the cell, for example, to a tumor-associated antigen. In some embodiments, the 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 that may vary in length and includes a disease or disorder-associated antigen binding region, for example, a tumor antigen binding region. In a particular aspect, the 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. The specificity of other CAR designs may be derived from the ligand of a receptor (e.g., a peptide), or a pattern recognition receptor, such as a dectin. In certain cases, the spacing of the antigen recognition domains can be adjusted to reduce activation-induced cell death. In certain cases, the CAR includes 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.

[0051] 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" (about 25 kDa) and one "heavy chain" (about 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 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. In general, antibody molecules obtained from humans are associated with 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 include IgG, IgM, IgA, IgE, IgD ... 1 , IgG 2 etc. Furthermore, in humans, the light chain can be a kappa chain or a lambda chain.

[0052] The term "Fab" refers to the entire light chain (V L and C L ) plus the H chain variable domain (V H ) 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 Fab light and heavy chain fragments produced by papain digestion are held together by interchain disulfide bonds.

[0053] 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 the antibody are determined by the sequence of the Fc region, which is also the portion recognized by Fc receptors (FcR) found on certain types of cells. An Fc fragment can be obtained by papain digestion of a complete antibody.

[0054] "F(ab') 2 The term F(ab')" refers to an antibody fragment produced by pepsin digestion of a whole antibody. 2The F(ab') fragment contains two Fab fragments and part of the hinge region held together by disulfide bonds. 2 The fragment has divalent antigen-binding activity and is capable of cross-linking antigen.

[0055] The term Fab' stands for F(ab') 2 Refers to antibody fragments that are the products of reduction of the fragment. Fab' fragments differ from Fab fragments in that they have 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.

[0056] The term "Fv" refers to an antibody fragment consisting of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, non-covalent association. The folding of these two domains results in 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 an antigen, although usually with a lower affinity than the complete binding site.

[0057] The term "single-chain Fv", also abbreviated as "sFv" or "scFv", refers to an antibody fragment comprising the VH and VL antibody domains linked as a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For reviews of scFvs, see, e.g., Pluckthun, 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.

[0058] 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 Linear antibodies are used to refer to a polypeptide comprising the amino acid sequence of any one of the following: 1) Linear antibodies can be bispecific or monospecific, and are described, for example, in Zapata et al., Protein Eng. 8(10):1057-1062 (1995).

[0059] 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 to its particular antigen. However, the variability is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light 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, which are 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 contribute to the formation of the antigen-binding site of antibodies with the hypervariable regions from the other chain (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).

[0060] The term "antigen-binding site" or "binding portion" refers to the portion of an immunoglobulin molecule that is involved in antigen binding. The antigen-binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. The three regions of greatest variance within the V regions of the heavy and light chains are called "hypervariable regions" and are flanked by flanking, more conserved regions known as "framework regions" or "FRs". Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to 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 with respect to each other to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity determining regions" or "CDRs". The assignment of amino acids to each domain is based on the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or the definitions in Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989).

[0061] The term "hypervariable region", "HVR" or "HV" refers to the region of an antibody variable domain that is sequence hypervariable and / or forms structurally defined loops. Generally, an antibody contains six HVRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 are thought to exhibit the greatest diversity among the six HVRs, and H3 in particular plays 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, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0062] "Framework Regions" (FR) are those variable domain residues other than the CDR residues. Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4. When the CDRs are defined by Kabat, the light chain FR residues are located at about residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3) and 98-107 (LCFR4) and the heavy chain FR residues are located at about residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3) and 103-113 (HCFR4) of the heavy chain. 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 as defined by Kabat and that of a hypervariable loop, the FR residues will be adjusted accordingly. For example, when CDRH1 comprises amino acids H26-H35, the heavy chain FR1 residues are at positions 1-25 and the FR2 residues are at positions 36-49.

[0063] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues of a selection of human immunoglobulin VL or VH framework sequences. Usually, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Usually, the subgroup of sequences is a subgroup as in Kabat. In some cases, for VL, the subgroup is subgroup kappa I as in Kabat. In some cases, for VH, the subgroup is subgroup III as in Kabat.

[0064] The antibodies described herein can be humanized. "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capacity. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are FRs of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol., 1:105-115 (1998); Harris, Biochem. Soc. Transactions, 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech., 5:428-433 (1994).

[0065] A "human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human and / or is made using any of the techniques for making human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J.Mol.Biol., 227:381(1991); Marks et al., J.Mol.Biol., 222:581(1991). Human monoclonal antibodies can also be prepared using the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77(1985); Boerner et al., J.Immunol., 147(1):86-95(1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenogeneic mice, that have been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), regarding human antibodies generated via human B cell hybridoma technology.

[0066] The antigen-binding moieties described herein useful for activating γδ T cells, such as, for example, the antibodies or antigen-binding fragments thereof described herein, can be multivalent. For example, F(ab') 2The fragment has bivalent antigen-binding activity and can crosslink antigens. Similarly, an antigen-binding moiety, e.g., an IgG or other canonical antibody architecture, can have a bivalent structure. In some cases, the antigen-binding moiety is more than bivalent. In some cases, the antigen-binding moiety can be a trivalent moiety, such as a trivalent antibody. In some cases, the antigen-binding moiety can be tetravalent, such as a tetravalent antibody, e.g., an IgA antibody. In some cases, the antigen-binding moiety can have a valency of 10. For example, the antigen-binding moiety can be an IgM antibody. Preferred multivalent antigen-binding moieties, e.g., antibodies or fragments thereof, described herein typically bind to the same antigen, and in some cases the same epitope of the same antigen, at each antigen-binding site. In some cases, the multivalent antigen-binding moiety comprises at least one antigen-binding site that is different from one other antigen-binding site of the multivalent antigen-binding moiety.

[0067] As used herein, "Kd" or "Kd value" refers to the dissociation constant measured by surface plasmon resonance assay, for example, by using BIAcore.TM.-2000 or BIAcore.TM.-3000 (BIAcore, Inc., Piscataway, NJ) with a CM5 chip immobilized with about 10 response units (RU) of antigen or antibody at 25°C. In the case of bivalent or other multivalent antibodies, it is typical to immobilize the antibody to avoid interference with the measurement of the dissociation constant induced by avidity. For further details, see, for example, Chen et al., J.Mol.Biol.293:865-881 (1999).

[0068] As used herein to refer to binding affinity, "greater than" refers to stronger binding between a molecule and its binding partner. As used herein, "greater than" refers to a smaller numerical value K D For example, an antibody may have an affinity for an antigen of "0.6 nM or greater," where the affinity of the antibody for the antigen is ≦0.6 nM, i.e., 0.59 nM, 0.58 nM, 0.57 nM, etc., or any value equal to or less than 0.6 nM.

[0069] The term "epitope" includes any protein, lipid, or carbohydrate determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitope determinants often consist of active surface groupings of molecules such as amino acids, lipids, or sugar side chains and often have unique three dimensional structural characteristics as well as unique charge characteristics. Antibodies are characterized by the equilibrium dissociation constant (K D ) is 10 -6 ~10 -12 A specific binding is said to be in the range of 10-fold, preferably 100-fold, or more preferably 1,000-fold tighter dissociation constant (lower K M ) than the dissociation constant for binding to other non-target epitopes. D ) in a non-targeted region of a γδ-TCR. In some cases, the non-targeted epitope is an epitope of the δ1, δ2, or δ3 chain of a delta3 TCR. In some cases, the non-targeted epitope is an αβ TCR. In some cases, the non-targeted epitope is a delta chain of a different subtype. The specificity of binding can be determined in the context of binding to the extracellular region of a γδ-TCR and / or an αβ-TCR (e.g., as an Fc fusion immobilized on an ELISA plate or expressed on a cell).

[0070] An "activating epitope" is capable of activating a specific γδ T cell population upon binding. T cell proliferation is indicative of T cell activation and proliferation.

[0071] An antibody binds to "essentially the same epitope" as a reference antibody when 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 competitive assay that can be configured in a variety of ways, using either labeled antigen or labeled antibody. In some embodiments, antigen is immobilized on a 96-well plate, and the ability of unlabeled antibody to block the binding of labeled antibody is measured using radioactive or enzyme labeling. Alternatively, the competitive test using labeled and unlabeled antibodies is performed using flow cytometry on antigen-expressing cells.

[0072] "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 are brought together by folding of the protein into its three-dimensional structure.

[0073] "Epitope binning" as defined herein 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.

[0074] An "agent" or "compound" according to the present invention includes small molecules, polypeptides, proteins, antibodies or antibody fragments. Small molecules in the context of the present invention in one embodiment refer to chemicals 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.

[0075] As used herein, the term "cell culture" refers to any in vitro culture of cells. This term includes continuous cell lines (e.g., having 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, and the like.

[0076] The term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures aimed at preventing or slowing (reducing) undesirable physiological changes or disorders. 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 the expected survival in the absence of treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to having the condition or disorder or those in whom the condition or disorder is to be prevented.

[0077] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0078] 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 a percentage of sequence units that are the same when compared and aligned for maximum correspondence over a specified region as measured using a comparison window or comparison 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 particular region. Such percentages represent the "percent identity" of two or more sequences. Sequence identity may exist over a region that is at least about 75-100 sequence units in length, over a region that is at least about 50 sequence units in length, or over the entire sequence if not specified. 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 particular 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.

[0079] The term "pharmacologically 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 is relatively non-toxic, i.e., that may be administered to an individual without causing undesirable biological effects or interacting adversely with any of the components contained in the composition.

[0080] 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, farm animals (e.g., cows), sport animals, and pets (e.g., cats, dogs, and horses). In certain embodiments, the mammal is a human.

[0081] The term antigen-presenting cell (APC) refers to wild-type APC or engineered or artificial antigen-presenting cell (aAPC). APC can be provided as an irradiated population of APC. APC can be provided from immobilized cell line (e.g., K562 or engineered aAPC derived from immobilized cell line) or as a fraction of cells from a donor (e.g., PBMC).

[0082] As used herein, the terms "structurally distinct" and "structurally distinct" with respect to a protein or a polypeptide fragment thereof or an 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.

[0083] 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, a γδ T cell that exhibits 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.

[0084] 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 a specific receptor. The presence or absence of such receptor-dependent antitumor cytotoxicity can be determined by performing a standard in vitro cytotoxicity assay in the presence or absence of an antagonist against the specific receptor, such as that performed in Example 48 of PCT / US17 / 32530. 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 in the presence of an anti-NKp30 antagonist with the results obtained in the absence of an anti-NKp30 antagonist.

[0085] It is further understood that γδ T cells or γδ T cell populations can be assayed for mRNA expression of one or more of the cytotoxic receptors NKp30, NKp44, and / or NKp46. In such cases, the expression assay can indicate the presence or absence of receptor-dependent antitumor cytotoxicity. For example, the measured mRNA expression of γδ T cells or γδ T cell populations can be compared to a positive control using cells or cell lines that do not exhibit a particular receptor-dependent cytotoxicity (e.g., as verified by in vitro cytotoxicity assays in the presence and absence of an antagonist).

[0086] A γδ T cell population comprising anti-tumor cytotoxicity, where at least a certain "%" of the anti-tumor cytotoxicity is "independent" of a particular receptor activity (e.g., NKp30 activity, NKp44 activity, and / or NKp46 activity), as used herein, refers to cells whose measured anti-tumor cytotoxicity is not reduced by more than that numerical percentage value upon blocking a particular receptor. Thus, a γδ T cell population comprising anti-tumor cytotoxicity, where at least 50% of the anti-tumor cytotoxicity is independent of NKp30 activity, will exhibit no more than a 50% reduction in in vitro anti-tumor cytotoxicity in the presence of an NKp30 antagonist compared to the absence of an NKp30 antagonist.

[0087] Overview In humans, γδ T cell(s) 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), and γδ T cell(s) 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 functions. 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, and 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.

[0088] TS-1, TS8.2, B6 and 15D are capable of activating 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, and in particular, can activate specific γδ T cell populations in enriched cultures to clinically relevant levels, i.e., 10 8 It has been found that not all binding epitopes of a γδ T cell population are activating epitopes, i.e., capable of activating a particular γδ T cell population upon binding.

[0089] The inventors have identified specific γδ variable TCR binding regions associated with strong activation of specific γδ T cell subtypes, thus enabling in vivo or ex vivo activation and expansion of γδ T cell subtypes. Ex vivo activation and expansion by binding to the identified TCR binding regions can be used to generate highly enriched γδ T cell populations and mixtures thereof with improved purity and clinically relevant levels that can be administered to patients. In vivo activation can be used to expand and / or maintain the administered γδ T cell population (or one or more subtypes thereof) and induce expansion of an endogenous γδ T cell population (or one or more subtypes thereof), or a combination thereof. In some cases, in vivo activation can be used to expand an endogenous γδ T cell population in vivo, and a portion of the expanded population can be isolated and expanded ex vivo using the methods described herein. The in vivo expanded, isolated, and ex vivo expanded γδ T cells can be stored, optionally manipulated, and / or administered to a subject in need thereof. Manipulation can be performed before or after ex vivo expansion. The administered cells may be expanded and / or maintained in vivo by administering to the subject one or more agents that selectively expand γδ T cells.

[0090] Activating ligands, including antibodies or other binding agents, 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. In some embodiments, the activating agents used in the methods and compositions described herein, including methods for in vivo expansion of γδ T cells and / or administration to a subject in need thereof, are agents described in PCT / US2015 / 061189 and / or PCT / US17 / 32530 for ex vivo expansion. In some embodiments, the activating agents used in the methods and compositions described herein, including methods for in vivo expansion of γδ T cells and / or administration to a subject in need thereof, are δ3-specific activating agents described in PCT / US18 / 061384 for ex vivo expansion. PCT / US17 / 32530 and PCT / US18 / 061384 are incorporated by reference in their entirety for all purposes, including all disclosures relating to γδ T cell activators, γδ T cell compositions, and methods of γδ T cell activation, γδ T cell proliferation, treatment, administration, and dosing.

[0091] In some aspects, the invention provides an in vitro method of expanding engineered or non-engineered γδ T cells. In general, the in vitro expansion method is used in combination with an in vivo expansion and / or maintenance method for expanding engineered or non-engineered γδ T cells as described herein. For example, γδ T cells can be selectively expanded in vivo by administration of one or more agents that selectively expand γδ T cells or one or more subpopulations thereof. A portion of the γδ T cells that have been selectively expanded in vivo can be isolated and then further expanded, e.g., selectively, in vitro. In some cases, the in vitro expanded γδ T cells can be administered to a subject in need thereof, regardless of whether they have been previously expanded in vivo. In some cases, the in vitro expanded γδ T cells, or a portion thereof, are administered to the same subject from which the initial population was isolated. In some cases, the in vitro expanded γδ T cells, or a portion thereof, are administered to a different subject from which the initial population was isolated. In some cases, the administered in vitro expanded γδ T cells are further expanded or maintained in vivo by administering to the subject one or more agents that selectively expand γδ T cells.

[0092] In vivo expansion of γδ T cells The present disclosure provides methods for in vivo expansion and / or maintenance of populations of non-engineered and engineered γδ T cells. The non-engineered or engineered γδ T cells of the present disclosure may be additionally activated and / or expanded ex vivo before and / or after in vivo expansion or maintenance. In some embodiments, the in vivo activation, expansion, and / or maintenance of the non-engineered or engineered γδ T cells of the present disclosure may be performed without administering an aminophosphonate or prenylphosphonate. In some embodiments, the in vivo activation, expansion, and / or maintenance of the non-engineered or engineered γδ T cells of the present disclosure may be performed, at least in part, by administering an aminophosphonate or prenylphosphonate. For example, the in vivo activation and / or expansion may be performed by a method comprising administering one or more agents that selectively expand the non-engineered or engineered γδ T cells of the present disclosure by binding to an epitope unique to δ1, δ2, or δ3 γδ T cells, or a combination thereof, the method further comprising administering an aminophosphonate or prenylphosphonate.

[0093] Generally, the method comprises administering to the subject an effective amount of one or more agents that selectively expand γδ T cells. In some embodiments, the method comprises providing a pharmaceutical composition comprising a γδ T cell population and one or more agents that selectively expand γδ T cells, and administering the provided pharmaceutical composition to the subject. In some embodiments, the method comprises administering γδ T cells to the subject and then administering one or more agents that selectively expand γδ T cells. In some embodiments, the method comprises administering one or more agents that selectively expand γδ T cells and then administering γδ T cells to the subject. In certain embodiments, the γδ T cells are administered one or more times and the one or more agents that selectively expand γδ T cells are administered periodically to the subject in an effective amount to expand or maintain the administered γδ T cells.

[0094] In some cases, the method includes administering one or more agents that selectively expand γδ T cells to the subject multiple times, e.g., at least two times, at least three times, or at least four times. In some cases, the method includes administering one or more agents that selectively expand γδ T cells to the subject 1-2 times, 1-3 times, 1-5 times, 1-10 times, 2-4 times, 2-10 times, or 5-10 times. In some cases, the method includes administering one or more agents that selectively expand γδ T cells to the subject for a dosing period of 2-4 weeks, 2-6 weeks, 1 month (e.g., about 30 days), 2 months, or 1-2 months. In some cases, the method includes administering one or more agents that selectively expand γδ T cells to the subject periodically for at least 2 months (e.g., about 60 days), at least 3 months (e.g., about 90 days), at least 4 months (e.g., about 120 days), or at least 6 months (e.g., about 180 days).

[0095] The methods of the invention include those that involve various γδ T cell populations, e.g., Vγ1 + , Vγ2 + , or Vγ3 + The γδ T cell population can be expanded in vivo. In some cases, the methods of the invention include, but are not limited to, Vδ1 + 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.

[0096] In some cases, the γδ T cell population can be expanded in vivo a detectable amount (e.g., at least 10%, at least 25%, at least 50%, at least 75%, at least 2-fold, or about 10% to about 2-fold) in less than 45 days, less than 40 days, less than 35 days, or less than 30 days. In some cases, the γδ T cell population can be expanded in vivo about 10% to about 10-fold, about 50% to about 10-fold or more in less than 45 days, less than 40 days, less than 35 days, or less than 30 days. In some cases, the γδ T cell population can be expanded in vivo about 10 to about 50-fold in less than 45 days, less than 40 days, less than 35 days, or less than 30 days.

[0097] In some aspects, methods are provided for selectively expanding various γδ T cells in vivo, including engineered and non-engineered γδ T cells, by contacting γδ T cells with an activating agent. In some cases, the activation or activating agent binds to a unique epitope on a cell surface receptor of a γδ T cell. 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; δ3; δ1 and δ3; δ1 and δ4; δ1 and δ5; δ1, δ3, and δ4; or δ1, δ3, δ4, and δ5 cell populations, or combinations thereof. In some embodiments, the activating agent specifically activates the growth of a δ1 cell population to expand or maintain a δ1 T cell population in vivo. In other cases, the activating agent specifically activates the growth of a δ2 cell population to expand or maintain a δ2 T cell population in vivo. In other cases, the activating agent specifically activates the growth of a δ3 cell population to expand or maintain a δ3 T cell population in vivo. In other cases, the activating agent specifically activates the growth of a δ1 and δ3 cell population to expand or maintain a δ1 and δ3 T cell population in vivo. In other cases, the activating agent specifically activates the growth of a δ1 and δ4 cell population to expand or maintain a δ1 and δ3 T cell population in vivo. In other cases, the activating agent specifically activates the growth of a δ1 and δ5 cell population to expand or maintain a δ1 and δ5 T cell population in vivo.

[0098] In some cases, the agent that selectively expands δ1 T cells, δ2 T cells, or δ3 T cells, or a combination thereof, is multivalent, preferably the multivalent agent comprises at least two antigen binding sites that specifically bind to the same antigen, or the multivalent agent comprises at least two antigen binding sites that specifically bind to the same epitope of the same antigen. In some cases, the agent that selectively expands δ1 T cells, δ2 T cells, or δ3 T cells, or a combination thereof, comprises at least three antigen binding sites that specifically bind to the same antigen, or the multivalent agent comprises at least three antigen binding sites that specifically bind to the same epitope of the same antigen. In some cases, the agent that selectively expands δ1 T cells, δ2 T cells, or δ3 T cells, or a combination thereof, is bivalent, trivalent, tetravalent, or pentavalent, or is at least bivalent, trivalent, tetravalent, or pentavalent.

[0099] One or more activators may contact γδ T cells in vivo (e.g., activator γδ T cell natural receptors), and simultaneously or sequentially, costimulatory molecules may contact γδ T cells to provide further stimulation and expand γδ T cells. 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 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).

[0100] 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.

[0101] 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.

[0102] The activators and co-stimulatory agents described herein can be used to perform in vivo activation, proliferation and / or maintenance of γδ T cells to induce specific γδ T cell proliferation and persistent populations. In one embodiment, the agents providing specific γδ T cell activation signals can be various monoclonal antibodies (MAbs) directed against the γδ TCR.

[0103] In one embodiment, the MAbs can bind to different epitopes on the constant or variable regions of the δTCR and / or γTCR. In one embodiment, the MAbs can comprise γδTCR pan-MAbs. In one embodiment, the γδTCR pan-MAbs can recognize domains common to various γ and δTCRs on either the γ or δ chain or both chains, including δ1, δ2, and δ3 T cell populations. In one embodiment, the antibodies can be 5A6.E9 (Thermo scientific), B1 (Biolegend), IMMU510 and / or 11F2 (11F2) (Beckman Coulter).

[0104] In one embodiment, the MAb can be directed to a specific domain unique to the variable region of the gamma chain (7A5Mab (Thermo Scientific #TCR1720) directed against the Vg9TCR), or to a domain on the V51 variable region (Mab TS8.2 (Thermo Scientific #TCR1730; MAb TS-1 (ThermoFisher #TCR 1055), MAb R9.12 (Beckman Coulter #IM1761)), or to the V52 chain (MAb 15D (Thermo Scientific #TCR1732 or Life technologies #TCR2732), B6 ​​(Biolegend #331402), one of the 51-# antibodies described in Figures 1-2, one of the 52-# antibodies described in Figures 3-4, or one of the 53-# antibodies described in Figure 5.

[0105] In certain embodiments, the activator is an agent that binds to the same or essentially the same epitope as one of the δ1-# antibodies described in Figures 1-2, one of the δ2-# antibodies described in Figures 3-4, or one of the δ3-# antibodies described in Figure 5. In certain embodiments, the activator is an agent that competes with one of the δ1-# antibodies described in Figures 1-2, one of the δ2-# antibodies described in Figures 3-4, or one of the δ3-# antibodies described in Figure 5. Additional activators are described herein, as well as in PCT / US17 / 32530, filed May 12, 2017, and Attorney Docket No. ADC-0003-PR1, U.S. Provisional Patent Application No. 62 / 586,782, which was filed concurrently herewith.

[0106] In certain embodiments, bioactivation, proliferation, and / or maintenance may be further supported by simultaneous or sequential administration of cytokines or other stimulants such as 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), or granulocyte colony-stimulating factor (G-CSF). In some cases, the cytokine is IL-2, IL-15, IL-12, or IL-21. In some cases, the cytokine is IL-2. In some cases, the cytokine is IL-15. In some cases, the cytokine is IL-4. In some cases, the cytokine is a general gamma chain cytokine selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, or a combination thereof.

[0107] In certain embodiments, in vivo expansion or maintenance can be further supported by administering a lymphodepletion protocol to the subject prior to administration of the activating agent and / or prior to administration of the γδ T-cells (e.g., γδ CAR-T cells). In some cases, the lymphodepletion protocol includes administering fludarabine and cyclophosphamide. In some cases, lymphodepletion includes or further includes leukapheresis. In some cases, lymphodepletion includes or further includes bendamustine. In some cases, lymphodepletion is not administered to patients diagnosed with or suspected of having lymphopenia at the time the CART cells are administered.

[0108] Isolation of γδ T cells In some embodiments, the invention provides an in vitro method 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 TCRs, respectively, by binding to epitopes unique to the δ1 TCR; δ1 and δ4 TCR; to result in an enriched γδ T cell population. In another aspect, the invention provides an in vitro method for producing an enriched γδ2 T cell population from an isolated mixed cell population, 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, to result in an enriched γδ2 T cell population. In another aspect, the invention provides an in vitro method for producing an enriched γδ3 T cell population from an isolated mixed cell population, comprising contacting the mixed cell population with one or more agents that selectively expand δ3 T cells by binding to epitopes unique to the δ3TCR, resulting in an enriched γδ3 T cell population.

[0109] In another aspect, the disclosure provides a method of genetically engineering γδ T cells isolated from a subject. The methods of enrichment, expansion, purification, e.g., by positive and / or negative selection, or genetic engineering can be performed alone or in combination, in any order. In one embodiment, γδ T cells can be expanded in vivo in a subject, isolated from a subject, genetically engineered, then expanded ex vivo, and optionally administered to a subject. In another embodiment, γδ T cells can be isolated from a subject, genetically engineered, optionally activated and expanded ex vivo, administered to a subject, and then expanded or maintained in vivo. In some cases, the subject from which the γδ T cells are isolated and the subject to which the γδ T cells are administered are the same subject. In some cases, the subject from which the γδ T cells are isolated and the subject to which the γδ T cells are administered are different subjects.

[0110] A γδ T cell population, engineered or unengineered, can be expanded, for example, directly from a composite sample of a subject. In some cases, the composite sample is isolated and expanded in vitro by directly contacting the composite sample with one or more agents that selectively expand the target γδ T cell population. In some cases, the composite sample is isolated and then purified by positive or negative selection before in vitro expansion is performed.

[0111] The composite sample may be from a peripheral blood sample (e.g., PBL or PBMC), a leukapheresis sample, an umbilical cord blood sample, a tumor, a stem cell precursor, a tumor biopsy, a tissue, a lymph, or an epithelial site of a subject in direct contact with the external environment, or derived from a stem progenitor cell. + cell, Vδ2 + cells, Vδ3 + 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 a cell, a mammalian cell line ...

[0112] Peripheral blood mononuclear cells can be collected from a subject with an apheresis machine, including, for example, a Ficoll-Paque™ PLUS (GE Healthcare) system or another suitable device / system. The 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.

[0113] γδ T cells may be expanded from an isolated complex sample or a mixed cell population cultured in vitro, for example 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 may be activated and expanded without prior removal of one or more specific cell populations, for example 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 may be performed using immobilized γδ T cell mitogens that bind to an activating epitope of the γδ TCR as provided herein, as well as other activating agents, including, for example, antibodies and lectins specific for an activating epitope of the γδ TCR.

[0114] In certain embodiments, the isolated mixed cell population, optionally purified, e.g., by positive and / or negative selection, 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 to about 4 days, about 2 to about 4 days, about 2 to about 5 days, about 3 to about 5 days, about 5 to about 21 days, about 5 to about 19 days, about 5 to about 15 days, about 5 to about 10 days, or about 5 to about 7 days to provide 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 to about 21 days, about 7 to about 19 days, about 7 to about 23 days, or about 7 to about 15 days to result in a first enriched γδ T cell population.

[0115] 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 (αβ T cells).

[0116] 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., 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 a second or further enriched γδ T cell population(s). For example, e.g., the first enriched γδ T cell population(s) may be depleted of αβ T cells, B cells and NK cells. A population of γδ T cells or γδ T cell(s) expressing similar cell surface markers may 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 harvested 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.

[0117] 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 1-3 days, about 1-4 days, about 1-5 days, about 2-5 days, about 2-4 days, or about 2-3 days in 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 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold or more expansion of γδ T-cells. In some cases, the cell density adjustment is performed contemporaneously (i.e., on the same day or at the same time) 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 in the second expansion step. Exemplary cell densities that support further expansion of γδ T cells include, but are not limited to, about 1×10 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 are included.

[0118] In some embodiments, the cell density is 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 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 ~About 20×10 6 cells / mL, or approximately 1 x 10 6 ~Approx. 30×10 6 Adjust to density of cells / mL.

[0119] In some embodiments, the second expansion step is performed under conditions that monitor 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 grown 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. 2×106 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 ~About 20×10 6 cells / mL, approximately 1×10 6 ~Approx. 30×10 6 The viable cell density can be maintained at 1000 cells / mL.

[0120] 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 throughout the first portion of the first or second expansion. As another example, culture volume may be most efficiently utilized at a higher cell concentration throughout the final portion of the first or second expansion. Thus, in some embodiments, cells are maintained at about 1×10 cells throughout at least a portion of the first or second expansion culture or throughout the entire first or second expansion culture. 6 ~About 20×10 6 The cells can be maintained at a viable cell density of 1000 cells / mL.

[0121] In another example, the cells may 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, the cells may 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, the 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.

[0122] Typically, the glucose content is maintained by adding fresh serum-containing or serum-free culture medium to the culture. In some embodiments, the cells may be maintained in culture medium with a predetermined glucose content interval at a predetermined viable cell density interval, for example by monitoring each parameter and adding fresh medium to maintain the parameter within a predetermined limit. In some embodiments, the glucose content is maintained by adding fresh serum-containing or serum-free culture medium to the culture during the removal of spent medium while keeping the cells inside in the perfusion bioreactor. In some embodiments, the pH, O2, and / or pH are adjusted 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. 2 Partial pressure of O 2 Saturation, CO 2 Partial pressure of CO 2 Additional parameters are monitored and / or maintained including, but not limited to, one or more of saturation, lactate, glutamine, glutamate, ammonium, sodium, potassium and calcium.

[0123] The γδ T cell subtype is 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; iv) selectively expanding δ1, δ3, δ4 and δ5 T cells by specifically binding to epitopes of δ1, δ3, δ4 and δ5 TCRs; or v) selectively expanded from an isolated complex sample or a mixed cell population cultured in vitro by contacting the mixed cell population with one or more agents that selectively expand δ3 T cells by specifically binding to an epitope of the δ3 TCR, For example, in a first enrichment step, an enriched γδ T cell population may be provided.

[0124] In some cases, the one or more agents specifically bind to 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 removal of specific cell populations, e.g., 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, activation and proliferation of γδ T cells from a tumor sample may be performed using immobilized γδ T cell mitogens including antibodies specific for activation epitopes unique to the δ1TCR; δ1, δ3, δ4, and δ5TCR; δ1 and δ4TCR; δ3TCR; or δ2TCR, as well as other activating agents including lectins that bind to activation epitopes unique to the δ1TCR; δ1, δ3, δ4, and δ5TCR; δ1 and δ4TCR; δ3TCR; or δ2TCR.

[0125] In certain embodiments, the isolated mixed cell population is contacted with one or more agents that selectively expand δ1, δ1 and δ4, δ2, δ3, δ1 and δ2, or δ1, δ2, and δ3 T cells for about 5 days, 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, 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 provide a first enriched γδ T cell population. In some embodiments, the selectively expanded δ1, δ1 and δ3, δ1 and δ4, δ2, δ3, δ1 and δ2, or δ1, δ2 and δ3 T cells are further expanded in a second expansion step as described herein.

[0126] In certain embodiments, the isolated starting mixed cell population, e.g., 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 not NK cells 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).

[0127] Thus, in some embodiments, the methods of the invention provide a clinically relevant number (10 8 Super, 10 9 Super, 10 10 Super, 10 11 More than or 10 12 More than or about 10 8 ~about 10 12 ) of expanded γδ T cells 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 More than or 10 12 More than or about 10 8 ~about 10 12) of expanded γδ T cells can be provided in less than 19 days or less than 21 days from the time the donor sample is obtained.

[0128] Following specific activation and expansion of specific γδ T cell subsets in a first enrichment step using activating agents that bind activation epitopes unique to 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, the first enriched γδ T cell population(s) may be depleted of αβ T cells, B cells, and NK cells. 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 harvested γδ T cell(s). For example, γδ T cells can be isolated from the first enriched γδ T cell population based on 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.

[0129] 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 comprises clinically relevant levels of γδ T cell subsets greater than 10 cells, 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. For example, the method of the present invention can be used in the following applications: 10-100mL, 25-100mL, 50-100mL, 75-100mL, 10-150mL, 25-150mL, 50-150mL, 75-150mL, 100-150mL, 10-200mL, 25-200mL, 50-200mL, 75-200mL, 100-200mL, 10-250mL, 25-250mL, 50-250mL, 75-2 >10 in a growth culture with a volume of 50 mL, 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 another embodiment, after the second, third, fourth, fifth, etc. enrichment step of the present invention, the enriched γδ T cell population can provide a clinically relevant level of cells of the γδ T cell subset. 8 Contains clinically relevant levels of the γδ T cell subset.

[0130] In some embodiments, γδ T cell(s) can rapidly proliferate in response to contact with one or more antigens. Some γδ T cell(s), e.g., Vγ9Vδ2+ 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 number of antigen-presenting, co-stimulatory, and adhesion molecules that can facilitate the isolation of γδ T cell(s) from a complex sample. The γδ T cell(s) in the combined 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 of time, either in combination with, prior to or following expansion with a selective γδ T cell expansion agent as described herein, such as an antibody or immobilized antibody. Stimulation of the γδ T cells with a suitable antigen can result in the expansion of the γδ T cell population in vivo or in vitro by administering one or more suitable agents to the subject.

[0131] Non-limiting examples of antigens that may 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, -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), isopentenyl pyrophosphate (IGP ... γ-Adenosine triphosphate (IPPPA), monoethyl phosphate (MEP), monoethyl pyrophosphate (MEPP), 3-formyl-1-butyl-pyrophosphate (TUBAg1), γ-pyrophosphate (TUBAg2), 3-formyl-1-butyl-uridine triphosphate (TUBAg3), 3-formyl-1-butyl-deoxythymidine triphosphate (TUBAg4), monoethyl alkylamines, allyl pyrophosphate, clotoyl pyrophosphate, dimethylallyl-γ-uridine triphosphate, clotoyl-γ-uridine triphosphate, allyl-γ-uridine triphosphate, ethylamines, isobutylamines, sec-butylamines, isoamylamines, and nitrogen-containing bisphosphonates (e.g., aminophosphonates).

[0132] Activation and expansion of γδ T cells can be performed using the activators and costimulatory agents described herein to induce populations of specific γδ T cell proliferation and persistence. 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, different 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 different monoclonal antibodies (MAbs) directed against γδ TCR.

[0133] In one embodiment, the MAbs can bind to different epitopes on the constant or variable regions of the γTCR and / or δTCR. In one embodiment, the MAbs can comprise γδTCR pan-MAbs. In one embodiment, the γδTCR pan-MAbs can recognize domains common to various γ and δTCRs on either the γ or δ chain or both chains, including the δ3 cell population. In one embodiment, the antibodies can be 5A6.E9 (Thermo scientific), B1 (Biolegend), IMMU510 and / or 11F2 (11F2) (Beckman Coulter). In one embodiment, the MAbs are directed against specific domains unique to the variable region of the gamma chain (7A5 MAb (Thermo Scientific #TCR1720) directed against the Vg9 TCR analogue), or against domains on the V51 variable region (Mab TS8.2 (Thermo Scientific #TCR1730); MAb TS-1 (ThermoFisher #TCR1055), MAb R9.12 (Beckman Coulter #IM1761)), or against the V52 chain (MAb 15D (Thermo Scientific #TCR1732 or Life technologies #TCR2732), B6 ​​(Biolegend #331402), one or more of the δ1-# antibodies described in Figures 1-2, one or more of the δ2-# antibodies described in Figures 3-4, or one or more of δ3-08, δ3-20, δ3-23, δ3-31, δ3-42, δ3-47, and δ3-58 described in Figure 5.

[0134] In some embodiments, antibodies against different domains of 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 may 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 may be specific antibodies for unique epitopes on CD2 and CD3 molecules. CD2 and CD3 may have different conformational structures when expressed on αβ or γδ T cell(s), and in some cases, specific antibodies for CD3 and CD2 may result in selective activation of γδ T cells.

[0135] A 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.

[0136] Genetic engineering of the 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 a 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 the proliferation, survival, and function of the T cells in vitro and in vivo. In some cases, the cytokine is IL-2, IL-15, IL-12, or IL-21. In some cases, the cytokine is IL-2. In some cases, the cytokine is IL-15. In some cases, the cytokine is IL-4. In some cases, the cytokine is a common gamma chain cytokine selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, or a combination thereof. Genetic engineering of the isolated γδ T-cell can also include deleting or disrupting gene expression from one or more endogenous genes in the genome of the isolated γδ T-cell, e.g., the MHC locus(s).

[0137] Ex vivo expansion of γδ T cells In other aspects, the present disclosure provides methods for ex vivo expansion of populations of non-engineered and engineered γδ T cells for adoptive transfer therapy. Non-engineered or engineered γδ T cells of the present disclosure may be expanded ex vivo. Ex vivo expansion may be performed after in vivo expansion, isolation, and optional purification. Ex vivo expansion may additionally or alternatively be performed prior to in vivo expansion to provide a γδ T cell population, which is then administered to a subject and subjected to one or more in vivo expansion or maintenance methods described herein. Ex vivo expansion may be performed on a mixed cell population, for example, by directly contacting an isolated sample containing γδ T cells with one or more agents that selectively expand γδ T cells. Additionally or alternatively, ex vivo expansion may be performed after positive selection for γδ T cells or one or more subtypes thereof, and / or negative selection to remove one or more of αβ T cells, B cells, or NK cells.

[0138] The non-engineered or engineered γδ T-cells of the disclosure can be expanded in vitro without activation by APCs or without co-culture with APCs and / or aminophosphonates. Additionally or alternatively, the non-engineered or engineered γδ T-cells of the disclosure can be expanded in vitro with at least one expansion step that includes activation 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 APC in a first γδ T-cell expansion and then expanded in vitro with activation by APC 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 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 a 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 a 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 activation epitopes 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 a structurally distinct γδ TCR epitope) compared to 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, e.g., 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 a culture medium containing a soluble second agent and an APC, optionally expressing on its cell surface or bound to its 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 activation epitopes unique to δ1 TCR; δ2 TCR; δ1 and δ4 TCR; or δ1, δ3, δ4 and δ5 TCR, respectively.

[0143] The skilled artisan will appreciate that in certain embodiments the methods of the second expansion step described herein may be performed as the first expansion step and the methods of the first step described herein may be performed as the second expansion step. By way of a non-limiting example, in some embodiments a mixed population of cells (e.g. PBMCs) may be expanded in a first step by contacting with APCs and then expanded in the absence of APCs, for example by contacting the expanded population from the first expansion step with an immobilizing 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 activation epitopes unique to δ1 TCR; δ2 TCR; δ1 and δ4 TCR; or δ1, δ3, δ4 and δ5 TCR, respectively.

[0144] The methods of the invention include various γδ T cell(s) populations, e.g., Vγ1 + , Vγ2 + or Vγ3 + In some cases, 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 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. In other cases, the activating agent specifically activates the growth of a δ3 cell population resulting in an enriched δ3 T cell population.

[0147] Activators may stimulate the proliferation of engineered and non-engineered γδ T cells at high growth rates. For example, agents may stimulate the proliferation of engineered and non-engineered γδ T cells at high growth rates, such as 1 cell division in less than 30 hours, 1 cell division in less than 29 hours, 1 cell division in less than 28 hours, 1 cell division in less than 27 hours, 1 cell division in less than 26 hours, 1 cell division in less than 25 hours, 1 cell division in less than 24 hours, 1 cell division in less than 23 hours, 1 cell division in less than 22 hours, 1 cell division in less than 21 hours, 1 cell division in less than 20 hours, 1 cell division in less than 19 hours, 1 cell division in less than 18 hours, 1 cell division in less than 17 hours, 1 cell division in less than 16 ... stimulate 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 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 about 21 hours, an average rate of about 1 division every about 22 hours, an average rate of about 1 division every about 23 hours, an average rate of about 1 division every about 24 hours, an average rate of about 1 division every about 25 hours, an average rate of about 1 division every about 26 hours, an average rate of about 1 division every about 27 hours, an average rate of about 1 division every about 28 hours, an average rate of about 1 division every about 29 hours, an average rate of about 1 division every about 30 hours, an average rate of about 1 division every about 31 hours, an average rate of about 1 division every about 32 hours, an average rate of about 1 division every about 33 hours, a rate of about 1 division every about 34 hours, a rate of about 1 division every about 35 hours, an average rate of about 1 division every about 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 consecutive day to about 19 consecutive days, about 1 consecutive day to about 14 consecutive days, about 1 consecutive day to about 7 consecutive days, about 1 consecutive day to about 5 consecutive days, about 2 consecutive days to about 19 consecutive days, about 2 consecutive days to about 14 consecutive days, about 2 consecutive days to about 7 consecutive days, about 2 consecutive days to about 5 consecutive days, about 4 consecutive days to about 19 consecutive days, about 4 consecutive days to about 14 consecutive days, about 4 consecutive days to about 7 consecutive days, or about 4 consecutive days to about 5 consecutive days.

[0150] In some cases, the activator may be configured to induce an average rate of about 1 division every about 12 hours (e.g., 10-12 hours), an average rate of about 1 division every about 13 hours (e.g., 10-13 hours), an average rate of about 1 division every about 14 hours (e.g., 10-14 hours), an average rate of about 1 division every about 15 hours (e.g., 10-15 hours), an average rate of about 1 division every about 16 hours (e.g., 10-16 hours), an average rate of about 1 division every about 17 hours (e.g., 10-17 hours or 12-17 hours), an average rate of about 1 division every about 18 hours (e.g., 10-18 hours or 12-18 hours), an average rate of about 1 division every about 19 hours (e.g., 20-22 hours), or an average rate of about 20 divisions every about 24 hours (e.g., 20-22 hours). The engineered and / or non-engineered γδ T cells may be stimulated to proliferate at an average rate of about one division every about 10-19 hours or 12-19 hours), at an average rate of about one division every about 20 hours (e.g., 12-20 hours, 16-20 hours or 18-20 hours), at an average rate of about one division every about 21 hours (e.g., 12-21 hours, 16-21 hours or 18-21 hours), at a rate of about one division every about 22 hours (e.g., 12-22 hours, 16-22 hours or 18-22 hours), at a rate of about one division every about 23 hours or less (e.g., 12-23 hours, 16-23 hours or 18-23 hours), at an average rate of about one division every about 24 hours (e.g., 12-24 hours, 16-24 hours or 18-24 hours).

[0151] In some cases, the activator 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, 16-27 hours, 18-26 hours, or 20-26 hours), or an average rate of about 1 division every 27 hours (e.g., 12-27 hours, 16-27 hours, 18-27 hours, or 20-27 hours) in a γδ T cell expansion culture 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, 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 1 division every 0 hours), an average rate of approximately 1 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 1 division every 32 hours (e.g., 18-32 hours, 20-32 hours, 22-32 hours or 24-32 hours), an average rate of approximately 1 division every 33 hours (e.g., 18-33 hours, 20-33 hours, 22-33 hours or 24-33 hours) The invention may stimulate the proliferation of engineered and / or non-engineered γδ T cells at an average rate of about one division every about 34 hours (e.g., 18-34 hours, 20-34 hours, 22-34 hours or 24-34 hours), about one division every about 35 hours (e.g., 18-35 hours, 20-35 hours, 22-35 hours or 24-35 hours), about one division every about 36 hours (e.g., 18-36 hours, 20-36 hours, 22-36 hours or 24-36 hours).

[0152] In some cases, the activator may induce an average rate of about 1 division every about 12 hours (e.g., 10-12 hours), an average rate of about 1 division every about 13 hours (e.g., 10-13 hours), an average rate of about 1 division every about 14 hours (e.g., 10-14 hours), an average rate of about 1 division every about 15 hours (e.g., 10-15 hours), an average rate of about 1 division every about 16 hours (e.g., 10-16 hours), an average rate of about 1 division every about 17 hours (e.g., 10-17 hours or 12-17 hours), an average rate of about 1 division every about 18 hours (e.g., 10-18 hours or 12-18 hours), an average rate of about 1 division every about 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 about 9 hours or 12-19 hours), an average rate of about one division every about 20 hours (e.g., 12-20 hours, 16-20 hours or 18-20 hours), an average rate of about one division every about 21 hours (e.g., 12-21 hours, 16-21 hours or 18-21 hours), a rate of about one division every about 22 hours (e.g., 12-22 hours, 16-22 hours or 18-22 hours), a rate of about one division every about 23 hours or less (e.g., 12-23 hours, 16-23 hours or 18-23 hours), or an average rate of about one division every about 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 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, 16-27 hours, 18-27 hours, or 20-26 hours), or an average rate of about 1 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 about one division every 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 about 1 division, an average rate of about 1 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 1 division every 32 hours (e.g., 18-32 hours, 20-32 hours, 22-32 hours, or 24-32 hours), an average rate of about 1 division every 33 hours (e.g., 18-33 hours, 20-33 hours, 22-33 hours, or 24-33 hours), an average rate of about 1 division every 3 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 about 35 hours (e.g., 18-35 hours, 20-35 hours, 22-35 hours or 24-35 hours), about one division every about 36 hours (e.g., 18-36 hours, 20-36 hours, 22-36 hours or 24-36 hours).

[0154] In some cases, the activator may induce an average rate of about 1 division every about 12 hours (e.g., 10-12 hours), an average rate of about 1 division every about 13 hours (e.g., 10-13 hours), an average rate of about 1 division every about 14 hours (e.g., 10-14 hours), an average rate of about 1 division every about 15 hours (e.g., 10-15 hours), an average rate of about 1 division every about 16 hours (e.g., 10-16 hours), an average rate of about 1 division every about 17 hours (e.g., 10-17 hours or 12-17 hours), an average rate of about 1 division every about 18 hours (e.g., 10-18 hours or 12-18 hours), an average rate of about 1 division every about 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 about 9 hours or 12-19 hours), an average rate of about one division every about 20 hours (e.g., 12-20 hours, 16-20 hours or 18-20 hours), an average rate of about one division every about 21 hours (e.g., 12-21 hours, 16-21 hours or 18-21 hours), a rate of about one division every about 22 hours (e.g., 12-22 hours, 16-22 hours or 18-22 hours), a rate of about one division every about 23 hours or less (e.g., 12-23 hours, 16-23 hours or 18-23 hours), or an average rate of about one division every about 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 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, 16-27 hours, 18-27 hours, or 20-26 hours), or an average rate of about 1 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 about one division every 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 about 1 division, an average rate of about 1 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 1 division every 32 hours (e.g., 18-32 hours, 20-32 hours, 22-32 hours, or 24-32 hours), an average rate of about 1 division every 33 hours (e.g., 18-33 hours, 20-33 hours, 22-33 hours, or 24-33 hours), an average rate of about 1 division every 3 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 about 35 hours (e.g., 18-35 hours, 20-35 hours, 22-35 hours or 24-35 hours), about one division every about 36 hours (e.g., 18-36 hours, 20-36 hours, 22-36 hours or 24-36 hours).

[0156] An activating agent may stimulate the proliferation of a subpopulation of engineered or non-engineered γδ T cells at various growth rates. For example, an agent may 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 an αβ T cell population in culture. In some embodiments, stimulation may be at a faster rate such that stimulation 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 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. In some embodiments, the cells may be stimulated at a faster rate such that the proliferation 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.

[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. In some embodiments, the cells may be stimulated at a faster rate such that the proliferation 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.

[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 proliferation 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 the growth of the δ2 ​​population at a faster rate such that the 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) 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 the period of 1 day to 90 days of culture (e.g., from about 1 day to about 19, 21 or 23 days of culture).

[0161] In some aspects, the disclosure provides engineered or non-engineered γδ T cell populations in contact 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, for example, comprise 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 an APC or bound to an Fc receptor expressed on the surface of an 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 a particular embodiment, the invention provides a mixture of expanded γδ T cell populations comprising 10-90% δ1 T cells and 90-10% δ2 T cells. In a particular embodiment, 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 a particular embodiment, 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 a particular embodiment, 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 the γδ T cells (e.g., activator γδ T cell natural receptors), and then a costimulatory molecule may contact the γδ T cells to provide further stimulation and proliferate the γδ T cells. 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 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 can be generated by various methods known in the art. Engineered γδ T-cells can be engineered to stably express a particular tumor recognition moiety. A polynucleotide encoding an expression cassette comprising a tumor recognition moiety or another type of recognition moiety can be delivered to the cell by transposon / transposase systems or virus-based gene transfer systems, such as lentivirus or retrovirus systems, or another suitable method, such as transfection, electroporation, transduction, lipofection, calcium phosphate (CaPO 4), nanoengineered materials such as Ormosil, viral delivery methods including 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 comprising the genetic code of the antigen-specific TCR into the genome of the γδ T cell. A polynucleotide encoding a CAR with 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 incorporated into the genome of the engineered γδ T cell comprises a polynucleotide encoding an engineered TCRα (TCR alpha) gene, an engineered TCRβ (TCR beta) gene, a TCRδ (TCR delta) gene, or an engineered TCRγ (TCR gamma) gene. In some cases, the expression cassette incorporated into the genome of the engineered γδ T cell comprises a polynucleotide encoding an antibody fragment or an antigen-binding portion thereof. In some cases, the antibody fragment or antigen-binding fragment thereof is a whole antibody, antibody fragment, single chain variable fragment (scFv), single domain antibody (sdAb), Fab, F(ab) or IgG antibody that binds to a cell surface tumor antigen as part of a chimeric antigen receptor (CAR) construct or a bispecific construct that includes a CAR and a T cell receptor (TCR) or includes a CAR with an antibody directed against a different antigen. 2The polynucleotide is a polynucleotide that encodes the Fc, Fc, light or heavy chains on the antibody, variable or constant regions of the antibody, or any combination thereof. In some cases, the polynucleotide is derived from a human or from another species. The antibody fragment or antigen-binding fragment polynucleotides from a non-human species can be modified to improve their similarity to antibody variants that naturally occur in humans, and the antibody fragment or antigen-binding fragment can be partially or fully humanized. The antibody fragment or antigen-binding fragment polynucleotide can also be chimeric, such as a mouse-human antibody chimera. The engineered γδ T-cell expressing the 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 the tumor recognition moiety 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, respectively, as described in WO201409370, WO2003087341, WO2014134412, and WO2011090804, each of which is incorporated herein by reference in its entirety, can be used to provide 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 result in the knock-out of one gene and the knock-in of another. For example, a polynucleotide comprising an expression cassette of the present disclosure can be inserted into a genomic region that encodes an MHC gene. Such manipulations can result in the simultaneous knock-in of one or more genes, for example a gene 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 (hereby incorporated by reference in its entirety), may be used to introduce the polynucleotide into the engineered γδ T-cells.

[0172] In some cases, a polynucleotide comprising a tumor recognition moiety is introduced into the genome of engineered γδ T cells using a viral method. 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 retrovirus, adenovirus, lentivirus, herpes simplex virus, vaccinia virus, poxvirus, or adenovirus-associated virus methods.

[0173] The polynucleotide containing the genetic code for the tumor recognition moiety may include a mutation or other transgene that affects the growth, proliferation, 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 may be engineered to express a polynucleotide that includes an activation domain linked to an antigen recognition moiety, e.g., a molecule in the TCR-CD3 complex, or a costimulatory factor. The engineered γδ T-cells may express an intracellular signaling domain that is a T lymphocyte activation domain. The γδ T-cells may be engineered to express an intracellular activation domain gene or an intracellular signaling domain. The intracellular signaling domain gene may 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, cellular receptors, or other immune-modulating molecules.

[0174] A suitable 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 a cancer cell. 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 include a ligand, such as an IL-13 ligand, or a ligand mimic for a tumor antigen, such as an IL-13 mimic for IL13R.

[0175] γδ T cells can be engineered to express chimeric tumor recognition moieties that include 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-Met, C The antibody binds to one or more of the following antigens: SPG4, CD44v6, PVRL-4, VEGFR2, C4.4a, PSCA, folate binding protein / receptor, SLC44A4, Cripto, CTAG1B, AXL, IL-13Rα2, IL-3R, IL-3, EPHA3, SLTRK6, gp100, MART1, tyrosinase, SSX2, SSX4, NYESO-1, epithelial tumor antigen (ETA), MAGEA family genes (e.g., MAGEA3.MAGEA4), KKLC1, mutated 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 MHC class I molecules (HLA-A, HLA-B, or HLA-C) complexed with tumor-associated peptides. Methods and compositions for generating and using tumor recognition moieties that target tumor-associated peptides complexed with MHC class I molecules include, for example, those described 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; US9,540,448, and WO2017 / 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 from 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 one 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 an 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 a peptide-MHC complex, each antibody directed against the same or different peptide complexed with the same or different MHC haplotype. In some cases, the tumor recognition moiety can be an antibody that binds to a 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 mismatching within the engineered cells. The tumor recognition moieties expressed from two or more expression cassettes preferably have different polynucleotide sequences, e.g., encoding 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 TCR and CAR tumor recognition moieties. 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 activation and inactivation domains of target cells, thereby providing improved target specificity. A γδ T-cell can be engineered to express at least one tumor recognition moiety, at least two tumor recognition moieties, at least three tumor recognition moieties, at least four tumor recognition moieties, at least five tumor recognition moieties, at least six tumor recognition moieties, at least seven tumor recognition moieties, at least eight tumor recognition moieties, at least nine tumor recognition moieties, at least ten tumor recognition moieties, at least eleven tumor recognition moieties, at least twelve 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 include genetic coding for the tumor recognition moiety, the linker moiety, and the activation domain. Translation of the polynucleotide by the engineered γδ T cell can provide the tumor recognition moiety and the activation domain joined together by a protein linker. In most cases, the linker includes amino acids that do not interfere with folding of the tumor recognition moiety and the 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 be, for example, 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 made to modify the encoded amino acid into a polar, non-polar, basic or acidic amino acid. Nucleic acid mutations can be made 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 gamma delta T cell may have more than one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, seven amino acid mutations, eight amino acid mutations, nine amino acid mutations, ten amino acid mutations, eleven amino acid mutations, twelve amino acid mutations, thirteen amino acid mutations, fourteen amino acid mutations, fifteen amino acid mutations, sixteen amino acid mutations, seventeen amino acid mutations, eighteen amino acid mutations, nineteen amino acid mutations, twenty amino acid mutations, twenty one amino acid mutations, twenty two amino acid mutations, twenty three amino acid mutations, twenty four amino acid mutations, twenty four amino acid mutations, twenty five ... 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, one or more of the MHC genes are completely deleted, specific exons are deleted, or β2 microglobulin (B2m) is deleted. 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 be universal donors for human subjects with any HLA haplotype.

[0184] γδ T-cells can be engineered to lack one or various HLA loci(s). Engineered γδ T-cells can be engineered to lack HLA-A alleles, HLA-B alleles, HLA-C alleles, HLA-DR alleles, HLA-DQ alleles, or HLA-DP alleles. In some cases, the HLA alleles are associated with human conditions, such as autoimmune conditions. For example, HLA-B27 alleles are associated with arthritis and uveitis, HLA-DR2 alleles are associated with systemic lupus erythematosus and multiple sclerosis, HLA-DR3 alleles are 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 immediately 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 a 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, FcεRIγ.

[0186] Any portion of the genome of the engineered γδ T cell can be deleted to disrupt the 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 repeats. In some cases, the deleted portion of the genome ranges from 1 nucleic acid to about 10 nucleic acids, 1 nucleic acid to about 100 nucleic acids, 1 nucleic acid to about 1,000 nucleic acids, 1 nucleic acid to about 10,000 nucleic acids, 1 nucleic acid to about 100,000 nucleic acids, 1 nucleic acid 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 engineered γδ T cell genomes or disrupt gene expression of at least one HLA locus in engineered γδ T cells. 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 nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases technologies, as described in WO201409370, WO2003087341, WO2014134412, and WO2011090804, each of which is incorporated herein 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, e.g., isolated from intratumoral 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. The homing molecules can be, e.g., lymphocyte homing receptors or cell adhesion molecules. The homing molecules can help the engineered γδ T cells migrate and infiltrate solid tumors, including targeted solid tumors, when the engineered γδ T cells are 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, integrins, e.g., VLA-4 and LFA-1. Non-limiting examples of cell adhesion molecules include ICAM, N-CAM, VCAM, PE-CAM, L1-CAM, nectin (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 metalloproteases (MMPs), such as MMP9 or MMP2.

[0190] The 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 damage to tissues when the immune system responds to disease conditions, such as cell transformation or infection. The balance of costimulatory and inhibitory signals used to control immune responses from either γδ 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 immune responses "on" or "off". Tumors can dysregulate checkpoint protein function as an immune tolerance mechanism, especially for 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), e.g., 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 a tumor. In some cases, the immune checkpoint gene is a CTLA-4 gene. In some cases, the immune checkpoint gene is a 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 suppress 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 of the present disclosure lack the PD-1 gene locus. In some cases, the expression of the PD-1 gene in the engineered γδ T cells is disrupted by gene editing techniques.

[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 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 with increased affinity for CD80 and CD86 during the early stages of T cell activation. The major 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, the 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 the function of regulatory T cells, 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 tolerance of T cell proliferation and homeostasis. Mitigation of LAG-3 / class II interaction using 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 locus of the LAG3 gene. In some cases, expression of the LAG3 gene in the engineered γδ T cells is disrupted by gene editing technology.

[0195] Phenotype of non-engineered and engineered γδ T cells Engineered γδ T cells can home to specific physical locations in the 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 affect the homing of engineered γδ T cells, including but not limited to CXCR3 (whose ligands are expressed by IP-10 / CXCL10 and 6Ckine / SLC / CCL21), CCR4+CXCR5+ (receptor for RANTES, MIP-1α, MIP-1β), CCR6+ and CCR7. In some cases, engineered γδ T cells can home to sites of inflammation and injury as well as 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 may 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 the subject's body. The engineered γδ T-cells may express one or more homing moieties, e.g., specific TCR alleles and / or lymphocyte homing molecules.

[0196] The engineered γδ T-cells may have a particular phenotype, which may be expressed in terms of cell surface marker expression. Various types of γδ T-cells may 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, e.g., mammalian or synthetic cells.

[0197] The immunophenotype of the activated and / or expanded cell populations can be determined using markers including, but not limited to, CD137, CD27, CD45RA, CD45RO, CCR7 and CD62L (Klebanoff et al., Immunol Rev. 211:214 2006). CD137, or 4-1BB, is an activation-induced costimulatory molecule and a key regulator of the immune response. Pollok et al., J. Immunol. 150, 771-81 (1993). 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 following 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). Additional or alternative activation markers include, but are not limited to, one or more of CD25, PD-1, and CD69.

[0198] antigen The invention disclosed herein provides engineered γδ T cells expressing an antigen recognition moiety, where the antigen recognition moiety recognizes an epitope specific to a disease. The antigen can be a molecule that elicits an immune response. The immune response can involve either antibody production or activation of specific cells capable of immune response, or both. The antigen can be, for example, a peptide, a protein, a hapten, a lipid, a carbohydrate, a bacterium, a pathogen, or a virus. The antigen can be a tumor antigen. The tumor epitope can be presented on the surface of a tumor cell by the MHCI or MHCII complex. The epitope can be a part 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, 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, mutated ras, VRaf, 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 intracellular or extracellular tumor antigens. In some cases, the αβ TCR of the engineered γδ T cell recognizes peptides derived from either intracellular or extracellular tumor antigens. 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 may recognize antigens from cells under stress, such as cancer cells or cells infected with a virus. 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 may be highly expressed by cancer cells as stress markers. The engineered γδ T cells can recognize the MICA or MICB tumor epitopes.

[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 20 nM, at least 1 ... M, at least 30 nM, 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, 40 ...7 nM or less, 8 nM or less, 9 nM or less, 10 nM or less, 20 nM or less, 30 nM or less, 40 nM or less, 5 nM or less, 6 nM or less, 7 nM or less, 8 nM The antigen may be engineered to recognize an antigen with a dissociation constant of M or less, 50nM or less, 60nM or less, 70nM or less, 80nM or less, 90nM or less, 100nM or less, 200nM or less, 300nM or less, 400nM or less, 500nM or less, 600nM or less, 700nM or less, 800nM or less, 900nM or less, 1μM or less, 2μM or less, 3μM or less, 4μM or less, 5μM or less, 6μM or less, 7μM or less, 8μM or less, 9μM or less, 10μM or less, 20μM or less, 30μM or less, 40μM or less, 50μM or less, 60μM or less, 70μM or less, 80μM or less, 90μM or less or 100μM or less.

[0204] Treatment The pharmaceutical compositions described herein containing non-manipulated enriched γδ T cell populations, engineered enriched γδ T cell populations and / or mixtures thereof may be administered for prophylactic and / or therapeutic treatments. Additionally or alternatively, pharmaceutical compositions containing one or more agents that selectively expand γδ T cell populations as described herein may be administered for prophylactic and / or therapeutic treatments. In therapeutic applications, a subject already suffering from a disease or condition may be administered an amount of the composition sufficient to cure or at least partially arrest the symptoms of the disease or condition. The composition may also be administered to reduce the likelihood of developing, contracting, or worsening the condition. The effective amount of non-manipulated enriched γδ T cell populations, engineered enriched γδ T cell populations, mixtures thereof, and / or one or more agents that selectively expand γδ T cell populations used for treatment may 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.

[0205] The compositions of the present disclosure can be used to treat subjects 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 cows, horses, sheep, goats, pigs; a domestic animal, such as rabbits, dogs, and cats; a laboratory animal, such as a rodent, such as rats, mice, and guinea pigs. 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.

[0206] The compositions and combinations thereof of the present disclosure may be administered in various regimens (e.g., timing, concentration, dosage, treatment interval, and / or dosage form). The subject may also be pretreated, for example, with chemotherapy, radiation, or a combination of both, prior to receiving the in vivo expansion agent and / or enriched γδ T cell population of the present disclosure, or a mixture thereof. As part of the treatment, the composition may be administered to the subject in a first regimen, and the subject may be monitored to determine whether the treatment meets a given level of therapeutic efficacy in the first regimen. In some cases, the engineered γδ T cells or another engineered γδ T cell may be administered to the subject in a second regimen.

[0207] In some embodiments, the first procedure involves administering at least one composition as described herein to a subject having or suspected of having a given condition (e.g., cancer). The composition may be administered in a first regimen. In the second procedure, the subject may be monitored, for example, by a health care provider (e.g., a treating physician or nurse). In some instances, the subject is monitored to determine or measure the effectiveness of the composition in treating the subject's condition. In some circumstances, the subject may be monitored to determine the in vivo activation, proliferation, or cell count of the γδ T cell population in the subject. Then, in a third procedure, at least one composition as described herein is administered to the subject in a second regimen. The second regimen may be the same as the first regimen or may be different from the first regimen. In some circumstances, the third procedure is not performed, for example, if administration of the composition in the first procedure 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, the population of engineered γδ T cells may be administered to a variety of subjects with different MHC haplotypes. The engineered γδ T-cells may be frozen or cryopreserved prior to administration to a subject.

[0208] Enriched populations and / or mixtures of γδ T-cells (i.e., engineered or non-engineered) may be frozen or cryopreserved prior to administration to a subject, and may optionally be further activated and expanded and / or maintained in vivo by administration of one or more agents that selectively expand the administered γδ T-cells. In certain embodiments, a population of engineered enriched γδ T-cells may comprise 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.

[0209] For example, a population of engineered enriched γδ T cells can 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 can express the NY-ESO-1 oncogene. Infected cells in humans can process the NY-ESO-1 oncoprotein into smaller fragments and present different portions of the NY-ESO-1 protein for antigen recognition. A population of engineered enriched γδ T cells can contain various engineered γδ T cells expressing different tumor recognition moieties designed to recognize different portions of the NY-ESO-1 protein.

[0210] In some embodiments, the invention provides 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 operation, 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 may include two or more cells expressing different tumor recognition moieties that recognize different parts of the NY-ESO-1 protein. In a second operation, the population of engineered γδ T cells may be administered in a first regimen. In a second operation, the subject may be monitored, for example, by a health care provider (e.g., a treating physician or nurse). In a third operation, the subject may be administered one or more agents that selectively expand the administered γδ T cells in vivo, thereby expanding and / or maintaining the administered γδ T cell population in vivo. In a fourth operation, the subject may be monitored to determine the effectiveness of the in vivo expansion and / or maintenance. In some embodiments, the second operation is not performed. In some embodiments, the fourth operation is not performed.

[0211] One or more compositions of the present disclosure may be used to treat a variety of conditions. In some cases, the compositions of the present disclosure may 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, appendix cancer, astrocytoma, neuroblastoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors, such as 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, and the like. 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, cardiac cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liposarcoma, liver cancer, lung cancer, e.g. 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 cavity cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myeloid leukemia, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, epithelial ovarian cancer, ovarian germ cell tumor, pancreatic cancer, 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.

[0212] In some cases, the compositions 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, Clostridium tetani; g) the genus Corynebacterium, such as the species Corynebacterium diphtheria species; h) the genus Enterococcus, such as the species Enterococcus faecalis and / or Enterococcus faecium; i) the genus Escherichia, such as the species Escherichia coli; j) the genus Francisella, such as the species Francisella tularensis; k) the genus Haemophilus, such as the species Haemophilus influenza; l) the genus Helicobacter, such as 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 the species Salmonella typhi and / or Salmonella typhimurium; v) the genus Shigella, for example the species Shigella sonnei species; w) the genus Staphylococcus, such as the species Staphylococcus aureus, Staphylococcus epidermidis, and / or Staphylococcus saprophyticus; x) the genus Streptpcoccus, such as the species Streptococcus agalactiae, Streptococcus pneumonia, and / or Streptococcus pyogenes; y) the genus Treponema, such as the species Treponema pallidum; z) the genus Vibrio, such as the species Vibrio cholera; and / or aa) the genus Yersinia, such as the species Yersinia pestis;

[0213] In some cases, the compositions of the present disclosure may be used to treat infectious diseases, which may be caused by viruses. Non-limiting examples of viruses may be found in the following virus 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 herpes virus type 8 species; c) Papillomaviridae, e.g., Human papillomavirus species; d) Polyomaviridae, e.g., BK virus. , JC virus species; e) the family Poxviridae, e.g., Variola species; f) the family Hepadnaviridae, e.g., Hepatitis B virus species; g) the family Parvoviridae, e.g., Human Bocavirus, Parvovirus B19 species; h) the family Astroviridae, e.g., Human Astrovirus species; i) the family Caliciviridae, e.g., Norwalk virus species; j) the family Flaviviridae, e.g., 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 s) 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 instances, the virus is one that cannot be assigned to a virus family, such as Hepatitis D.

[0214] In some cases, the compositions of the present disclosure can be used to treat immune diseases, such as autoimmune diseases. 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, ulcerative colitis, 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 disorders, Myasthenia gravis, 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-A 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.

[0215] Treatment with the compositions of the present disclosure may be provided to a subject before, during and after clinical onset of symptoms. Treatment may be provided to a subject 1 day, 1 week, 6 months, 12 months or 2 years after clinical onset of disease. Treatment may 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 clinical onset of disease. Treatment may be provided to a subject less than 1 day, 1 week, 1 month, 6 months, 12 months or 2 years after clinical onset of disease. Treatment may also include treating humans in clinical trials. Treatment may include administering to a subject a pharmaceutical composition comprising one or more agents that selectively expand a γδ T cell population. Treatment may include administering to a subject a non-manipulated enriched γδ T cell population of the present disclosure, an engineered enriched γδ T cell population and / or a mixture thereof. In some cases, the pharmaceutical composition comprises one or more agents of the present disclosure that selectively expand a γδ T cell population of the present disclosure and a non-manipulated enriched γδ T cell population, an engineered enriched γδ T cell population, and / or a mixture thereof.

[0216] In some cases, administration of the compositions of the present disclosure to a subject modulates the activity of endogenous lymphocytes in the subject. In some cases, administration of the compositions of the present disclosure to a subject can provide antigens 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 composition of the present disclosure to a subject activates the cytotoxicity of another immune cell. In some cases, the other immune cell is a CD8+ T cell. In some cases, the other immune cell is a natural killer T cell. In some cases, administration of the composition to a subject suppresses a regulatory T cell. In some cases, the regulatory T cell is a Fox3+ T cell. In some cases, the regulatory T cell is a Fox3- T cell. Non-limiting examples of cells whose activity can be regulated by a γδ T cell population include hematopoietic stem cells, B cells, CD4, CD8, red blood cells, white blood cells, dendritic cells, such as 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.

[0217] During most bone marrow transplants, a combination of cyclophosphamide and total body irradiation is routinely employed to prevent rejection of 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 co-administration of γδ T cells with interleukin-2. However, both low and high doses of IL-2 may have toxic side effects. IL-2 toxicity may be manifested in multiple organs / systems, most notably the heart, lungs, kidneys and central nervous system. In some cases, the disclosure provides methods of administering a non-engineered enriched γδ T cell population, an engineered enriched γδ T cell population, and / or a mixture thereof to a subject without co-administration of a cytokine, e.g., IL-2, IL-15, IL-12, or IL-21. In some cases, a 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, a 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.

[0218] In some cases, the disclosure provides methods of administering non-engineered enriched γδ T cell populations, engineered enriched γδ T cell populations, and / or mixtures thereof to a subject with simultaneous or sequential co-administration of a cytokine or other stimulant, such as 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), or granulocyte colony stimulating factor (G-CSF). In some cases, the cytokine is IL-2, IL-15, IL-12, or IL-21. In some cases, the cytokine is IL-2. In some cases, the cytokine is IL-15. In some cases, the cytokine is IL-4. In some cases, the cytokine is a common gamma chain cytokine selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, or a combination thereof.

[0219] Method of administration One or more compositions of the invention comprising selective expansion agents, non-manipulated enriched γδ T cell populations, engineered enriched γδ T cell populations, and / or mixtures thereof can be administered to a subject in any order or simultaneously. If simultaneously, the compositions can be provided in a single combined form, such as an intravenous injection, or as a multiple form, for example, as multiple intravenous infusions, sc, injections, or tablets. The compositions can be packaged together or separately in a single package or multiple packages. One or all of the compositions of the invention can be given in multiple doses. If not simultaneously, the timing between multiple doses can vary from about one week, one month, two months, three months, four months, five months, six months, or even up to about one year. In some cases, the administered γδ T cell populations, engineered enriched γδ T cell populations, and / or mixtures thereof can be expanded in vivo in the subject's body after administration to the subject. The pharmaceutical composition comprising γδ T cells and / or activating agents can be packaged as a kit. The kits can include, in addition to one or more of the compositions described herein, instructions (eg, instructions) for use of the composition.

[0220] In some cases, the method of treating cancer comprises administering a composition described herein, whereby the cancer is treated. In some embodiments, a therapeutically effective amount of the composition 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.

[0221] One or more compositions described herein can be administered before, during or after the onset of a disease or condition, and the timing of administering the pharmaceutical composition can vary. For example, one or more compositions can be used as a prophylactic and can be administered continuously to a subject with a tendency to a condition or disease to reduce the likelihood of the onset of the disease or condition. One or more compositions can be administered to a subject during the onset of symptoms or as soon as possible thereafter. Administration of one or more compositions can begin as soon as possible, up to the time of 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 from onset of symptoms, or by any time from onset of symptoms. Initial administration can be via any practical route, for example, by any route described herein using any formulation described herein. In some examples, administration of one or more compositions of the present disclosure is intravenous administration. Single or multiple dosages of one or more compositions may be administered as soon as practicable after onset of cancer, infectious disease, immune disease, sepsis, or in conjunction with a bone marrow transplant and for the length of time required to treat the immune disease, for example, 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, from about 1 month to about 3 months. For the treatment of cancer, single or multiple dosages of one or more compositions may be administered several years after onset of the cancer and before or after other treatments. In some examples, one or more compositions described herein 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 for each subject.

[0222] Dosage The non-manipulated enriched γδ T cell populations, the manipulated enriched γδ T cell populations and / or mixtures thereof disclosed herein may be formulated as unit dosage forms suitable for single administration of precise dosages. In some cases, the unit dosage forms include additional lymphocytes. In unit dosage forms, the formulation is divided into unit doses containing appropriate amounts of one or more compounds. The unit dosages may be in the form of packages containing individual amounts of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions may be in single-dose non-reclosable containers. Multi-dose reclosable containers may be used, for example, in combination with or without preservatives. In some examples, the pharmaceutical composition does not include preservatives. Formulations for parenteral injection may be provided in unit dosage forms, for example, in ampoules, or in multi-dose containers together with a preservative.

[0223] The non-engineered enriched γδ T cell populations, engineered enriched γδ T cell populations and / or mixtures thereof described herein may comprise 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 104 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 6 Cells, 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 108 Cells, at least 8 x 10 8 Cells, at least 9 x 10 8 Cells, at least 1 x 10 9 Cells or greater amounts may be present in the composition.

[0224] 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 3 Cells, about 1 cell ~ about 6 x 10 3 Cells, about 1 cell ~ about 7 x 10 3 Cells, about 1 cell ~ about 8 x 10 3 Cells, about 1 cell ~ about 9 x 10 3 Cells, about 1 cell ~ about 1 x 10 4 Cells, about 1 cell ~ about 2 x 10 4 Cells, about 1 cell ~ about 3 x 10 4 Cells, about 1 cell ~ about 4 x 10 4 Cells, about 1 cell ~ about 5 x 10 4 Cells, about 1 cell ~ about 6 x 10 4 Cells, about 1 cell ~ about 7 x 10 4 Cells, about 1 cell ~ about 8 x 10 4 Cells, about 1 cell ~ about 9 x 10 4 Cells, about 1 cell ~ about 1 x 10 5 Cells, about 1 cell ~ about 2 x 10 5 Cells, about 1 cell ~ about 3 x 10 5 Cells, about 1 cell ~ about 4 x 10 5 Cells, about 1 cell ~ about 5 x 10 5 Cells, about 1 cell ~ about 6 x 10 5 Cells, about 1 cell ~ about 7 x 10 5 Cells, about 1 cell ~ about 8 x 105 Cells, about 1 cell ~ about 9 x 10 5 Cells, about 1 cell ~ about 1 x 10 6 Cells, about 1 cell ~ about 2 x 10 6 Cells, about 1 cell ~ about 3 x 10 6 Cells, about 1 cell ~ about 4 x 10 6 Cells, about 1 cell ~ about 5 x 10 6 Cells, about 1 cell ~ about 6 x 10 6 Cells, about 1 cell ~ about 7 x 10 6 Cells, about 1 cell ~ about 8 x 10 6 Cells, about 1 cell ~ about 9 x 10 6 Cells, about 1 cell ~ about 1 x 10 7 Cells, about 1 cell ~ about 2 x 10 7 Cells, about 1 cell ~ about 3 x 10 7 Cells, about 1 cell ~ about 4 x 10 7 Cells, about 1 cell ~ about 5 x 10 7 Cells, about 1 cell ~ about 6 x 10 7 Cells, about 1 cell ~ about 7 x 10 7 Cells, about 1 cell ~ about 8 x 10 7 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 about 1 cell to about 1 x 10 9 It may be a cell.

[0225] In some cases, a therapeutically effective dose of the non-manipulated enriched γδ T cell population, the engineered 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 3Cells, about 1×10 3 cells~about 5×10 3 Cells, about 1×10 3 cells~about 6×10 3 Cells, about 1×10 3 cells~about 7×10 3 Cells, about 1×10 3 cells~about 8×10 3 Cells, about 1×10 3 cells~about 9×10 3 Cells, about 1×10 3 cells~about 1×10 4 Cells, about 1×10 3 cells~about 2×10 4 Cells, about 1×10 3 cells~about 3×10 4 Cells, about 1×10 3 cells~about 4×10 4 Cells, about 1×10 3 cells~about 5×10 4 Cells, about 1×10 3 cells~about 6×10 4 Cells, about 1×10 3 cells~about 7×10 4 Cells, about 1×10 3 cells~about 8×10 4 Cells, about 1×10 3 cells~about 9×10 4 Cells, about 1×10 3 cells~about 1×10 5 Cells, about 1×10 3 cells~about 2×10 5 Cells, about 1×10 3 cells~about 3×10 5 Cells, about 1×10 3 cells~about 4×10 5 Cells, about 1×10 3 cells~about 5×10 5 Cells, about 1×10 3 cells~about 6×10 5 Cells, about 1×10 3 cells~about 7×10 5 Cells, about 1×10 3 cells~about 8×10 5 Cells, about 1×10 3 cells~about 9×10 5 Cells, about 1×10 3 cells~about 1×106 Cells, about 1×10 3 Cells ~ about 2×10 6 Cells, about 1×10 3 Cells ~ about 3×10 6 Cells, about 1×10 3 Cells ~ about 4×10 6 Cells, about 1×10 3 Cells ~ about 5×10 6 Cells, about 1×10 3 Cells ~ about 6×10 6 Cells, about 1×10 3 Cells ~ about 7×10 6 Cells, about 1×10 3 Cells ~ about 8×10 6 Cells, about 1×10 3 Cells ~ about 9×10 6 Cells, about 1×10 3 Cells ~ about 1×10 7 Cells, about 1×10 3 Cells ~ about 2×10 7 Cells, about 1×10 3 Cells ~ about 3×10 7 Cells, about 1×10 3 Cells ~ about 4×10 7 Cells, about 1×10 3 Cells ~ about 5×10 7 Cells, about 1×10 3 Cells ~ about 6×10 7 Cells, about 1×10 3 Cells ~ about 7×10 7 Cells, about 1×10 3 Cells ~ about 8×10 7 Cells, about 1×10 3 Cells ~ about 9×10 7 Cells, about 1×10 3 Cells ~ about 1×10 8 Cells, about 1×10 3 Cells ~ about 2×10 8 Cells, about 1×10 3 Cells ~ about 3×10 8 Cells, about 1×10 3 Cells ~ about 4×10 8 Cells, about 1×10 3 Cells ~ about 5×10 8 Cells, about 1×10 3 Cells ~ about 6×10 8 Cells, about 1×10 3cells ~ 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.

[0226] In some cases, a therapeutically effective dose of the non-manipulated enriched γδ T cell population, the engineered 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 10 6 cells ~ approx. 7 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 8 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 9 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 1 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 2 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 3 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 4 x 10 7 cells, approximately 1 x 10 6 Cells ~ approx. 5 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 6 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 7 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 8 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 9 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 1 x 108 Cells, about 1×10 6 cells ~ about 2×10 8 Cells, about 1×10 6 cells ~ about 3×10 8 Cells, about 1×10 6 cells ~ about 4×10 8 Cells, about 1×10 6 cells ~ about 5×10 8 Cells, about 1×10 6 cells ~ about 6×10 8 Cells, about 1×10 6 cells ~ about 7×10 8 Cells, about 1×10 6 cells ~ about 8×10 8 Cells, about 1×10 6 cells ~ about 9×10 8 Cells, about 1×10 6 cells ~ about 1×10 9 Cells, about 1×10 6 cells ~ about 2×10 9 Cells, about 1×10 6 cells ~ about 3×10 9 Cells, about 1×10 6 cells ~ about 4×10 9 Cells, about 1×10 6 cells ~ about 5×10 9 Cells, about 1×10 6 cells ~ about 6×10 9 Cells, about 1×10 6 cells ~ about 7×10 9 Cells, about 1×10 6 cells ~ about 8×10 9 Cells, about 1×10 6 cells ~ about 9×10 9 Cells, about 1×10 7 cells ~ about 1×10 9 Cells, about 1×10 7 cells ~ about 2×10 9 Cells, about 1×10 7 cells ~ about 3×10 9 Cells, about 1×10 7 cells ~ about 4×10 9 Cells, about 1×10 7 cells ~ about 5×10 9 Cells, about 1×10 7 cells ~ about 6×10 9 Cells, about 1×10 7cells ~ approx. 7 x 10 9 cells, approximately 1 x 10 7 cells ~ approx. 8 x 10 9 cells, approximately 1 x 10 7 cells ~ approx. 9 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 1 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 2 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 3 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 4 x 10 9 cells, approximately 1 x 10 8 Cells ~ approx. 5 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 6 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 7 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 8 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 9 x 10 9 cells, or approximately 1 x 10 9 cells ~ approx. 1 x 10 10 It may be a cell.

[0227] When an antibody or other activator is administered, e.g., an agent that binds to or competes with the same or essentially the same epitope as an antibody described in any one of Figures 1-5, typical dosages may vary from about 10 ng / kg to up to 100 mg / kg or more of mammalian body weight per day, preferably from about 1 μg / kg / day to 10 mg / kg / day, depending on the route of administration. Guidance regarding specific dosages and delivery methods is provided in the literature, see, e.g., U.S. Pat. Nos. 4,657,760, 5,206,344, or 5,225,212. It is expected that different formulations will be effective for different therapeutic compounds and different disorders, e.g., administration targeted to one organ or tissue may require delivery to another organ or tissue in a different manner.

[0228] For treating or reducing the severity of immune-related diseases, the appropriate dosage of the compositions of the invention will depend on the type of disease being treated, the severity and course of the disease, whether the agent is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the compound, and the discretion of the attending physician, as defined above. The compositions may be suitably administered to the subject at one time or over a series of treatments.

[0229] For example, depending on the type and severity of the disease, about 1 mg / kg to 15 mg / kg (e.g., 0.1 to 20 mg / kg) of activator (e.g., polypeptide or antibody) is an initial candidate dosage for administration to a subject, whether by one or more separate administrations or by continuous infusion. Typical daily dosages may range from about 1 mg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or more, treatment is maintained until a desired suppression of disease symptoms occurs, depending on the condition. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0230] keep In some embodiments, enriched γδ T cell populations obtained by in vivo activation or ex vivo expansion of expanded γδ T cell populations, and / or mixtures thereof, may be formulated with freezing medium and placed in a cryopreservation unit such as a liquid nitrogen freezer (−195° C.) or a deep 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 may contain dimethyl sulfoxide (DMSO), and / or sodium chloride (NaCl), and / or dextrose, and / or dextran sulfate, and / or hydroxyethyl starch (HES) with physiological pH buffers 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. In some cases, non-manipulated γδ T-cells can be expanded by methods described herein, which methods include a step of in vivo expansion, genetically modified and cryopreserved.

[0231] In this manner, the engineered and / or non-engineered γδ T cells can be further cryopreserved to produce at least about 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 Or at least about 10 10A cell bank in the amount of at least 1, 5, 10, 100, 150, 200, 500 vials of cells can be generated. The cryopreserved cell bank can retain its functionality and can be thawed and further stimulated and expanded. In some embodiments, the thawed cells can be stimulated and expanded in a suitable closed vessel, such as a cell culture bag and / or a bioreactor, to generate a large amount of cells as an allogeneic cell product. The 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, the formulation is preservative-free. The cryopreserved γδ T cells can be thawed and administered (e.g., infused) to multiple patients as an allogeneic commercial cell product. The infused cells can be expanded and / or maintained in the subject(s) to which they are administered by administering one or more agents described herein that selectively expand γδ T cells.

[0232] 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 methodologies described therein that may 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. EXAMPLES

[0233] Example 1. Treatment of tumors by in vivo expansion of γδ T cells with one or more agents that selectively bind to the δ1TCR, δ2TCR, and / or δ3TCR The effect of administration of γδ T cell activators on the proliferation and activation of human γδ T cells and the treatment of tumors is examined in xenograft mouse models. Different hematological tumor cell lines (e.g., Raji, Daudi, Mino, NALM6, JVM-2, HL-60, MOLM-13, K562, KG-1a, Mv4-11, MOLT-4, or others) and solid tumor cell lines (e.g., HCT116, colon cancer COLO205, melanoma SK-MEL5, pancreatic BcPC3 or ASPC-1, breast cancer, e.g., MDA-MB-231, prostate cancer, e.g., PC3 or LNCAP, liver cancer HepG2 and Huh7, etc.) were injected subcutaneously, intraperitoneally, orthotopically, or intravenously (e.g., 1×10) into SCID / SCID, NOD-SCID, NSG, NOG® (Taconic), NCG® (Charles River Laboratories), or CD34hu-NSG® (Jackson Labs) mice. 5 ~1×10 7 Subcutaneous, orthotopic, or disseminated tumor growth is measured twice weekly by caliper or imaging on the day of tumor cell administration or when tumors are established (50–200 mm 3 ), or δ1, δ2, or δ3 γδ T cells (1–100×10 6 γδ T cell) are adoptively transferred in PBS via tail vein or intraperitoneal injection into tumor-bearing mice in the absence or presence of cytokines such as IL-2, IL-15, or IL-7. Each group of animals is divided into treatment groups (treated with different activating agents) or vehicle control groups. At predefined time points (e.g., day -1, day 0, day 1, day 2, day 3 or later) before or after γδ T cell adoptive transfer, animals are administered vehicle control or δ1 or δ2 or δ3 specific activating agents. The effect of activating agents in combination with aminobisphosphonates (at the same or different doses and / or dosing schedules) is also tested. Activating agents are administered 1-2 times per week based on drug half-life and potency in mice, for one or more cycles until the end of the study. Activating agents can be given 1-4 times per week at doses of 0.001-1 mg per animal.

[0234] The use of CD34+ hu-NSG® or equivalent mice humanized at birth with engrafted CD34+ progenitor cells shows the effect of activating agents on endogenous γδ T cell populations that arise in such model animals in the presence or absence of human xenograft tumors.

[0235] Example 2. Expansion of γδ T cell populations in vivo Activators are used to activate and expand distinct γδ T cell populations of a subject's endogenous γδ T cells and / or γδ T cells that are administered to the subject, e.g., after ex vivo expansion, as non-manipulated or engineered cells.

[0236] For an in vivo expanded γδ1 T cell population, the subject receives one or more δ1 γδ T cell activators. For an in vivo expanded γδ2 T cell population, the patient receives one or more δ2 γδ T cell activators, and for an in vivo expanded γδ3 T cell population, the patient receives one or more δ3 γδ cell activators. Activators such as MAbs are formulated to be administered via any route capable of delivering the antibody to blood, tumor tissue, and other tissues where the γδ T cell population resides. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumoral, intradermal, etc. Treatment generally involves repeated administration of the activated MAb preparation via an acceptable route of administration, typically at a dose ranging from, but not limited to, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg / kg body weight. Generally, doses ranging from 10 to 1000 mg of MAb per week are effective and well tolerated. Preferably, the initial loading dose is administered as a 90-minute or longer infusion. Periodic maintenance doses are administered as a 30-minute or longer infusion, provided that the initial dose was well tolerated. As will be appreciated by those skilled in the art, various factors may affect the ideal dosing regimen in a particular case. Such factors include, for example, the binding affinity and half-life of the MAb used, the number of circulating or target tissue-resident γδ T cells in the subject, the MAb isotype, the desired steady-state antibody concentration level, frequency of treatment, and the effect of chemotherapeutic or other agents used in combination with the in vivo expansion methods of the invention, as well as the health status of the particular patient.

[0237] An initial loading dose of about 4 mg / kg patient body weight IV, followed by weekly doses of about 2 mg / kg MAb preparation IV, represents an exemplary dosing regimen. One or more activators are administered weekly, biweekly, or monthly, based on the considerations listed above, for one or more cycles of treatment. Non-limiting preferred human unit doses are, for example, 500 μg to 1 mg, 1 mg to 50 mg, 50 mg to 100 mg, 100 mg to 200 mg, 200 mg to 300 mg, 400 mg to 500 mg, 500 mg to 600 mg, 600 mg to 700 mg, 700 mg to 800 mg, 800 mg to 900 mg, 900 mg to 1 g, or 1 mg to 700 mg. In certain embodiments, the dose ranges from 2-5 mg / kg body weight, e.g., a weekly dose of 1-3 mg / kg, followed by, e.g., 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / kg body weight, e.g., 0.5-10 mg / kg body weight, followed by, e.g., 2, 3, or 4 weeks of weekly doses, 225, 250, 275, 300, 325, 350, 375, 400 mg / m of body surface area every week. 2 , 1-600 mg / m2 of body surface area weekly 2 , 225-400 mg / m2 of body surface area weekly 2 These doses can be followed by weekly doses for 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, or 12 or more weeks.

[0238] The dose and frequency of administration are selected to support the expansion or maintenance of a relevant γδ T cell population. The effect on the frequency, phenotype, and activation status of circulating γδ T cells in the patient is examined 1, 2, 3, 7, 14, or 28 days after administration of the activating agent. In addition, the effect on the frequency, phenotype, and activation status of αβ T cells, NK cells, and / or dendritic cells can be examined.

[0239] In some treatment regimens, the activating agent is administered in conjunction with other cytokines, including, but not limited to, IL-2, or IL-15. When the activating agent is administered with an aminobisphosphonate, the aminobisphosphonate (e.g., pamidronate or zoledronic acid) is administered prior to, in conjunction with, or after administration of the activating agent.

[0240] The therapeutic effect of the activator is evaluated based on clinical response. For example, for the treatment of solid tumors, the Recist criteria can be used. Response is measured at 1, 3, 6, 9, and 12 months after the start of treatment. Safety is evaluated using vital signs, electrocardiograms, clinical laboratory results, and adverse events. Tumor assessments, such as computed tomography (CT) scans, are performed at baseline and every 8 weeks while the subject is participating in the study. Serial blood samples are taken from each subject for the presence of infused cells. All statistical tests used in the analysis of efficacy and safety data are two-sided and performed at the 0.05 level of significance, with 90% confidence intervals calculated.

[0241] The described study will be a multicenter trial and will be performed in subjects with locally advanced and / or metastatic hematological or solid cancers who have been previously treated or for which no effective standard treatment is available. Non-manipulated or engineered γδ T cells (1 × 10 at each dose level) will be administered. 8 , 2×10 8 , 5×10 8 , and 1 × 10 9 -1 cohort) will be administered intravenously over 4 hours once or on day 1 of each cycle every 4 weeks.

[0242] Example 3. Expansion of γδ T cell populations in vivo NOG non-tumor bearing mice expressing the hIL-15 transgene were cultured using Taconic (NOD.Cg-Prkdc scid IL2rg tm1SugThe mice were inoculated with Vδ1, Vδ2, or Vδ3 γδ CAR-T cells obtained from the Tg(CMV-IL2 / IL15)1-1Jic / JicTac) and expanded as described in WO2017 / 197347 or WO2019 / 099744. Cells were cryopreserved after expansion and thawed 1 day before administration in cell culture medium containing IL-2. On the day of administration, cells were labeled with CellTrace Violate for 30 min according to the manufacturer's instructions and diluted with 20 × 10 6 Cells / animal were administered i.v. A subset of animals also received an i.p. injection of a nonspecific mouse IgG fraction 4–5 h prior to administration of the cells.

[0243] On day 2 after cell administration, animals were administered Vδ subtype-specific activators as indicated. Control animals received no activators. On days 4 or 5, animals were bled and circulating human γδ T cells were characterized for identity, activation markers, and proliferation. On day 7 after cell administration, animals were sacrificed and various organs were harvested. Spleens and bone marrow were disrupted using a GentleMax (Miltenyi) device and single cell suspensions were analyzed by flow cytometry.

[0244] As shown in FIG. 6, both doses of activator significantly increased total human CD45 in the circulation and lungs compared to control untreated animals. + The number of cells detected was reduced (at 3 and 10 μg per animal). The number of cells found in other tissues tested (bone marrow, spleen) did not experience a significant reduction.

[0245] As shown in Figure 7, cells in blood, bone marrow, and spleen are proliferating by day 5 after treatment with activators, as evidenced by a left shift in the CellTrace Violet profile toward decreased fluorescence intensity due to dye dilution in cell progeny at both dose levels. We also detected CD137 upregulation in circulating cells treated with D1-35 MAb activators, providing a further indication of activation.

[0246] As shown in Figure 8, animals that received the D1-35 activating MAb but not the IgG fraction (row 3) showed increased activation compared to animals that received both the IgG fraction and D1-35 (row 2). Similarly, the activator D1-08 MAb (row 5), a Vδ1-specific antibody that binds to a different epitope than D1-35, also has activating properties in vivo and induces cell proliferation as detected by CellTrace Violet. In contrast, changing the isotype of the D1-35 antibody to hIgG4 significantly impaired the ability of the MAb to induce cell proliferation (row 4). hIgG4 shows significantly reduced affinity for Fc receptors. Thus, the reduced ability to induce cell proliferation may be due to reduced fixation to Fc receptors. Without wishing to be bound by theory, the inventors hypothesize that immobilization on the surface of cells expressing Fc receptors increases the activating effect provided by the administered antibody on the administered γδ T cells, possibly by increasing TCR aggregation by cross-linking with the immobilized antibody. The inventors further hypothesize that this increased TCR aggregation can also be achieved using antigen-binding moieties with high valency, e.g., higher valency than IgG. Thus, one object of the present invention is, for example, a monospecific, highly multivalent γδ T cell activator. For example, robust γδ T cell activation, proliferation, and / or maintenance can be obtained by contacting γδ T cells with, for example, a trivalent, tetravalent, pentavalent, etc. activator. In some cases, the contacting is performed in vivo (e.g., by administering the activator to a subject).

[0247] As shown in Figures 9A-B, proliferation of V52 and V53 cells was detected in the bone marrow and spleen of animals treated with D2-37 and D3-23 MAbs, respectively, as detected by CellTerace Violet dye dilution.

[0248] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerical variations, changes and substitutions will now occur to those skilled in the art without departing from the present invention. It should be understood that various modifications to the embodiments of the present invention described herein may be employed in the practice of the invention. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims and their equivalents are thereby covered.

Claims

1. 1. An in vivo method for activating, expanding, and / or maintaining a γδ T cell population in a subject, comprising administering to the subject an effective amount of one or more agents that selectively expand δ1 T cells, δ2 T cells, or δ3 T cells, or a combination thereof; the one or more agents that selectively expand δ1 T cells bind to an activation epitope that is unique to the δ1 TCR; the one or more agents that selectively expand δ2 T cells bind to an activation epitope that is unique to the δ2 ​​TCR; said one or more agents that selectively expand δ3 T cells bind to an activation epitope that is unique to the δ3 TCR; thereby activating, expanding and / or maintaining the γδ T cell population in the subject.

2. 2. The method of claim 1, wherein the method comprises administering to the subject an effective amount of one or more agents that selectively expand δ1 T cells.

3. 3. The method of claim 2, wherein the agent that selectively expands δ1 T cells is selected from an agent that binds to the same epitope as an antibody selected from TS-1 and TS8.

2.

4. The method of claim 2, wherein the agent that selectively expands δ1 T cells is selected from agents that do not compete with TS-1, TS8.2, or R9.

12.

5. 3. The method of claim 2, wherein the agent that selectively expands δ1 T cells is selected from agents that specifically bind to an epitope comprising the δ1 variable region.

6. 3. The method of claim 2, wherein the agent that selectively expands δ1 T cells binds to an epitope comprising residues Arg71, Asp72, and Lys120 of the δ1 variable region.

7. 3. The method of claim 2, wherein the agent that selectively expands δ1 T cells has reduced binding to mutant δ1 TCR polypeptides comprising a mutation at K120 of deltaJ1 and deltaJ2.

8. 3. The method of claim 2, wherein the agent that selectively expands δ1 T cells is an agent that binds to a δ1 TCR 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 of a human δ1 TCR.

9. 3. The method of claim 2, wherein the agent that selectively expands δ1 T cells is selected from agents that bind to the same or essentially the same epitope as or compete 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.

10. 3. The method of claim 2, wherein 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, δ1-199, δ1-201, δ1-203, δ1-239, δ1-253, δ1-257, δ1-278, δ1-282, and δ1-285.

11. 3. The method of claim 2, wherein the agent that selectively expands δ1 T cells selectively expands δ1 T cells and δ3 T cells.

12. The method of claim 2, wherein said agent that selectively expands δ1 T cells comprises the CDRs of antibody δ1-35 or δ1-08 or binds to the same epitope as antibody δ1-08 or δ1-35.

13. The method of claim 2, wherein the agent that selectively expands δ1 T cells comprises the CDRs of antibody δ1-35.

14. 3. The method of claim 2, wherein the agent selectively expands δ1 T cells selectively expands δ1, δ3, δ4, and δ5 γδ T cells.

15. 2. The method of claim 1, wherein the method comprises administering to the subject an effective amount of one or more agents that selectively expand δ2 T cells.

16. 16. The method of claim 15, wherein the agent that selectively expands δ2 T cells is selected from an agent that binds to the same epitope as an antibody selected from 15D and B6.

17. 16. The method of claim 15, wherein the agent that selectively expands δ2 T cells is selected from agents that specifically bind to an epitope comprising the δ2 ​​variable region.

18. 16. The method of claim 15, wherein the agent that selectively expands δ2 T cells has reduced binding to a mutant δ2 TCR polypeptide comprising a mutation at G35 in the δ2 ​​variable region.

19. 16. The method of claim 15, wherein the agent that selectively expands δ2 T cells binds to a δ2 TCR bin 1 δ2 epitope, a bin 2 δ2 epitope, a bin 3 δ2 epitope or a bin 4 δ2 epitope of a human δ2 TCR.

20. 16. The method of claim 15, wherein the agent that selectively expands δ2 T cells is an agent that binds to or competes with the same or essentially the same epitope as 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.

21. 16. The method of claim 15, wherein the agent that selectively expands δ2 T cells is 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.

22. The method of claim 15, wherein the agent that selectively expands δ2 T cells comprises the CDRs of antibody δ2-37 or binds to the same epitope as antibody δ2-37.

23. 2. The method of claim 1, wherein the method comprises administering to the subject an effective amount of one or more agents that selectively expand δ3 T cells.

24. 24. The method of claim 23, wherein said agent that selectively expands δ3 T cells is selected from agents that bind to the same or essentially the same epitope as or compete with an antibody selected from the group consisting of δ3-08, δ3-20, δ3-23, δ3-31, δ3-42, δ3-47, and δ3-58.

25. 24. The method of claim 23, wherein said agent that selectively expands δ3 T cells is an antibody selected from the group consisting of δ3-08, δ3-20, δ3-23, δ3-31, δ3-42, δ3-47, and δ3-58.

26. The method of claim 23, wherein said agent that selectively expands δ3 T cells comprises the CDRs of antibody δ3-23 or binds to the same epitope as antibody δ3-23.

27. The method according to any one of claims 1 to 26, wherein the method comprises administering to the patient a population of engineered and / or non-engineered γδ T-cells.

28. 28. The method of claim 27, wherein the method comprises administering the population of engineered and / or non-engineered γδ T-cells prior to administering the one or more agents that selectively expand δ1 T cells, δ2 T cells, or δ3 T cells.

29. 28. The method of claim 27, wherein the administered population of engineered and / or non-engineered γδ T-cells is a population of cells autologous to the subject.

30. 28. The method of claim 27, wherein the administered population of engineered and / or non-engineered γδ T-cells is a population of cells allogeneic to the subject.

31. 28. The method of claim 27, wherein the administered population of engineered and / or non-engineered γδ T-cells is a population comprising at least 60% δ1 γδ T-cells, and the method comprises administering the γδ T-cells and sequentially or simultaneously administering the one or more agents that selectively expand δ1 T-cells.

32. 28. The method of claim 27, wherein the administered population of engineered and / or non-engineered γδ T-cells is a population comprising at least 60% δ2 γδ T-cells, and the method comprises administering γδ T-cells and sequentially or simultaneously administering one or more agents that selectively expand δ2 T-cells.

33. 28. The method of claim 27, wherein the administered population of engineered and / or non-engineered γδ T-cells is a population comprising at least 60% δ3 γδ T-cells, and the method comprises administering γδ T-cells and sequentially or simultaneously administering one or more agents that selectively expand δ3 T-cells.

34. 34. The method of any one of claims 1 to 33, wherein the method comprises administering an aminophosphonate or a prenylphosphonate.

35. 35. The method of claim 34, wherein the method comprises administering an aminophosphonate, the aminophosphonate being selected from the group consisting of zoledronate, pamidronic acid, alendronic acid, risedronic acid, ibandronic acid, and incadronic acid, or salts thereof and / or hydrates thereof.

36. 36. The method of any one of claims 1 to 9, 12 to 24, or 25 to 35, wherein the agent that selectively expands δ1 T cells, δ2 T cells, or δ3 T cells, or a combination thereof, is multivalent, preferably wherein the multivalent agent comprises at least two antigen binding sites that specifically bind to the same antigen, or wherein the multivalent agent comprises at least two antigen binding sites that specifically bind to the same epitope of the same antigen.

37. 37. The method of claim 36, wherein the agent that selectively expands δ1 T cells, δ2 T cells, or δ3 T cells, or a combination thereof, is bivalent, trivalent, or tetravalent, or is at least bivalent, trivalent, or tetravalent.

38. The method of any one of claims 1 to 37, wherein the method comprises simultaneously or sequentially administering cytokines to the subject.

39. 39. The method of claim 38, wherein the method comprises administering to the subject an engineered γδ T-cell, wherein the engineered γδ T-cell comprises a transgene encoding a secreted cytokine.

40. 40. The method of claim 38 or 39, wherein the cytokine is a general gamma chain cytokine or a cytokine selected from the group consisting of IL-2, IL-15, and IL-4, preferably the cytokine is IL-2, IL-15, and / or IL-4.

41. The method of any one of claims 1 to 40, wherein the method comprises administering a lymphodepletion protocol to the subject prior to administering γδ T-cells.

42. 42. The method of any one of claims 1 to 41, wherein the method comprises repeatedly administering the one or more agents that selectively expand δ1 T cells, δ2 T cells, and / or δ3 T cells.

43. The method of any one of claims 1 to 42, wherein the method comprises expanding or maintaining the population of administered γδ T cells in the subject.

44. The method of any one of claims 1 to 43, wherein the method comprises expanding or maintaining a population of endogenous γδ T cells in the subject.

45. 45. The method of any one of claims 1 to 44, wherein said method comprises expanding the population of endogenous and / or administered γδ T cells in said subject by at least 10% relative to the amount of γδ T cells in a subject who has not been administered said one or more agents that selectively expand δ1 T cells, δ2 T cells, and / or δ3 T cells.

46. 46. ​​The method of any one of claims 1 to 45, wherein said method comprises maintaining a population of endogenous and / or administered γδ T cells in said subject that is at least 10% greater relative to the number of γδ T cells in a subject that has not been administered said one or more agents that selectively expand δ1 T cells, δ2 T cells, and / or δ3 T cells.

47. 47. A method of treating cancer, an infectious disease, an inflammatory disease, or an autoimmune disease in a subject in need thereof by carrying out any one of the methods according to claims 1 to 46.

48. Use of an agent that selectively expands δ1 T cells, δ2 T cells, or δ3 T cells in the manufacture of a medicament for the in vivo expansion of γδ T cells in a subject in need thereof.

49. 49. The use of claim 48, wherein in vivo expansion of said γδ T cells in a subject comprises treating cancer, an infectious disease, an inflammatory disease, or an autoimmune disease in said subject.

Images