Materials and methods for improving immune cell proliferation and uses of immune cells

Abstract

Provided herein are, inter alia, improved materials and methods for obtaining and using immune cells (e.g., T cells). In particular, the disclosure provides improved materials and methods for ex vivo immune cell activation, immune cell expansion, and / or enrichment of immune cells, immune cell subsets, more particularly Vy9V52 cells. The disclosure further provides isolated or purified populations of such Vy9V52 cells, and ex vivo and in vivo uses thereof. Also provided herein, in various embodiments, are compositions comprising the improved cells, etc., including isolated or purified populations of produced Vy9V52 cells, CAR cells, etc., and uses of the compositions and cells, etc., in treating a subject in need thereof, e.g., having a disease or disorder.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Application No. 63 / 323,040, filed March 23, 2022, and U.S. Provisional Application No. 63 / 279,042, filed November 12, 2021, the contents of each of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION Provided herein are, inter alia, improved materials and methods for obtaining and using immune cells (e.g., T cells). In particular, the present disclosure provides improved materials and methods for ex vivo immune cell activation, immune cell expansion, and / or enrichment of immune cells, immune cell subsets, and more particularly Vy9V52 cells. The present disclosure further provides isolated or purified populations of such Vy9V52 cells, and ex vivo and in vivo uses thereof. Also provided herein, in various embodiments, are compositions comprising the cells etc., including the isolated or purified populations of Vy9V52 cells, CAR cells etc. produced, and uses of the compositions and cells etc. in treating a subject in need thereof, e.g., having a disease or disorder. Summary of the Invention

[0003] Provided herein are, inter alia, improved materials and methods for obtaining and using immune cells (e.g., T cells). In one aspect, provided herein is a method for the activation and expansion of Vy9V52 T cells ex vivo, the method comprising: (a) contacting a cell population comprising T cells with a culture system comprising IL-2, IL-15, and a bisphosphonate or mevalonate pathway intermediate; and (b) culturing the cell population ex vivo in the culture system under hypoxic conditions to activate and expand Vy9V52 T cells.

[0004] In some embodiments, the method further comprises obtaining the cell population from a subject. In certain embodiments, the subject is healthy. In certain embodiments, the subject is unhealthy.

[0005] In some embodiments, the cell population is a mammalian cell population. In certain embodiments, the mammalian cells are human cells. In certain embodiments, the human cells are genetically engineered cells. In certain embodiments, the human cells are non-genetically engineered cells.

[0006] In some embodiments, the cell population is a peripheral blood mononuclear cell (PBMC) population. In certain embodiments, the PBMCs are freshly obtained PBMCs. In certain embodiments, the PBMCs are frozen PBMCs.

[0007] In some embodiments, the cell population is derived from human tissue. In certain embodiments, the human tissue is fresh. In certain embodiments, the human tissue is frozen.

[0008] In some embodiments, the cell population comprises tumor-infiltrating lymphocytes (TILs). In certain embodiments, the TILs are freshly obtained TILs. In certain embodiments, the TILs are frozen TILs.

[0009] In some embodiments, the cell population is cultured in the culture system under hypoxic conditions for at least 3 days, or at least 5 days, or at least 7 days, or at least 9 days, or at least 11 days, or at least 13 days, or at least 15 days, or at least 17 days, or at least 19 days, or at least 21 days. In certain embodiments, the cell population is cultured in the culture system under hypoxic conditions for 3 to 25 days, or 4 to 23 days, or 5 to 21 days, or 6 to 19 days, or 7 to 17 days, or 8 to 15 days, or 9 to 14 days, or 10 to 14 days, or 11 to 14 days, or 12 to 14 days. In certain embodiments, the cell population is cultured in the culture system under hypoxic conditions for about 15 days. In certain embodiments, the cell population is cultured in the culture system under hypoxic conditions for 14 days.

[0010] In some embodiments, the hypoxic oxygen concentration is less than 15%, or less than 13%, or less than 11%, or less than 9%, or less than 7%, or less than 5%, or less than 3%, or less than 1%, or less than 0.5%. In certain embodiments, the hypoxic oxygen concentration is 0.1%-15%, or 0.5%-13%, or 1%-13%, or 1%-11%, or 1%-9%, or 1%-7%, or 2%-5%. In certain embodiments, the hypoxic oxygen concentration is at or about 2%, or 5% or about 5%, or 12% or about 12%. In certain embodiments, the hypoxic oxygen concentration is about 5%.

[0011] In some embodiments, the method further comprises culturing the cell population in the culture system under normoxic conditions to activate and expand Vy9V52 T cells. In certain embodiments, the cell population is cultured under normoxic conditions for at least 1 hour before being cultured under hypoxic conditions. In certain embodiments, the cell population is cultured under normoxic conditions for 0.5 to 7 days, or 1 to 6 days, or 1 to 5 days, or 1 to 4 days, or 1 to 3 days, or 1 to 2 days before being cultured under hypoxic conditions.

[0012] In certain embodiments, the normoxic oxygen concentration is 18.2% or 18.6%, or about 18.2% or about 18.6%.

[0013] In some embodiments, the IL-2 concentration in the culture system is 10 IU / mL to 1200 IU / mL. In some embodiments, the IL-2 concentration in the culture system is 10 IU / mL or about 10 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is adjusted to gradually decrease during the culture process. In certain embodiments, the IL-2 concentration in the culture system is 1000 IU / mL or less on days 0 and 1, 800 IU / mL or less on days 2, 3, and 4, and 100 IU / mL or less on days 5 and thereafter.

[0014] In some embodiments, the IL-15 concentration in the culture system is 5 ng / mL to 25 ng / mL, or 50 ng / mL to 300 ng / mL. In some embodiments, the IL-15 concentration in the culture system is 100 ng / mL or about 100 ng / mL, or 200 ng / mL or about 200 ng / mL. In certain embodiments, the IL-15 concentration in the culture system is adjusted during the culture process. In certain embodiments, the IL-15 concentration in the culture system is 10 ng / mL or less on days 0 and 1, 20 ng / mL or less on days 2, 3, and 4, and 10 ng / mL or less on days 5 and thereafter.

[0015] In some embodiments, the bisphosphonate is selected from the group consisting of zoledronic acid, risedronate, ibandronic acid, alendronate, pamidronate, tiludronic acid, etidronic acid, and clodronate. In some embodiments, the mevalonate pathway intermediate is selected from the group consisting of HMBPP, BrHPP, and isopentenyl pyrophosphate.

[0016] In some embodiments, the method increases the percentage of Vy9V52 T cells in the cell population to greater than 10%, or greater than 15%, or greater than 20%, or greater than 25%, or greater than 30%, or greater than 35%, or greater than 40%, or greater than 45%, or greater than 50%, or greater than 55%, or greater than 60%. In certain embodiments, the method increases the percentage of Vy9V52 T cells in the cell population to between 10% and 99%, or between 20% and 95%, or between 30% and 95%, or between 35% and 95%, or between 40% and 95%, or between 45% and 95%, or between 50% and 95%, or between 55% and 95%, or between 60% and 95%, or between 65% and 95%.

[0017] In some embodiments, the method increases the total number of Vy9V52 T cells in the cell population by at least 10 fold, or at least 30 fold, or at least 50 fold, or at least 100 fold, or at least 150 fold, or at least 200 fold, or at least 250 fold, or at least 300 fold, or at least 350 fold, or at least 400 fold, or at least 450 fold, or at least 500 fold, or at least 550 fold, or at least 600 fold compared to the total number of Vy9V52 T cells in the cell population before the contacting. In certain embodiments, the method increases the total number of Vy9V52 T cells in the cell population by 10x to 700x, or 30x to 650x, or 50x to 600x, or 100x to 600x, or 150x to 600x, or 200x to 600x, or 250x to 600x, or 300x to 600x, or 350x to 600x, or 400x to 600x, or 450x to 600x, or 500x to 600x relative to the total number of Vy9V52 T cells in the cell population before the contacting.

[0018] In some embodiments, the method further comprises enriching the ex vivo expanded Vy9V52 T cells from the cell population.

[0019] In another embodiment, there is provided herein a method for the ex vivo activation and expansion of Vy9V52 T cells, comprising: a) contacting a cell population comprising T cells with a culture system comprising IL-2, IL-15, and zoledronic acid; and b) culturing the cell population ex vivo in the culture system under hypoxic conditions for about 14 days to activate and expand Vy9V52 T cells, wherein (i) the IL-2 concentration in the culture system is (i) equal to or less than 1000 IU / mL on days 0 and 1, equal to or less than 800 IU / mL on days 2, 3, and 4, and equal to or less than 100 IU / mL on days 5 and beyond, or (2) 10 or about 10. (ii) the IL-15 concentration in the culture system is (1) 10 ng / mL or less on days 0 and 1, 20 ng / mL or less on days 2, 3, and 4, and 10 ng / mL or less on days 5 and thereafter, or (2) 100 ng / mL or about 100 ng / mL, or 200 ng / mL or about 200 ng / mL, and (iii) the hypoxic oxygen concentration is about 5%. In certain embodiments, optionally, (iv) the zoledronic acid concentration in the culture system is about 350 nM.

[0020] In other embodiments, there is provided herein a method for the ex vivo activation and expansion of Vy9V52 T cells, comprising: a) contacting a cell population comprising T cells with a culture system comprising IL-2, IL-15, and zoledronic acid; b) culturing the cell population under normoxic conditions for at least 1 hour to activate and expand Vy9V52 T cells before being cultured under hypoxic conditions; and c) culturing the cell population in the culture system under hypoxic conditions for about 14 days to further activate and expand Vy9V52 T cells, wherein (i) the IL-2 concentration in the culture system is (i) equal to or less than 1000 IU / mL on days 0 and 1, equal to or less than 800 IU / mL on days 2, 3, and 4, and equal to or less than 100 IU / mL on days 5 and beyond, or (ii) 10 or about 10. (ii) the IL-15 concentration in the culture system is (1) 10 ng / mL or less on days 0 and 1, 20 ng / mL or less on days 2, 3, and 4, and 10 ng / mL or less on days 5 and thereafter, or (2) 100 ng / mL or about 100 ng / mL, or 200 ng / mL or about 200 ng / mL; (iii) the hypoxic oxygen concentration is about 5%; (iv) the normoxic oxygen concentration is 18.2% or about 18.2% (e.g., 18.2% or about 18.6%); and optionally, (v) the zoledronic acid concentration in the culture system is about 350 nM.

[0021] In other embodiments, provided herein is an isolated population of Vy9V52 T cells produced by the methods disclosed herein.

[0022] In other embodiments, provided herein is an isolated cell population, wherein the percentage of Vy9V52 T cells in the isolated cell population is (a) greater than 10%, or greater than 15%, or greater than 20%, or greater than 25%, or greater than 30%, or greater than 35%, or greater than 40%, or greater than 45%, or greater than 50%, or greater than 55%, or greater than 60%, or (b) between 10% and 99%, or between 20% and 95%, or between 30% and 95%, or between 35% and 95%, or between 40% and 95%, or between 45% and 95%, or between 50% and 95%, or between 55% and 95%, or between 60% and 95%, or between 65% and 95%.

[0023] In yet another aspect, provided herein is a pharmaceutical composition comprising an isolated population of Vy9V52 T cells as disclosed herein and a pharmaceutically acceptable excipient.

[0024] In yet another aspect, provided herein are methods for treating a disease or disorder in a subject using the cells or compositions of the disclosure. In certain embodiments, the method comprises administering to the subject (i) a therapeutically effective amount of a Vy9V52 T cell or pharmaceutical composition disclosed herein, and (ii) a therapeutically effective amount of one or more multispecific antibodies. In some embodiments, each of the multispecific antibodies comprises a first binding domain that binds to an antigen expressed on a Vy9V52 T cell and a second binding domain that binds to an antigen expressed on an unhealthy cell. In certain embodiments, the antigen expressed on a Vy9V52 T cell is T cell receptor gamma variable 9 (TRGV9) or CD3. In certain embodiments, the unhealthy cell is a cancer cell and the antigen expressed on the unhealthy cell is a tumor-associated antigen (TAA). In certain embodiments, the cancer cell is a hematological cancer cell or a solid tumor cancer cell. In some embodiments, the method comprises administering to a subject (i) a therapeutically effective amount of a Vy9V52 T cell or pharmaceutical composition disclosed herein, and (ii) a therapeutically effective amount of a Vy9xTAA and / or CD3xTAA bispecific antibody. In some embodiments, the disease or disorder is cancer. In certain embodiments, the cancer is a hematological cancer. In certain embodiments, the cancer is a solid tumor cancer. In some embodiments, the subject is a human subject in need thereof.

[0025] In yet another aspect, provided herein is a process for producing a chimeric antigen receptor (CAR) T cell product, the process comprising: (i) performing the functions disclosed herein to obtain an isolated Vy9V52 T cell population; and (ii) expressing a CAR in the Vy9V52 T cells. In some embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In certain embodiments, the extracellular domain binds to an antigen expressed on an unhealthy cell. In certain embodiments, the unhealthy cell is a cancer cell. In certain embodiments, the cancer cell is a hematological cancer cell or a solid tumor cancer cell.

[0026] In yet another aspect, provided herein is a method for producing a chimeric antigen receptor (CAR) T cell product, the method comprising: (i) obtaining a cell population comprising a Vy9V52 T cell as disclosed herein; and (ii) introducing a nucleic acid encoding the CAR into the cell population. In some embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In certain embodiments, the extracellular domain binds to an antigen expressed on an unhealthy cell. In certain embodiments, the unhealthy cell is a cancer cell. In certain embodiments, the cancer cell is a hematological cancer cell or a solid tumor cancer cell.

[0027] In yet another aspect, provided herein is a CAR T cell product produced by the methods disclosed herein.

[0028] In yet another aspect, provided herein is a CAR T cell comprising a CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the CAR T cell is a Vy9V52 T cell.

[0029] In yet another aspect, provided herein is a pharmaceutical composition comprising a CAR T cell product or CAR T cells disclosed herein and a pharmaceutically acceptable excipient.

[0030] In yet another aspect, provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a CAR T cell disclosed herein, or a CAR T cell product or pharmaceutical composition comprising a CAR T cell disclosed herein.

[0031] In yet another aspect, provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a CAR T cell, wherein the CAR T cell comprises a CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain, and wherein the CAR T cell is a Vy9V52 T cell.

[0032] In yet another aspect, provided herein are methods for establishing in vivo engraftment of Vy9V52 T cells in a recipient subject. In certain embodiments, the method comprises (i) obtaining a cell population comprising Vy9V52 T cells as disclosed herein, and (ii) adoptively transferring the cell population into the recipient subject.

[0033] In some embodiments, the cell population comprising Vy9V52 T cells is produced by ex vivo activation and expansion of a cell population comprising T cells obtained from the recipient subject.

[0034] In some embodiments, a cell population comprising Vy9V52 T cells comprises more than 10%, or more than 15%, or more than 20%, or more than 25%, or more than 30%, or more than 35%, or more than 40%, or more than 45%, or more than 50%, or more than 55%, or more than 60% Vy9V52 T cells in the cell population.

[0035] In some embodiments, the cell population comprising Vy9V52 T cells comprises 10% to 99%, or 20% to 95%, or 30% to 95%, or 35% to 95%, or 40% to 95%, or 45% to 95%, or 50% to 95%, or 55% to 95%, or 60% to 95%, or 65% to 95% Vy9V52 T cells in the cell population.

[0036] In some embodiments, a cell population comprising Vy9V52 T cells comprises less than 95%, or less than 90%, or less than 85%, or less than 80%, or less than 75%, or less than 70%, or less than 65%, or less than 60%, or less than 55%, or less than 50%, or less than 45%, or less than 40%, or less than 35%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 10%, or less than 5%, or less than 1% or no αβ T cells in the cell population.

[0037] In some embodiments, the cell population comprises at least about or about 5 x 10 6 cells, at least about or about 1 x 10 7 cells, at least about or about 5 x 10 7cells, at least about or about 1 x 10 8 cells, at least about or about x 10 8 cells, at least about or about 3 x 10 8 Cells, at least approximately 4 x 10 8 cells, at least about or about 5 x 10 8 cells, at least approximately 6 x 10 8 cells, at least about or about 7 x 10 8 cells, at least about or about 8 x 10 8 cells, at least approximately 9 x 10 8 cells, at least about or about 1 x 10 9 cells, at least about or about 2 x 10 9 cells, at least about or about 3 x 10 9 Cells, at least approximately 4 x 10 9 cells, at least about or about 5 x 10 9 cells, at least approximately 6 x 10 9 cells, at least about or about 7 x 10 9 cells, at least about or about 8 x 10 9 cells, at least approximately 9 x 10 9 cells, or at least about or about 1 x 10 10 Contains cells.

[0038] In some embodiments, the cell population comprising Vy9V52 T cells is a purified Vy9V52 T cell population.

[0039] In some embodiments, the cell population comprising Vy9V52 T cells is enriched to contain greater than 95% Vy9V52 T cells one day prior to adoptive transfer of the cell population into a recipient subject.

[0040] In some embodiments, adoptively transferring comprises administering the cell population to the recipient subject. In some embodiments, the method further comprises (iii) administering, simultaneously or sequentially with the adoptive transfer of the cell population, an effective amount of a composition comprising IL-2, IL-15, a bisphosphonate, or a mevalonate pathway intermediate, or a combination thereof. In some embodiments, the bisphosphonate is selected from the group consisting of zoledronic acid, risedronate, ibandronic acid, alendronate, pamidronate, tiludronic acid, etidronic acid, and clodronate. In some embodiments, the mevalonate pathway intermediate is selected from the group consisting of HMBPP, BrHPP, and isopentenyl pyrophosphate. In some embodiments, the bisphosphonate is zoledronic acid. In some embodiments, the zoledronic acid is administered at a dose of 2.5 mg / kg body weight. In some embodiments, the administration of the cell population and / or the administration of the effective amount of the composition is intravenous or intraperitoneal. In some embodiments, IL-2 is administered at 2×10 4 IU / kg body weight. In some embodiments, the recipient subject expresses IL-15. In some embodiments, the recipient subject is an animal model.

[0041] In some embodiments of the methods for establishing in vivo engraftment of Vy9V52 T cells in a recipient subject described herein, the adoptively transferred Vy9V52 T cells produce progeny cells in the subject. In some embodiments, the progeny cells express CD45 + In some embodiments, the progeny cells are CD56 + In some embodiments, the progeny cells are CD69 +In some embodiments, the adoptively transferred Vy9V52 T cells are present in the blood of the recipient subject at least 7 days, at least 14 days, at least 21 days, or at least 28 days after adoptive transfer of the cell population into the recipient subject. In some embodiments, the adoptively transferred Vy9V52 T cells infiltrate a tissue in the recipient subject. In some embodiments, the tissue is spleen tissue, liver tissue, lung tissue, intestinal tissue, skin tissue, or a combination thereof. In some embodiments, the tissue comprises unhealthy cells. In some embodiments, the unhealthy cells are cancer cells. In some embodiments, the cancer cells are blood cancer cells or solid tumor cancer cells. In some embodiments, the recipient subject does not develop any symptoms of graft-versus-host disease (GvHD) at least 7 days, at least 14 days, at least 21 days, or at least 28 days after adoptive transfer of the cell population into the recipient subject.

[0042] In some embodiments of the methods for establishing in vivo engraftment of Vy9V52 T cells in a recipient subject described herein, the Vy9V52 T cells are chimeric antigen receptor (CAR) T cells comprising an extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the extracellular domain binds to an antigen expressed on an unhealthy cell. In some embodiments, the unhealthy cell is a cancer cell. In some embodiments, the cancer cell is a blood cancer cell or a solid tumor cancer cell.

[0043] In yet another aspect, a method for treating a disease or disorder in a subject comprises administering to the subject (i) a therapeutically effective amount of a population of Vy9V52 T cells and (ii) a therapeutically effective amount of one or more multispecific antibodies. In some embodiments, each of the multispecific antibodies comprises a first binding domain that binds to an antigen expressed on a Vy9V52 T cell and a second binding domain that binds to an antigen expressed on an unhealthy cell. In certain embodiments, the antigen expressed on the Vy9V52 T cell is T cell receptor gamma variable 9 (TRGV9) or CD3. In certain embodiments, the unhealthy cell is a cancer cell and the antigen expressed on the unhealthy cell is a tumor-associated antigen (TAA). In certain embodiments, the cancer cell is a hematological cancer cell or a solid tumor cancer cell. In some embodiments, the method comprises administering to the subject (i) a therapeutically effective amount of a Vy9V52 T cell or pharmaceutical composition disclosed herein, and (ii) a therapeutically effective amount of a Vy9xTAA and / or CD3xTAA bispecific antibody. In some embodiments, the disease or disorder is cancer. In certain embodiments, the cancer is a hematological cancer. In certain embodiments, the cancer is a solid tumor cancer. In some embodiments, the subject is a human subject in need thereof.

[0044] In yet another aspect, the present disclosure provides a pharmaceutical composition as disclosed herein, an isolated Vy9V52 T cell population as disclosed herein, or an isolated cell population as disclosed herein, for use in treating a disease or disorder in a subject. In certain embodiments, the treatment comprises administering to the subject (i) a therapeutically effective amount of the isolated Vy9V52 T cell population, isolated cell population, or pharmaceutical composition, and (ii) a therapeutically effective amount of at least one multispecific antibody.

[0045] In some embodiments, at least one multispecific antibody comprises (a) a first binding domain that binds to an antigen expressed on a Vy9V52 T cell, and (b) a second binding domain that binds to an antigen expressed on an unhealthy cell.

[0046] In some embodiments, (a) the antigen expressed on the Vy9V52 T cell is T cell receptor gamma variable 9 (TRGV9) or CD3, and (b) the unhealthy cell is a cancer cell and the antigen expressed on the unhealthy cell is a tumor-associated antigen (TAA). In certain embodiments, the cancer cell is a blood cancer cell or a solid tumor cancer cell.

[0047] In some embodiments, at least one multispecific antibody comprises a Vγ9xTAA and / or a CD3xTAA bispecific antibody.

[0048] In some embodiments, the disease or disorder is cancer. In certain embodiments, the cancer is a hematological cancer or a solid tumor cancer. In certain embodiments, the subject is a human subject in need of treatment thereof. [Brief explanation of the drawings]

[0049] [Figure 1]

[0033] Figure 1 shows an exemplary schematic diagram of the process of the present disclosure, the selective in vitro expansion of Vy9V52 T cells. Briefly, peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured in complete growth medium containing IL-2, IL-15, and zoledronic acid (zol). Monocytes selectively took up zol and zol, which was subsequently metabolized to IPP (isoprenyl diphosphate) and DMAPP (dimethylallyl diphosphate). The generated phosphate bound to the cytoplasmic tail of CD277, leading to its conformational change on the surface of monocytes. Upon conformational change, CD277 was recognized by Vy9V52 T cell receptors (TCRs), which subsequently triggered their activation and proliferation, while other immune cell types underwent apoptosis. [Figure 2A] Starting Vy9V52 T cell populations in PBMCs prior to in vitro expansion. Figure 2A shows the identification of Vy9V52 T cell populations by flow cytometry gating on single viable CD3+ cells on PBMCs from a single healthy donor on day 0 prior to in vitro expansion. Figure 2B shows the distribution of CD3+ Vy9V52 T cells prior to in vitro expansion across a cohort of 68 healthy donors. [Figure 2B] Starting Vy9V52 T cell populations in PBMCs prior to in vitro expansion. Figure 2A shows the identification of Vy9V52 T cell populations by flow cytometry gating on single viable CD3+ cells on PBMCs from a single healthy donor on day 0 prior to in vitro expansion. Figure 2B shows the distribution of CD3+ Vy9V52 T cells prior to in vitro expansion across a cohort of 68 healthy donors. [Figure 3A] Figure 3A shows the expansion of Vy9V52 T cells from 68 healthy donors after 14 days of in vitro expansion under normoxic conditions (18.2% oxygen). Figure 3A shows Vy9V52 T cell expansion values ​​(black line) plotted against the frequency of the starting CD3+ Vy9V52 T cell population (gray bars). The left Y-axis shows the range of frequencies of starting CD3+ Vy9V52 T cells, and the right Y-axis shows the expansion efficiency as fold change. Each individual bar / line point represents a single donor. Figure 3B shows Vy9V52 T cell expansion (black bars) plotted against the total number of Vy9V52 T cells obtained after expansion (gray line). The left Y-axis shows the expansion efficiency as fold change, and the right Y-axis shows the total number of Vy9V52 T cells obtained after expansion, normalized to the starting amount of 50 x 106 PBMCs. Each individual bar / line point represents a single donor. Figure 3C shows the frequency of the Vy9V52 T cell population at day 0 (grey line) and day 14 (black line). The left Y-axis shows the frequency of the Vy9V52 T cell population at day 0 and the right Y-axis shows the frequency of the Vy9V52 T cell population at day 14. [Figure 3B]Figure 3A shows the expansion of Vy9V52 T cells from 68 healthy donors after 14 days of in vitro expansion under normoxic conditions (18.2% oxygen). Figure 3A shows Vy9V52 T cell expansion values ​​(black line) plotted against the frequency of the starting CD3+ Vy9V52 T cell population (gray bars). The left Y-axis shows the range of frequencies of starting CD3+ Vy9V52 T cells, and the right Y-axis shows the expansion efficiency as fold change. Each individual bar / line point represents a single donor. Figure 3B shows Vy9V52 T cell expansion (black bars) plotted against the total number of Vy9V52 T cells obtained after expansion (gray line). The left Y-axis shows the expansion efficiency as fold change, and the right Y-axis shows the total number of Vy9V52 T cells obtained after expansion, normalized to the starting amount of 50 x 106 PBMCs. Each individual bar / line point represents a single donor. Figure 3C shows the frequency of the Vy9V52 T cell population at day 0 (grey line) and day 14 (black line). The left Y-axis shows the frequency of the Vy9V52 T cell population at day 0 and the right Y-axis shows the frequency of the Vy9V52 T cell population at day 14. [Figure 3C] Figure 3A shows the expansion of Vy9V52 T cells from 68 healthy donors after 14 days of in vitro expansion under normoxic conditions (18.2% oxygen). Figure 3A shows Vy9V52 T cell expansion values ​​(black line) plotted against the frequency of the starting CD3+ Vy9V52 T cell population (gray bars). The left Y-axis shows the range of frequencies of starting CD3+ Vy9V52 T cells, and the right Y-axis shows the expansion efficiency as fold change. Each individual bar / line point represents a single donor. Figure 3B shows Vy9V52 T cell expansion (black bars) plotted against the total number of Vy9V52 T cells obtained after expansion (gray line). The left Y-axis shows the expansion efficiency as fold change, and the right Y-axis shows the total number of Vy9V52 T cells obtained after expansion, normalized to the starting amount of 50 x 106 PBMCs. Each individual bar / line point represents a single donor. Figure 3C shows the frequency of the Vy9V52 T cell population at day 0 (grey line) and day 14 (black line). The left Y-axis shows the frequency of the Vy9V52 T cell population at day 0 and the right Y-axis shows the frequency of the Vy9V52 T cell population at day 14. [Figure 4A]Figure 4 shows the characteristics of Vy9V52 T cell proliferation in low and high proliferators after 14 days of in vitro expansion under normoxic conditions. Figure 4A shows immune cell clusters within PBMCs on day 0 from five low proliferators (left panel) and five high proliferators (right panel). Figure 4B compares the presence of αβ T cells, Vy9V52 T cells, NK cells, and monocytes between high proliferators (solid line, n=10) and low proliferators (dashed line, n=10) throughout the 14-day expansion period. Figure 4C compares the proliferation capacity of high proliferators (n=10) and low proliferators (n=10) by measuring the frequency of Vy9V52 T cells expressing the proliferation marker Ki-67 at day 14. [Figure 4B] Figure 4 shows the characteristics of Vy9V52 T cell proliferation in low and high proliferators after 14 days of in vitro expansion under normoxic conditions. Figure 4A shows immune cell clusters within PBMCs on day 0 from five low proliferators (left panel) and five high proliferators (right panel). Figure 4B compares the presence of αβ T cells, Vy9V52 T cells, NK cells, and monocytes between high proliferators (solid line, n=10) and low proliferators (dashed line, n=10) throughout the 14-day expansion period. Figure 4C compares the proliferation capacity of high proliferators (n=10) and low proliferators (n=10) by measuring the frequency of Vy9V52 T cells expressing the proliferation marker Ki-67 at day 14. [Figure 4C] Figure 4 shows the characteristics of Vy9V52 T cell proliferation in low and high proliferators after 14 days of in vitro expansion under normoxic conditions. Figure 4A shows immune cell clusters within PBMCs on day 0 from five low proliferators (left panel) and five high proliferators (right panel). Figure 4B compares the presence of αβ T cells, Vy9V52 T cells, NK cells, and monocytes between high proliferators (solid line, n=10) and low proliferators (dashed line, n=10) throughout the 14-day expansion period. Figure 4C compares the proliferation capacity of high proliferators (n=10) and low proliferators (n=10) by measuring the frequency of Vy9V52 T cells expressing the proliferation marker Ki-67 at day 14. [Figure 5A]Figure 5A shows a linear regression analysis of Vy9V52 T cell expansion efficiency plotted against donor age. Figure 5B compares Vy9V52 T cell expansion efficiency between genders. Figure 5C compares Vy9V52 T cell expansion efficiency across ethnic identity. [Figure 5B] Figure 5A shows a linear regression analysis of Vy9V52 T cell expansion efficiency plotted against donor age. Figure 5B compares Vy9V52 T cell expansion efficiency between genders. Figure 5C compares Vy9V52 T cell expansion efficiency across ethnic identity. [Figure 5C] Figure 5A shows a linear regression analysis of Vy9V52 T cell expansion efficiency plotted against donor age. Figure 5B compares Vy9V52 T cell expansion efficiency between genders. Figure 5C compares Vy9V52 T cell expansion efficiency across ethnic identity. [Figure 6A] Figure 6A shows immune cell clusters identified based on the transcriptomes of cells collected from high and low proliferators under different experimental conditions. Figure 6A shows a uniform manifold approximation and projection (UMAP) plot of immune cell clusters representing 358,370 sequenced cells collected from 10 donors (5 high proliferators and 5 low proliferators) across three experimental conditions (day 0 whole blood PBMCs, day 14 expanded non-enriched Vy9V52 T cells, and day 0 non-expanded enriched γδ T cells). Figure 6B shows a UMAP plot of immune cell clusters representing cells collected and sequenced from PBMCs of 10 donors on day 0. Figure 6C shows a UMAP plot of immune cell clusters representing cells collected and sequenced from expanded non-enriched Vy9V52 T cells of 10 donors on day 14. [Figure 6B]Figure 6A shows immune cell clusters identified based on the transcriptomes of cells collected from high and low proliferators under different experimental conditions. Figure 6A shows a uniform manifold approximation and projection (UMAP) plot of immune cell clusters representing 358,370 sequenced cells collected from 10 donors (5 high proliferators and 5 low proliferators) across three experimental conditions (day 0 whole blood PBMCs, day 14 expanded non-enriched Vy9V52 T cells, and day 0 non-expanded enriched γδ T cells). Figure 6B shows a UMAP plot of immune cell clusters representing cells collected and sequenced from PBMCs of 10 donors on day 0. Figure 6C shows a UMAP plot of immune cell clusters representing cells collected and sequenced from expanded non-enriched Vy9V52 T cells of 10 donors on day 14. [Figure 6C] Figure 6A shows immune cell clusters identified based on the transcriptomes of cells collected from high and low proliferators under different experimental conditions. Figure 6A shows a uniform manifold approximation and projection (UMAP) plot of immune cell clusters representing 358,370 sequenced cells collected from 10 donors (5 high proliferators and 5 low proliferators) across three experimental conditions (day 0 whole blood PBMCs, day 14 expanded non-enriched Vy9V52 T cells, and day 0 non-expanded enriched γδ T cells). Figure 6B shows a UMAP plot of immune cell clusters representing cells collected and sequenced from PBMCs of 10 donors on day 0. Figure 6C shows a UMAP plot of immune cell clusters representing cells collected and sequenced from expanded non-enriched Vy9V52 T cells of 10 donors on day 14. [Figure 7A]Figure 7 shows the purity of γδ T cells in clusters of expanded non-enriched Vy9V52 T cells at day 14 ("expansion cohort") and non-expanded enriched Vy9V52 T cells at day 0 ("enriched cohort"). Figure 7A shows a UMAP plot showing the prevalence of γδ T cells corresponding to the TRGD signature (dark shading) in the expansion cohort. Figure 7B shows a UMAP plot showing the presence of αβ T cells corresponding to the TRDG signature (light shading) and the TRAB signature (dark shading) in the expansion cohort. Figure 7C shows a UMAP plot showing cells corresponding to the TRDG signature (light shading) and the TRAB signature (dark shading) in the expansion cohort. Figure 7D shows a UMAP plot showing the prevalence of γδ T cells corresponding to the TRGD signature (dark shading) in the purified cohort. Figure 7E shows a UMAP plot showing the presence of αβ T cells corresponding to the TRAB signature (dark shading) in the purified cohort. FIG. 7F shows a UMAP plot showing cells corresponding to the TRDG signature (light shading) and the TRAB signature (dark shading) in the purified cohort. [Figure 7B] Figure 7 shows the purity of γδ T cells in clusters of expanded non-enriched Vy9V52 T cells at day 14 ("expansion cohort") and non-expanded enriched Vy9V52 T cells at day 0 ("enriched cohort"). Figure 7A shows a UMAP plot showing the prevalence of γδ T cells corresponding to the TRGD signature (dark shading) in the expansion cohort. Figure 7B shows a UMAP plot showing the presence of αβ T cells corresponding to the TRDG signature (light shading) and the TRAB signature (dark shading) in the expansion cohort. Figure 7C shows a UMAP plot showing cells corresponding to the TRDG signature (light shading) and the TRAB signature (dark shading) in the expansion cohort. Figure 7D shows a UMAP plot showing the prevalence of γδ T cells corresponding to the TRGD signature (dark shading) in the purified cohort. Figure 7E shows a UMAP plot showing the presence of αβ T cells corresponding to the TRAB signature (dark shading) in the purified cohort. FIG. 7F shows a UMAP plot showing cells corresponding to the TRDG signature (light shading) and the TRAB signature (dark shading) in the purified cohort. [Figure 7C]Figure 7 shows the purity of γδ T cells in clusters of expanded non-enriched Vy9V52 T cells at day 14 ("expansion cohort") and non-expanded enriched Vy9V52 T cells at day 0 ("enriched cohort"). Figure 7A shows a UMAP plot showing the prevalence of γδ T cells corresponding to the TRGD signature (dark shading) in the expansion cohort. Figure 7B shows a UMAP plot showing the presence of αβ T cells corresponding to the TRDG signature (light shading) and the TRAB signature (dark shading) in the expansion cohort. Figure 7C shows a UMAP plot showing cells corresponding to the TRDG signature (light shading) and the TRAB signature (dark shading) in the expansion cohort. Figure 7D shows a UMAP plot showing the prevalence of γδ T cells corresponding to the TRGD signature (dark shading) in the purified cohort. Figure 7E shows a UMAP plot showing the presence of αβ T cells corresponding to the TRAB signature (dark shading) in the purified cohort. FIG. 7F shows a UMAP plot showing cells corresponding to the TRDG signature (light shading) and the TRAB signature (dark shading) in the purified cohort. [Figure 7D] Figure 7 shows the purity of γδ T cells in clusters of expanded non-enriched Vy9V52 T cells at day 14 ("expansion cohort") and non-expanded enriched Vy9V52 T cells at day 0 ("enriched cohort"). Figure 7A shows a UMAP plot showing the prevalence of γδ T cells corresponding to the TRGD signature (dark shading) in the expansion cohort. Figure 7B shows a UMAP plot showing the presence of αβ T cells corresponding to the TRDG signature (light shading) and the TRAB signature (dark shading) in the expansion cohort. Figure 7C shows a UMAP plot showing cells corresponding to the TRDG signature (light shading) and the TRAB signature (dark shading) in the expansion cohort. Figure 7D shows a UMAP plot showing the prevalence of γδ T cells corresponding to the TRGD signature (dark shading) in the purified cohort. Figure 7E shows a UMAP plot showing the presence of αβ T cells corresponding to the TRAB signature (dark shading) in the purified cohort. FIG. 7F shows a UMAP plot showing cells corresponding to the TRDG signature (light shading) and the TRAB signature (dark shading) in the purified cohort. [Figure 7E]Figure 7 shows the purity of γδ T cells in clusters of expanded non-enriched Vy9V52 T cells at day 14 ("expansion cohort") and non-expanded enriched Vy9V52 T cells at day 0 ("enriched cohort"). Figure 7A shows a UMAP plot showing the prevalence of γδ T cells corresponding to the TRGD signature (dark shading) in the expansion cohort. Figure 7B shows a UMAP plot showing the presence of αβ T cells corresponding to the TRDG signature (light shading) and the TRAB signature (dark shading) in the expansion cohort. Figure 7C shows a UMAP plot showing cells corresponding to the TRDG signature (light shading) and the TRAB signature (dark shading) in the expansion cohort. Figure 7D shows a UMAP plot showing the prevalence of γδ T cells corresponding to the TRGD signature (dark shading) in the purified cohort. Figure 7E shows a UMAP plot showing the presence of αβ T cells corresponding to the TRAB signature (dark shading) in the purified cohort. FIG. 7F shows a UMAP plot showing cells corresponding to the TRDG signature (light shading) and the TRAB signature (dark shading) in the purified cohort. [Figure 7F] Figure 7 shows the purity of γδ T cells in clusters of expanded non-enriched Vy9V52 T cells at day 14 ("expansion cohort") and non-expanded enriched Vy9V52 T cells at day 0 ("enriched cohort"). Figure 7A shows a UMAP plot showing the prevalence of γδ T cells corresponding to the TRGD signature (dark shading) in the expansion cohort. Figure 7B shows a UMAP plot showing the presence of αβ T cells corresponding to the TRDG signature (light shading) and the TRAB signature (dark shading) in the expansion cohort. Figure 7C shows a UMAP plot showing cells corresponding to the TRDG signature (light shading) and the TRAB signature (dark shading) in the expansion cohort. Figure 7D shows a UMAP plot showing the prevalence of γδ T cells corresponding to the TRGD signature (dark shading) in the purified cohort. Figure 7E shows a UMAP plot showing the presence of αβ T cells corresponding to the TRAB signature (dark shading) in the purified cohort. FIG. 7F shows a UMAP plot showing cells corresponding to the TRDG signature (light shading) and the TRAB signature (dark shading) in the purified cohort. [Figure 8A]Figure 8 shows the differential population distribution between high and low proliferators in clusters of expanded non-enriched Vy9V52 T cells at day 14 ("expansion cohort") and non-expanded enriched Vy9V52 T cells at day 0 ("enriched cohort"). Figure 8A shows a UMAP plot depicting pooled data of γδ T cell populations from five low proliferators (left panel) and five high proliferators (right panel) collected from the expansion cohort. High cell densities are indicated by solid black. Figure 8B shows a UMAP plot depicting pooled data of γδ T cell populations from five low proliferators (left panel) and five high proliferators (right panel) collected from the enrichment cohort. High cell densities are indicated by solid black. [Figure 8B] Figure 8 shows the differential population distribution between high and low proliferators in clusters of expanded non-enriched Vy9V52 T cells at day 14 ("expansion cohort") and non-expanded enriched Vy9V52 T cells at day 0 ("enriched cohort"). Figure 8A shows a UMAP plot depicting pooled data of γδ T cell populations from five low proliferators (left panel) and five high proliferators (right panel) collected from the expansion cohort. High cell densities are indicated by solid black. Figure 8B shows a UMAP plot depicting pooled data of γδ T cell populations from five low proliferators (left panel) and five high proliferators (right panel) collected from the enrichment cohort. High cell densities are indicated by solid black. [Figure 9A] Figure 9 shows differentially expressed genes (DEGs) analysis of high and low proliferators in a cluster of expanded, non-enriched Vy9V52 T cells at day 14 ("expansion cohort"). Figure 9A is a volcano plot depicting differential expression of genes between high and low proliferators. Figure 9B is a table listing the top 20 DEGs with the highest significance and / or highest fold change. Gene descriptions highlighted in grey are hypoxia-related genes. The gene list was adapted from Database for Annotation, Visualization, and Integrated Discovery (DAVID) (Dennis et al., Genome Biology volume 4, Article number: R60 (2003)). [Figure 9B] Figure 9 shows differentially expressed genes (DEGs) analysis of high and low proliferators in a cluster of expanded, non-enriched Vy9V52 T cells at day 14 ("expansion cohort"). Figure 9A is a volcano plot depicting differential expression of genes between high and low proliferators. Figure 9B is a table listing the top 20 DEGs with the highest significance and / or highest fold change. Gene descriptions highlighted in grey are hypoxia-related genes. The gene list was adapted from Database for Annotation, Visualization, and Integrated Discovery (DAVID) (Dennis et al., Genome Biology volume 4, Article number: R60 (2003)). [Figure 10A] Figure 10A shows hypoxia-related genes that colocalized with the unique γδT cluster in the high-growth cells identified in Figure 8A. Figure 10A shows HIF1A-AS3 expression plotted on a collected cell density plot from high-growth cells and low-growth cells in the expansion cohort. Figure 10B shows BNIP3L expression plotted on a collected cell density plot from high-growth cells and low-growth cells in the expansion cohort. Figure 10C shows MIF expression plotted on a collected cell density plot from high-growth cells and low-growth cells in the expansion cohort. [Figure 10B] Figure 10A shows hypoxia-related genes that colocalized with the unique γδT cluster in the high-growth cells identified in Figure 8A. Figure 10A shows HIF1A-AS3 expression plotted on a collected cell density plot from high-growth cells and low-growth cells in the expansion cohort. Figure 10B shows BNIP3L expression plotted on a collected cell density plot from high-growth cells and low-growth cells in the expansion cohort. Figure 10C shows MIF expression plotted on a collected cell density plot from high-growth cells and low-growth cells in the expansion cohort. [Figure 10C]Figure 10A shows hypoxia-related genes that colocalized with the unique γδT cluster in the high-growth cells identified in Figure 8A. Figure 10A shows HIF1A-AS3 expression plotted on a collected cell density plot from high-growth cells and low-growth cells in the expansion cohort. Figure 10B shows BNIP3L expression plotted on a collected cell density plot from high-growth cells and low-growth cells in the expansion cohort. Figure 10C shows MIF expression plotted on a collected cell density plot from high-growth cells and low-growth cells in the expansion cohort. [Figure 11A] Figure 11 shows an analysis of DEGs in high and low proliferators in a cluster of non-proliferating enriched Vy9V52 T cells on day 0 ("enriched cohort"). Figure 11A is a volcano plot depicting differential expression of genes between high and low proliferators. Figure 11B is a table listing the top 20 DEGs with the highest significance and / or highest fold change. Gene descriptions highlighted in grey are hypoxia-related genes. [Figure 11B] Figure 11 shows an analysis of DEGs in high and low proliferators in a cluster of non-proliferating enriched Vy9V52 T cells on day 0 ("enriched cohort"). Figure 11A is a volcano plot depicting differential expression of genes between high and low proliferators. Figure 11B is a table listing the top 20 DEGs with the highest significance and / or highest fold change. Gene descriptions highlighted in grey are hypoxia-related genes. [Figure 12A-1]Figure 12A shows flow cytometry plots (gated on single viable CD3+Vy9+ cells) demonstrating the increased purity of Vy9V52 T cells from a single donor cultured under hypoxic conditions. Figure 12A shows flow cytometry plots (gated on single viable CD3+Vy9+ cells) demonstrating the increased purity of Vy9V52 T cell populations in one donor after 14 days of in vitro expansion under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Figure 12B shows a histogram overlap of the plots shown in Figure 12A, demonstrating the increased Vy9V52 T cell population. Figure 12C shows that Vy9V52 T cells have increased purity with fold changes observed under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Each line represents a single donor. Figure 12D shows total Vy9V52 T cell frequency after 14 days of in vitro expansion under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Each line represents a single donor. Figure 12E shows total αβ T cell frequency after 14 days of in vitro expansion under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Each line represents a single donor. [Figure 12A-2]Figure 12A shows flow cytometry plots (gated on single viable CD3+Vy9+ cells) demonstrating the increased purity of Vy9V52 T cells from a single donor cultured under hypoxic conditions. Figure 12A shows flow cytometry plots (gated on single viable CD3+Vy9+ cells) demonstrating the increased purity of Vy9V52 T cell populations in one donor after 14 days of in vitro expansion under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Figure 12B shows a histogram overlap of the plots shown in Figure 12A, demonstrating the increased Vy9V52 T cell population. Figure 12C shows that Vy9V52 T cells have increased purity with fold changes observed under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Each line represents a single donor. Figure 12D shows total Vy9V52 T cell frequency after 14 days of in vitro expansion under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Each line represents a single donor. Figure 12E shows total αβ T cell frequency after 14 days of in vitro expansion under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Each line represents a single donor. [Figure 12B]Figure 12A shows flow cytometry plots (gated on single viable CD3+Vy9+ cells) demonstrating the increased purity of Vy9V52 T cells from a single donor cultured under hypoxic conditions. Figure 12A shows flow cytometry plots (gated on single viable CD3+Vy9+ cells) demonstrating the increased purity of Vy9V52 T cell populations in one donor after 14 days of in vitro expansion under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Figure 12B shows a histogram overlap of the plots shown in Figure 12A, demonstrating the increased Vy9V52 T cell population. Figure 12C shows that Vy9V52 T cells have increased purity with fold changes observed under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Each line represents a single donor. Figure 12D shows total Vy9V52 T cell frequency after 14 days of in vitro expansion under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Each line represents a single donor. Figure 12E shows total αβ T cell frequency after 14 days of in vitro expansion under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Each line represents a single donor. [Figure 12C]Figure 12A shows flow cytometry plots (gated on single viable CD3+Vy9+ cells) demonstrating the increased purity of Vy9V52 T cells from a single donor cultured under hypoxic conditions. Figure 12A shows flow cytometry plots (gated on single viable CD3+Vy9+ cells) demonstrating the increased purity of Vy9V52 T cell populations in one donor after 14 days of in vitro expansion under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Figure 12B shows a histogram overlap of the plots shown in Figure 12A, demonstrating the increased Vy9V52 T cell population. Figure 12C shows that Vy9V52 T cells have increased purity with fold changes observed under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Each line represents a single donor. Figure 12D shows total Vy9V52 T cell frequency after 14 days of in vitro expansion under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Each line represents a single donor. Figure 12E shows total αβ T cell frequency after 14 days of in vitro expansion under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Each line represents a single donor. [Figure 12D]Figure 12A shows flow cytometry plots (gated on single viable CD3+Vy9+ cells) demonstrating the increased purity of Vy9V52 T cells from a single donor cultured under hypoxic conditions. Figure 12A shows flow cytometry plots (gated on single viable CD3+Vy9+ cells) demonstrating the increased purity of Vy9V52 T cell populations in one donor after 14 days of in vitro expansion under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Figure 12B shows a histogram overlap of the plots shown in Figure 12A, demonstrating the increased Vy9V52 T cell population. Figure 12C shows that Vy9V52 T cells have increased purity with fold changes observed under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Each line represents a single donor. Figure 12D shows total Vy9V52 T cell frequency after 14 days of in vitro expansion under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Each line represents a single donor. Figure 12E shows total αβ T cell frequency after 14 days of in vitro expansion under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Each line represents a single donor. [Figure 12E]Figure 12A shows flow cytometry plots (gated on single viable CD3+Vy9+ cells) demonstrating the increased purity of Vy9V52 T cells from a single donor cultured under hypoxic conditions. Figure 12A shows flow cytometry plots (gated on single viable CD3+Vy9+ cells) demonstrating the increased purity of Vy9V52 T cell populations in one donor after 14 days of in vitro expansion under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Figure 12B shows a histogram overlap of the plots shown in Figure 12A, demonstrating the increased Vy9V52 T cell population. Figure 12C shows that Vy9V52 T cells have increased purity with fold changes observed under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Each line represents a single donor. Figure 12D shows total Vy9V52 T cell frequency after 14 days of in vitro expansion under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Each line represents a single donor. Figure 12E shows total αβ T cell frequency after 14 days of in vitro expansion under different culture conditions (normoxia control, 18.2% oxygen; hypoxia: 12% oxygen, 5% oxygen, 2% oxygen). Each line represents a single donor. [Figure 13] FIG. 1 is a schematic diagram of the process of the present disclosure in which bispecific antibodies such as Vy9xTAA or CD3xTAA are used to redirect Vy9V52 T cells to tumor-associated antigen (TAA)-expressing tumor cells. [Figure 14] Figure 1 shows that Vy9V52 T cells expanded under normoxic (18.2% oxygen, top panel) and hypoxic (5% oxygen, bottom panel) conditions maintained a robust effector profile in the presence of Vy9xHER2 or CD3xHER2 bispecific antibodies. [Figure 15A] Figure 1 shows the expansion of Vγ9Vδ2 T cells between different donors from fresh peripheral blood mononuclear cells (PBMCs). [Figure 15B] The total number of Vy9V52 T cells obtained after 14 days of expansion is shown. [Figure 16] The percentage of Vγ9+CD3+ cells during enrichment from day 0 to day 14 is shown, demonstrating that cells can be further enriched using negative selection. [Figure 17A] Figure 17 shows the gating strategy for Vy9V52 T cells. Figure 17A shows the gating strategy for determining the number of Vy9V52 T cells. Figure 17B shows the gating strategy for determining the number of Vy9V52 T cells using both Vy9 and V52 staining. [Figure 17B] Figure 17 shows the gating strategy for Vy9V52 T cells. Figure 17A shows the gating strategy for determining the number of Vy9V52 T cells. Figure 17B shows the gating strategy for determining the number of Vy9V52 T cells using both Vy9 and V52 staining. [Figure 18A] Figure 18 shows the study design and purity of Vy9V52 T cells of Example 4 disclosed herein. Figure 18A shows the protocol and timeline of the adoptive transfer process for NSG, NSG-IL15, and NOG-IL15 mice as disclosed in Example 4 of the present disclosure. Figure 18B shows the purity of non-enriched and enriched Vy9V52 T cells. [Figure 18B] Figure 18 shows the study design and purity of Vy9V52 T cells of Example 4 disclosed herein. Figure 18A shows the protocol and timeline of the adoptive transfer process for NSG, NSG-IL15, and NOG-IL15 mice as disclosed in Example 4 of the present disclosure. Figure 18B shows the purity of non-enriched and enriched Vy9V52 T cells. [Figure 19] Flow cytometry analysis of mice 7 days after adoptive transfer is shown. [Figure 20] Figure 1 shows flow cytometry analysis of Vy9V52 T cell engraftment among NSG, NSG-IL15, and NOG-IL15 mouse strains and treatment groups. [Figure 21] Figure 1 shows the kinetics of enriched Vy9V52 T cell engraftment between NSG, NSG-IL15, and NOG-IL15 mouse strains. [Figure 22-1] This shows the engraftment kinetics of human cells (CD45+) in mouse peripheral blood. [Figure 22-2] This shows the engraftment kinetics of human cells (CD45+) in mouse peripheral blood. [Figure 23]Shown is the percentage of Vy9V52 T cells in the mouse circulation normalized to the percentage of CD45+CD3+ cells. [Figure 24] Purity of Vy9V52 T cells in the CD45+CD3+ population is shown. [Figure 25] Figure 1 shows the cell counts of Vy9V52 T cells per microliter of mouse blood. [Figure 26-1] 1 shows the presence of αβ T cells in NOG-IL15 mice that received non-enriched Vγ9Vδ2 T cells. [Figure 26-2] 1 shows the presence of αβ T cells in NOG-IL15 mice that received non-enriched Vγ9Vδ2 T cells. [Figure 27-1] Shown is the body weight of NSG, NSG-IL15, and NOG-IL15 mice following administration of purified Vy9V52 T cells. [Figure 27-2] Shown is the body weight of NSG, NSG-IL15, and NOG-IL15 mice following administration of purified Vy9V52 T cells. [Figure 28-1] Figure 1 shows CD56 expression on adoptively transferred Vy9V52 T cells. [Figure 28-2] Figure 1 shows CD56 expression on adoptively transferred Vy9V52 T cells. [Figure 29-1] Figure 1 shows CD69 expression on adoptively transferred Vy9V52 T cells. [Figure 29-2] Figure 1 shows CD69 expression on adoptively transferred Vy9V52 T cells. [Figure 30] FIG. 1 shows the protocol and timeline of the adoptive transfer process in NOG-IL15 mice using fresh or frozen Vy9V52 T cells as disclosed in Example 5 of the present disclosure. [Figure 31] Vy9V52 T cell purity on the day of adoptive transfer is shown. [Figure 32] Figure 1 shows Vy9V52 T cell engraftment between different donor groups and fresh or frozen cells. [Figure 33-1] Figure 1 shows the kinetics of Vy9V52 T cell engraftment between different donor groups. [Figure 33-2]Figure 1 shows the kinetics of Vy9V52 T cell engraftment between different donor groups. [Figure 34] Shows the purity of negatively enriched Vy9V52 T cells. [Figure 35] Shown are the cell counts per microliter of donor Vy9V52 T cells in mouse blood. [Figure 36] Body weight of NOG-IL15 mice after administration of donor Vy9V52 T cells. [Figure 37] Shows infiltrating γδ T cells (dark staining) in mouse liver tissue. [Figure 38A] Figure 38A is a schematic diagram showing the study design for Example 6 of the present disclosure. Figure 38A shows the three study groups and model used in Example 6. Figure 38B is a schematic diagram showing the treatment schedule for Example 6. [Figure 38B] Figure 38A is a schematic diagram showing the study design for Example 6 of the present disclosure. Figure 38A shows the three study groups and model used in Example 6. Figure 38B is a schematic diagram showing the treatment schedule for Example 6. [Figure 39] 1 is a table showing the design of the three test groups of Example 6. [Figure 40A] Figure 40 shows the gating strategy and characterization of starting Vy9V52 T cells. Figure 40A shows the gating strategy used for detection of Vy9V52 T cells in mouse peripheral blood. Figure 40B shows the purity and phenotype of Vy9V52 T cells before adoptive transfer and subcutaneous implantation. [Figure 40B] Figure 40 shows the gating strategy and characterization of starting Vy9V52 T cells. Figure 40A shows the gating strategy used for detection of Vy9V52 T cells in mouse peripheral blood. Figure 40B shows the purity and phenotype of Vy9V52 T cells before adoptive transfer and subcutaneous implantation. [Figure 41A] Figure 41 shows the persistence of Vy9V52 T cells in mouse peripheral blood. Figure 41A shows a representative flow cytometry analysis of the percentage of Vy9V52 T cells in mouse peripheral blood. Figure 41B shows the percentage of Vy9V52 T cells in mouse peripheral blood in the three test groups over the course of the study. [Figure 41B]Figure 41 shows the persistence of Vy9V52 T cells in mouse peripheral blood. Figure 41A shows a representative flow cytometry analysis of the percentage of Vy9V52 T cells in mouse peripheral blood. Figure 41B shows the percentage of Vy9V52 T cells in mouse peripheral blood in the three test groups over the course of the study. [Figure 42A] Figure 42 shows the kinetic changes in the T cell memory phenotype of Vy9V52 T cells. Figure 42A shows a representative flow cytometry analysis of the percentage of memory phenotype Vy9V52 T cells. Figure 42B shows the percentage of central memory Vy9V52 T cells and effector memory T cells in the peripheral blood of mice over the course of the study. [Figure 42B] Figure 42 shows the kinetic changes in the T cell memory phenotype of Vy9V52 T cells. Figure 42A shows a representative flow cytometry analysis of the percentage of memory phenotype Vy9V52 T cells. Figure 42B shows the percentage of central memory Vy9V52 T cells and effector memory T cells in the peripheral blood of mice over the course of the study. [Figure 43] Figure 1 shows the dynamic changes in the phenotype of Vγ9Vδ2 T cells. [Figure 44] Tumor volume measurements over the course of the study in the three study groups are shown. [Figure 45A] Tumor volume measurements for each individual mouse over the study period in Group 1 (Figure 45A), Group 2 (Figure 45B), and Group 3 (Figure 45C) are shown. Each line represents the tumor volume measurement for one mouse. [Figure 45B] Tumor volume measurements for each individual mouse over the study period in Group 1 (Figure 45A), Group 2 (Figure 45B), and Group 3 (Figure 45C) are shown. Each line represents the tumor volume measurement for one mouse. [Figure 45C] Tumor volume measurements for each individual mouse over the study period in Group 1 (Figure 45A), Group 2 (Figure 45B), and Group 3 (Figure 45C) are shown. Each line represents the tumor volume measurement for one mouse. [Figure 46] Body weight measurements during the study period in the three study groups are shown. [Figure 47A]Shown are the body weight measurements of each individual mouse during the study period in Groups 1 (Figure 47A), 2 (Figure 47B), and 3 (Figure 47C). Each line represents the body weight measurement of one mouse. [Figure 47B] Shown are the body weight measurements of each individual mouse during the study period in Groups 1 (Figure 47A), 2 (Figure 47B), and 3 (Figure 47C). Each line represents the body weight measurement of one mouse. [Figure 47C] Shown are the body weight measurements of each individual mouse during the study period in Groups 1 (Figure 47A), 2 (Figure 47B), and 3 (Figure 47C). Each line represents the body weight measurement of one mouse. Detailed Description of the Invention

[0050] Adoptive transfer of immune cells genetically modified to recognize malignant tumor-associated antigens is a promising cancer treatment (see, e.g., Brenner et al., Current Opinion in Immunology, 22(2):251-257 (2010); Rosenberg et al., Nature Reviews Cancer, 8(4):299-308 (2008)). In particular, genetically engineered immune cells (e.g., T cells) expressing chimeric antigen receptors (CARs) are a potent and promising cancer treatment.

[0051] One known approach to generating clinical-grade autologous CAR T cells is to collect T cells from a patient (or peripheral blood) by leukapheresis, activate them, transduce them with a CAR construct using a viral vector, expand them, and then reinfuse them into the same patient as a single treatment after lymphodepleting chemotherapy (see Ruella et al., BioDrugs, 31(6):473-481 (2017)).

[0052] Despite the unprecedented therapeutic results of CAR T therapy, this approach faces challenges, including limitations due to its autologous characteristics. For example, T cells cannot be harvested and / or expanded from some patients, and the quality of T cells may not meet manufacturing standards. Furthermore, such highly personalized procedures inevitably incur high costs. While allogeneic cell therapy products are actively being explored and developed, one of the biggest obstacles to traditional T cell therapy is the inability of T cells to infiltrate and persist in the immunosuppressive tumor microenvironment or to effectively treat non-solid or semi-solid tumors. Furthermore, traditional CAR-T therapy has inherent manufacturing challenges, such as manufacturing failures, time delays, insufficient cell expansion, or heterogeneous products, which may be harmful to recipient patients.

[0053] Against this background, the present disclosure addresses various challenges in immune cell therapy.

[0054] The present disclosure is based in part on novel methods or processes for producing immune cells, such as Vy9V52 T cells, their improved advantageous properties, and their use to perform cell therapy for the treatment of subjects in need thereof, such as those having a disease or disorder.

[0055] 4.1.Definition The techniques and procedures described or referenced herein include those generally well understood by those of skill in the art and / or those routinely employed by those of skill in the art using conventional techniques in light of the teachings herein, such as the widely used techniques described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009), Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010), and Antibody Engineering Vols 1 and 2 (Kontermann and Dubel eds., 2nd ed. 2010). Unless otherwise defined herein, technical and scientific terms used herein have the meanings commonly understood by those of skill in the art. For purposes of interpreting this specification, the following terminology applies, and where appropriate, terms used in the singular also include the plural and vice versa. In the event that any explanation of a term provided herein conflicts with any document incorporated by reference, the explanation of the term provided below shall prevail.

[0056] The terms "antibody," "immunoglobulin," or "Ig" are used interchangeably herein and are used in the broadest sense, specifically encompassing, for example, monoclonal antibodies, antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single-chain antibodies, and fragments thereof (e.g., domain antibodies), as described below. Antibodies may be human, humanized, chimeric, and / or affinity-matured antibodies, as well as antibodies derived from other species, such as mouse, rabbit, llama, etc.

[0057] An "antigen" is a structure to which an antibody can selectively bind. In some embodiments, the target antigen is a polypeptide. In certain embodiments, the antigen is associated with a cell, e.g., present on or within a cell.

[0058] As used herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to graft one or more antigen specificities into immune cells, such as T cells. CARs are also known as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." In some embodiments, a CAR comprises an extracellular antigen-binding domain specific for one or more antigens (such as tumor antigens), a transmembrane domain, and an intracellular signaling domain of a T cell and / or other receptor. "CAR T cell" refers to a T cell that expresses a CAR.

[0059] An "isolated nucleic acid" is a nucleic acid, e.g., RNA, DNA, or mixed nucleic acid, that is substantially separated from other genomic DNA sequences, and proteins or complexes, such as ribosomes and polymerases, that are naturally associated with the natural sequence. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule.

[0060] The term "vector" refers to a substance used to carry or contain nucleic acid sequences, including, for example, nucleic acid sequences encoding binding molecules (e.g., antibodies) described herein, to introduce nucleic acid sequences into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain operable selection sequences or markers that allow for stable integration into a host cell chromosome.

[0061] As used herein, the term "host cell" refers to a particular subject cell into which a nucleic acid molecule can be transfected, and to the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur during the subsequent generation or integration of the nucleic acid molecule into the host cell genome.

[0062] As used herein, the term "autologous" is meant to refer to any material derived from the same individual that is later reintroduced into the individual.

[0063] As used herein, the term "allogeneic" refers to a graft derived from a different individual of the same species.

[0064] As used herein, the term "normoxia" refers to a state of normal oxygen concentration.

[0065] As used herein, the term "hypoxia" refers to a condition of oxygen concentrations below normal levels.

[0066] The terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. Such cells include the primary subject cell and its progeny.

[0067] As used herein, the term "isolation" or "isolating" refers to a process of increasing the percentage of a particular substance in a composition. For example, isolating a certain type of cell from a cell population refers to the process of creating a cell population in which the percentage of this type of cell is increased compared to the percentage of this type of cell in the original cell population. Thus, when used in the context of a certain type of cell, the term "isolated" does not mean that the isolated cell population contains 100% of this type of cell, but rather that the percentage of this type of cell in the cell population after the isolation process is increased.

[0068] As used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in animals, and more particularly for use in humans, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias.

[0069] In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other components of the pharmaceutical formulation, and suitable for use in contact with the tissues or organs of humans and animals without undue toxicity, irritation, allergic reaction, immunogenicity, or other problem or complication, commensurate with a reasonable benefit / risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005, Handbook of Pharmaceutical Excipients, 6 th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009, Handbook of Pharmaceutical Additives, 3 rd ed.;Ash and Ash Eds., Gower Publishing Company:2007, Pharmaceutical Preformulation and Formulation,2 nded.; Gibson Ed., CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, a pharmaceutically acceptable excipient is non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. In some embodiments, the pharmaceutically acceptable excipient is an aqueous pH buffered solution.

[0070] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a single domain antibody or therapeutic molecule, including drugs and single domain antibodies or pharmaceutical compositions provided herein, that is sufficient to bring about a desired result.

[0071] The terms "subject" and "patient" can be used interchangeably herein. As used herein, in certain embodiments, a subject is a mammal, either non-primate or primate (e.g., human). In specific embodiments, a subject is a human. In one embodiment, a subject is a mammal, e.g., a human, diagnosed with a disease or disorder. In another embodiment, a subject is a mammal, e.g., a human, at risk of developing a disease or disorder.

[0072] As used herein, the terms "treatment" and "treatment / treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or condition resulting from the administration of one or more therapies. Treatment may be determined by assessing whether there has been a reduction, alleviation, and / or alleviation of one or more symptoms associated with the underlying disease, such that an improvement is observed in the patient, even though the patient may still be suffering from the underlying disease. The term "treating" includes both disease management and remission. The terms "manage," "managing," and "management" refer to the beneficial effects a subject derives from treatment, which does not necessarily result in a cure of the disease.

[0073] The terms "prevent," "preventing," and "prevention" refer to reducing the likelihood of the occurrence (or recurrence) of a disease, disorder, condition, or associated symptom (e.g., diabetes or cancer).

[0074] The terms "about" and "approximately" refer to within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0075] As used in this disclosure and the claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0076] Whenever an embodiment is described herein with the term "comprising," it is understood that other similar embodiments otherwise described in terms of "consisting of" and / or "consisting essentially of" are also provided. Whenever an embodiment is described herein with the phrase "consisting essentially of," it is also understood that similar embodiments otherwise described in terms of "consisting of" are also provided.

[0077] The term "between" when used in phrases such as "between A and B" or "between A and B" refers to a range that includes both A and B.

[0078] The term "and / or" used herein in phrases such as "A and / or B" is intended to include both A and B, A or B, A alone, and B alone. Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A alone, B alone, and C alone.

[0079] 4.2. Methods for Ex Vivo Activation and Expansion of Vy9V52 T Cells In one aspect, provided herein are methods for the ex vivo activation and expansion of Vy9V52 T cells under hypoxic conditions. In some embodiments, the methods provided herein comprise contacting a cell population comprising T cells with a culture system comprising IL-2, IL-15, and a bisphosphonate or mevalonate pathway intermediate, and culturing the cell population comprising T cells ex vivo in the culture system under hypoxic conditions for enhanced ex vivo activation and expansion of Vy9V52 T cells in the cell population comprising T cells.

[0080] 4.2.1. Obtaining a cell population containing T cells Exemplary T cells include effector T cells, accessory T cells, cytotoxic T cells, helper T cells, regulatory T cells, and natural killer T cells. It is known in the art that T cell populations can be obtained from numerous sources. In some embodiments, the cell population comprising T cells is obtained from a cultured T cell line. In some embodiments, the cell population comprising T cells is collected, isolated, purified, or derived from a bodily fluid, tissue, or organ, including, but not limited to, peripheral blood, umbilical cord blood, bone marrow, lymph nodes, spleen, or other tissue or bodily fluid of a subject. In certain embodiments, the cell population comprising T cells is peripheral blood lymphocytes, T cell precursors (e.g., hematopoietic stem cells, lymphoid precursor cells, etc.), or cell populations containing the same. Immature T cells can be found in the thymus.

[0081] In certain embodiments, the cell population comprising T cells is peripheral blood mononuclear cells (PBMCs). In certain embodiments, the PBMCs are freshly obtained PBMCs. In certain embodiments, the PBMCs are frozen PBMCs. Various methods for collecting and preparing PBMCs are known in the art.

[0082] In certain embodiments, the cell population comprising T cells is derived from human tissue. In certain embodiments, the human tissue is fresh. In certain embodiments, the human tissue is frozen. Various methods for collecting and preparing human tissue are known in the art.

[0083] In certain embodiments, the cell population comprising T cells is tumor-infiltrating lymphocytes (TILs). In certain embodiments, the TILs are freshly obtained. In certain embodiments, the TILs are frozen. Various methods for collecting and preparing TILs are known in the art.

[0084] In some embodiments, the cell population comprising T cells is a mammalian cell. In certain embodiments, the mammalian cell is a human cell. In certain embodiments, the human cell is a genetically engineered cell. In certain embodiments, the human cell is a non-genetically engineered cell. In certain embodiments, the mammalian cell is a non-human cell. In certain embodiments, the non-human cell is a genetically engineered or non-genetically engineered cell.

[0085] In some embodiments, the cell population comprising T cells is obtained from a subject. In certain embodiments, the cell population comprising T cells is obtained from a healthy subject. In certain embodiments, the cell population comprising T cells is obtained from an unhealthy subject. In certain embodiments, the unhealthy subject has a solid tumor cancer. In certain embodiments, the unhealthy subject has a blood cancer. In certain embodiments, the unhealthy subject has both a solid tumor cancer and a blood cancer. In certain embodiments, the unhealthy subject has an autoimmune disease or an inflammatory disease. In certain embodiments, the unhealthy subject has a neurological disease.

[0086] 4.2.2. Activation and Growth Conditions The methods provided herein involve the ex vivo activation and expansion of Vy9V52 T cells in a culture system.

[0087] In some embodiments, the activation and proliferation conditions include cytokines. Non-limiting examples of cytokines that can be used with the presently disclosed subject matter include lectins, hepatic growth factors, prostaglandins, fibroblast growth factors, prolactin, placental lactogen, OB protein, tumor necrosis factor-α, tumor necrosis factor-β, Müllerian inhibitory substance, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrins, thrombopoietin (TPO), nerve growth factor (NGF), platelet growth factor, TGF-α, TGF-β, insulin-like growth factor-I, insulin-like growth factor-II, erythropoietin (EPO), bone morphogenetic factors, interferon-α, interferon-α, interferon-β ... Interferon-β, interferon-λ, macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF), interleukin-1 (IL-1), IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, LIF, kit-ligand, FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor, and LT (lymphotoxin).

[0088] In some embodiments, the activation and proliferation conditions include substances other than cytokines. In some embodiments, the activation and proliferation conditions include an anti-Vγ9 mAb. In some embodiments, the activation and proliferation conditions include a bisphosphonate. In some embodiments, the activation and proliferation conditions include a mevalonate pathway intermediate. In certain embodiments, the bisphosphonate is selected from the group consisting of zoledronic acid, risedronate, ibandronic acid, alendronate, pamidronate, tiludronic acid, etidronic acid, and clodronate. In certain embodiments, the mevalonate pathway intermediate is selected from the group consisting of HMBPP, BrHPP, and isopentenyl pyrophosphate. In certain embodiments, the bisphosphonate is zoledronic acid. In certain embodiments, the bisphosphonate is risedronate. In certain embodiments, the bisphosphonate is ibandronic acid. In certain embodiments, the bisphosphonate is alendronate. In certain embodiments, the bisphosphonate is pamidronate. In certain embodiments, the bisphosphonate is tiludronic acid. In certain embodiments, the bisphosphonate is etidronic acid. In certain embodiments, the bisphosphonate is clodronate. In certain embodiments, the mevalonate pathway intermediate is HMBPP. In certain embodiments, the mevalonate pathway intermediate is BrHPP. In certain embodiments, the mevalonate pathway intermediate is isopentenyl pyrophosphate.

[0089] In some embodiments, the activating and proliferative conditions include both a cytokine and a bisphosphonate. In some embodiments, the activating and proliferative conditions include both a cytokine and a mevalonate pathway intermediate.

[0090] In certain embodiments, the activation and proliferation conditions include IL-2, IL-15, and zoledronic acid.

[0091] In some embodiments, the IL-2 concentration in the culture system is 10 IU / mL to 1200 IU / mL. In some embodiments, the IL-2 concentration in the culture system is 50 IU / mL to 1200 IU / mL. In some embodiments, the IL-2 concentration in the culture system is 100 IU / mL to 1200 IU / mL. In some embodiments, the IL-2 concentration in the culture system is 100 IU / mL to 1100 IU / mL. In some embodiments, the IL-2 concentration in the culture system is 100 IU / mL to 1000 IU / mL. In some embodiments, the IL-2 concentration in the culture system is 100 IU / mL to 1000 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 10 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 50 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 100 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 200 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 300 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 400 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 500 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 600 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 700 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 800 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 900 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 1000 IU / mL.

[0092] In some embodiments, the IL-2 concentration in the culture system is 1 IU / mL to 100 IU / mL, 1 IU / mL to 50 IU / mL, 1 IU / mL to 40 IU / mL, 1 IU / mL to 30 IU / mL, 1 IU / mL to 20 IU / mL, 5 IU / mL to 50 IU / mL, 5 IU / mL to 30 IU / mL, 5 IU / mL to 20 IU / mL, or 5 IU / mL to 15 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is about 10 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 10 IU / mL.

[0093] In some embodiments, the IL-2 concentration in the culture system remains the same throughout the culture process. In some embodiments, the IL-2 concentration in the culture system is adjusted during the culture process. In certain embodiments, the IL-2 concentration in the culture system is adjusted to gradually decrease along the culture process. In certain embodiments, the IL-2 concentration in the culture system is highest on days 0 and 1, lower on days 2, 3, and 4, and lowest on days 5 and beyond. In certain embodiments, the IL-2 concentration in the culture system is 1000 IU / mL or less on days 0 and 1, 800 IU / mL or less on days 2, 3, and 4, and 100 IU / mL or less on days 5 and beyond.

[0094] In some embodiments, the IL-15 concentration in the culture system is 5 ng / mL to 25 ng / mL. In some embodiments, the IL-15 concentration in the culture system is 5 ng / mL to 20 ng / mL. In some embodiments, the IL-15 concentration in the culture system is 10 ng / mL to 20 ng / mL. In certain embodiments, the IL-15 concentration in the culture system is 5 ng / mL. In certain embodiments, the IL-15 concentration in the culture system is 8 ng / mL. In certain embodiments, the IL-15 concentration in the culture system is 10 ng / mL. In certain embodiments, the IL-15 concentration in the culture system is 13 ng / mL. In certain embodiments, the IL-15 concentration in the culture system is 15 ng / mL. In certain embodiments, the IL-15 concentration in the culture system is 18 ng / mL. In certain embodiments, the IL-15 concentration in the culture system is 20 ng / mL. In certain embodiments, the IL-15 concentration in the culture system is 23 ng / mL. In one specific embodiment, the IL-15 concentration in the culture system is 25 ng / mL.

[0095] In some embodiments, the IL-15 concentration in the culture system is at least 50 ng / mL, at least 100 ng / mL, at least 150 ng / mL, at least 200 ng / mL, at least 250 ng / mL, at least 300 ng / mL, at least 400 ng / mL, at least 500 ng / mL, at most 50 ng / mL, at most 100 ng / mL, at most 150 ng / mL, at most 200 ng / mL, at most 250 ng / mL, at most 300 ng / mL, at most 400 ng / mL, and / or at most 500 ng / mL. In some embodiments, the IL-15 concentration in the culture system is 50 ng / mL to 500 ng / mL, 50 ng / mL to 400 ng / mL, 300 ng / mL to 250 ng / mL, 50 ng / mL to 200 ng / mL, 50 ng / mL to 150 ng / mL, 100 ng / mL to 500 ng / mL, 100 ng / mL to 400 ng / mL, 100 ng / mL to 300 ng / mL, 100 ng / mL to 250 ng / mL, 100 ng / mL to 200 ng / mL, 150 ng / mL to 500 ng / mL, 150 ng / mL to 400ng / mL, 150ng / mL~300ng / mL, 150ng / mL~250ng / mL, 150ng / mL~200ng / mL, 200ng / mL~500ng / mL, 200ng / mL~400ng / mL, 200ng / mL~300ng / m L, 250ng / mL~500ng / mL, 250ng / mL~400ng / mL, 250ng / mL~300ng / mL, 300ng / mL~500ng / mL, 300ng / mL~400ng / mL, or 400ng / mL~500ng / mL. In some embodiments, the IL-15 concentration in the culture system is 50 ng / mL or about 50 ng / mL, 100 ng / mL or about 100 ng / mL, 150 ng / mL or about 150 ng / mL, 200 ng / mL or about 200 ng / mL, 250 ng / mL or about 250 ng / mL, 300 ng / mL or about 300 ng / mL, 400 ng / mL or about 400 ng / mL, or 500 ng / mL or about 500 ng / mL.

[0096] In some embodiments, the IL-15 concentration in the culture system is 50 ng / mL to 150 ng / mL, or 150 ng / mL to 250 ng / mL. In some embodiments, the IL-15 concentration in the culture system is at least 100 ng / mL. In some embodiments, the IL-15 concentration in the culture system is at least 200 ng / mL. In some embodiments, the IL-15 concentration in the culture system is up to 100 ng / mL. In some embodiments, the IL-15 concentration in the culture system is up to 200 ng / mL. In some embodiments, the IL-15 concentration in the culture system is 100 ng / mL or about 100 ng / mL. In some embodiments, the IL-15 concentration in the culture system is 200 ng / mL or about 200 ng / mL.

[0097] In some embodiments, the IL-15 concentration in the culture system remains the same throughout the culture process. In some embodiments, the IL-15 concentration in the culture system is adjusted during the culture process. In certain embodiments, the IL-15 concentration in the culture system is highest on days 2, 3, and 4 and lower on days 0, 1, 5, and thereafter. In certain embodiments, the IL-15 concentration in the culture system is 10 ng / mL or less on days 0 and 1, 20 ng / mL or less on days 2, 3, and 4, and 10 ng / mL or less on days 5 and thereafter.

[0098] In some embodiments, the concentration of zoledronic acid in the culture system is 100 nM to 1000 nM. In some embodiments, the concentration of zoledronic acid in the culture system is 200 nM to 500 nM. In some embodiments, the concentration of zoledronic acid in the culture system is 300 nM to 400 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 100 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 150 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 200 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 250 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 300 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 350 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 400 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 450 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 500 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 550 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 600 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 650 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 700 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 750 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 800 nM.

[0099] 4.2.3. Expansion of Vγ9Vδ2 T cells under hypoxic conditions The methods provided herein comprise culturing a cell population comprising T cells ex vivo in a culture system under hypoxic conditions for enhanced ex vivo activation and expansion of Vy9V52 T cells in the cell population comprising T cells.

[0100] In some embodiments, the cell population comprising T cells is activated and cultured ex vivo in a culture system under hypoxic conditions for at least 3 days, at least 5 days, at least 7 days, at least 9 days, at least 11 days, at least 13 days, at least 15 days, at least 17 days, at least 19 days, or at least 21 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in a culture system under hypoxic conditions for at least 3 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in a culture system under hypoxic conditions for at least 5 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in a culture system under hypoxic conditions for at least 7 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in a culture system under hypoxic conditions for at least 9 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in a culture system under hypoxic conditions for at least 11 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for at least 13 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for at least 15 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for at least 17 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for at least 19 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for at least 21 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 3-28 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 3-25 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo in a culture system under hypoxic conditions for 4 to 23 days.In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 5 to 21 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 6 to 19 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 7 to 17 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 8 to 15 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 9 to 14 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 10 to 14 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 11 to 14 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 12-14 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 10 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 11 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 12 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 13 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 14 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 15 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 16 days, hi certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 17 days.In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 18 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 19 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 20 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 21 days.

[0101] In some embodiments, the hypoxic oxygen concentration is less than 17%, less than 15%, less than 13%, less than 11%, less than 9%, less than 7%, less than 5%, less than 3%, less than 1%, or less than 0.5%. In some embodiments, the hypoxic oxygen concentration is less than 17%. In some embodiments, the hypoxic oxygen concentration is less than 15%. In some embodiments, the hypoxic oxygen concentration is less than 13%. In some embodiments, the hypoxic oxygen concentration is less than 11%. In some embodiments, the hypoxic oxygen concentration is less than 9%. In some embodiments, the hypoxic oxygen concentration is less than 7%. In some embodiments, the hypoxic oxygen concentration is less than 5%. In some embodiments, the hypoxic oxygen concentration is less than 3%. In some embodiments, the hypoxic oxygen concentration is less than 1%. In some embodiments, the hypoxic oxygen concentration is less than 0.5%. In certain embodiments, the hypoxic oxygen concentration is between 0.1% and 17%. In certain embodiments, the hypoxic oxygen concentration is 0.1% to 15%. In certain embodiments, the hypoxic oxygen concentration is 0.5% to 13%. In certain embodiments, the hypoxic oxygen concentration is 1% to 13%. In certain embodiments, the hypoxic oxygen concentration is 1% to 11%. In certain embodiments, the hypoxic oxygen concentration is 1% to 9%. In certain embodiments, the hypoxic oxygen concentration is 1% to 7%. In certain embodiments, the hypoxic oxygen concentration is 2% to 5%. In certain embodiments, the hypoxic oxygen concentration is about 15%. In certain embodiments, the hypoxic oxygen concentration is about 14%. In certain embodiments, the hypoxic oxygen concentration is about 13%. In certain embodiments, the hypoxic oxygen concentration is about 12%. In certain embodiments, the hypoxic oxygen concentration is about 11%. In certain embodiments, the hypoxic oxygen concentration is about 10%. In certain embodiments, the hypoxic oxygen concentration is about 9%. In certain embodiments, the hypoxic oxygen concentration is about 8%. In certain embodiments, the hypoxic oxygen concentration is about 7%.In certain embodiments, the hypoxic oxygen concentration is about 6%. In certain embodiments, the hypoxic oxygen concentration is about 5%. In certain embodiments, the hypoxic oxygen concentration is about 4%. In certain embodiments, the hypoxic oxygen concentration is about 3%. In certain embodiments, the hypoxic oxygen concentration is about 2%. In certain embodiments, the hypoxic oxygen concentration is about 1%.

[0102] In some embodiments, the methods provided herein further comprise culturing the cell population comprising T cells ex vivo in a culture system under normoxic conditions to activate and expand TVy9V52 T cells in the cell population comprising T cells prior to culturing the cell population comprising T cells ex vivo in a culture system under hypoxic conditions. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for at least 1 hour. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for at least 6 hours. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for at least 0.5 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for at least 1 day. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for at least 2 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for at least 3 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for at least 4 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for at least 5 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for 1 hour to 7 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for 6 hours to 7 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for 0.5 days to 7 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for 1 to 7 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for 1 to 6 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for 1 to 5 days.In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for 1-4 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for 1-3 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for 1-2 days.

[0103] In some embodiments, the normoxic oxygen concentration is at least 18%. In some embodiments, the normoxic oxygen concentration is at least 19%. In some embodiments, the normoxic oxygen concentration is at least 20%. In certain embodiments, the normoxic oxygen concentration is 18%-22%. In certain embodiments, the normoxic oxygen concentration is 18%-21%. In certain embodiments, the normoxic oxygen concentration is 18%-20%. In certain embodiments, the normoxic oxygen concentration is 18% or about 18%. In certain embodiments, the normoxic oxygen concentration is 18.2% or about 18.2%. In certain embodiments, the normoxic oxygen concentration is 18.6% or about 18.6%. In certain embodiments, the normoxic oxygen concentration is 19% or about 19%. In certain embodiments, the normoxic oxygen concentration is 19.5% or about 19.5%. In certain embodiments, the normoxic oxygen concentration is 20% or about 20%. In certain embodiments, the normoxic oxygen concentration is at or about 20.5%. In certain embodiments, the normoxic oxygen concentration is at or about 21%.

[0104] In some embodiments, the amount of a particular type of cell is measured by methods known to those of skill in the art, hi some embodiments, the amount of a particular type of cell is measured by flow cytometry analysis.

[0105] As disclosed herein, the total percentage of Vy9V52 T cells in a cell population is calculated by dividing the total number of Vy9V52 T cells, as measured by methods known in the art (e.g., flow cytometry analysis, such as those disclosed in section 6.1.8 of this disclosure), by the total number of cells in the population (i.e., total number of Vy9V52 T cells / total number of cells in the population).

[0106] In some embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, or more than 60%. In some embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by more than 10%. In some embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by more than 15%. In some embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by more than 20%. In some embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by more than 25%. In some embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by more than 30%. In some embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by more than 35%. In some embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by more than 40%. In some embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by more than 45%. In some embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by more than 50%. In some embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by more than 55%. In some embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by more than 60%, hi certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by 10% to 99%.In certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by 20% to 95%. In certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by 30% to 95%. In certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by 35% to 95%. In certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by 40% to 95%. In certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by 45% to 95%. In certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by 50% to 95%. In certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by 60% to 95%. In certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by 65% ​​to 95%. In certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by about 50%. In certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by about 55%. In certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by about 60%. In certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by about 65%. In certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by about 70%.In certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by about 75%. In certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by about 80%. In certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by about 85%. In certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by about 90%. In certain embodiments, the methods provided herein increase the total percentage of Vy9V52 T cells in a cell population comprising T cells by about 95%.

[0107] In some embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by at least 10-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 450-fold, at least 500-fold, at least 550-fold, or at least 600-fold compared to the total number of Vy9V52 T cells in the cell population comprising T cells prior to expansion. In some embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by at least 10-fold. In some embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by at least 30-fold. In some embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by at least 50-fold. In some embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by at least 100-fold. In some embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by at least 150-fold. In some embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by at least 200-fold. In some embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by at least 250-fold. In some embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by at least 300-fold. In some embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by at least 350-fold. In some embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by at least 400-fold.In some embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by at least 450-fold. In some embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by at least 500-fold. In some embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by at least 550-fold. In some embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by at least 600-fold. In certain embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by between 10-fold and 900-fold. In certain embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by between 10-fold and 800-fold. In certain embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by between 30-fold and 700-fold. In certain embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by 30-fold to 650-fold. In certain embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by 50-fold to 600-fold. In certain embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by 100-fold to 600-fold. In certain embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by 150-fold to 600-fold. In certain embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by 200-fold to 600-fold. In certain embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by 250-fold to 600-fold. In certain embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by 300- to 600-fold.In certain embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by 350- to 600-fold. In certain embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by 400- to 600-fold. In certain embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by 450- to 600-fold. In certain embodiments, the methods provided herein increase the total number of Vy9V52 T cells in a cell population comprising T cells by 500- to 600-fold.

[0108] 4.2.4. Enrichment of ex vivo expanded Vγ9Vδ2 T cells In some embodiments, the methods provided herein further comprise isolating or enriching Vy9V52 T cells from the cell population comprising T cells after culturing the cell population comprising T cells ex vivo in a culture system under hypoxic conditions for enhanced ex vivo activation and expansion of Vy9V52 T cells in the cell population comprising T cells.

[0109] In certain embodiments, the method comprises administering a Vγ9 + Isolation of cells followed by Vδ2 + and isolating V52 cells. In certain embodiments, the method comprises: + Isolating cells and subsequently Vγ9 + and isolating the Vγ9 cells. + cells and Vδ2 + In certain embodiments, methods for isolating or enriching Vy9V52 T cells are as described in Section 6.1.5 below.

[0110] Vγ9 + Methods for isolating and enriching cells are known to those of skill in the art. + The method of enriching cells comprises positive selection. +Methods for enriching cells include negative selection.

[0111] In some embodiments, Vγ9 + Methods for enriching cells include negative selection, which involves incubating a cell mixture with a reagent that binds to unwanted cells. + Methods for concentrating cells include density gradient centrifugation to remove unwanted cells using, for example, albumin, dextran, Ficoll, metrizamide, Percoll, and / or the like.

[0112] In some embodiments, Vγ9 + Methods for enriching cells include positive selection, which involves selecting or sorting for cells that have cell surface expression of Vγ9. + The method for enriching cells is Vγ9 + In some embodiments, the Vγ9 + The method for enriching cells includes using an anti-Vγ9 antibody that targets any of the Vγ9 chains. + The method for enriching cells includes an affinity column immobilized with a binding agent for Vγ9. In some embodiments, Vγ9 + Methods for enriching cells include a combination of two or more of the above methods.

[0113] In certain embodiments, Vγ9 + The method for enriching cells includes using magnetic beads coated with an anti-Vγ9 antibody. In certain embodiments, the magnetic beads coated with the anti-Vγ9 antibody are coated with a secondary reagent that binds to the anti-Vγ9 antibody. In certain embodiments, the Vγ9 + The method for enriching cells includes using magnetic microparticles coated with an anti-Vγ9 antibody. In certain embodiments, the magnetic microparticles coated with the anti-Vγ9 antibody are coated with a secondary reagent that binds to the anti-Vγ9 antibody. In certain embodiments, the Vγ9 +The method for enriching cells involves using magnetic nanoparticles coated with an anti-Vγ9 antibody. In certain embodiments, the anti-Vγ9 antibody-coated magnetic nanoparticles are coated with a secondary reagent that binds to the anti-Vγ9 antibody.

[0114] Vδ2 + Methods for isolating and enriching cells are known to those of skill in the art. + Methods for enriching cells include positive selection. + Methods for enriching cells include negative selection.

[0115] In some embodiments, V52 + Methods for enriching cells include negative selection, which involves incubating a cell mixture with a reagent that binds to undesired cells. + Methods for enriching cells include density gradient centrifugation to remove unwanted cells, for example, using albumin, dextran, Ficoll, metrizamide, Percoll, and / or the like. + Methods for enriching cells include positive selection, which involves selecting or sorting for cells with cell surface expression of V52. In some embodiments, V52 + The method for enriching cells is Vδ2 + In some embodiments, the method further comprises FACS sorting of cells. + Methods for enriching cells include using an anti-V52 antibody that targets either of the V52 chains. + Methods for enriching cells include affinity columns immobilized with a binding agent for V52. In some embodiments, V52 + Methods for enriching cells include a combination of two or more of the above methods.

[0116] In certain embodiments, V52 +The method for enriching cells includes using magnetic beads coated with an anti-V52 antibody. In certain embodiments, the magnetic beads coated with the anti-V52 antibody are coated with a secondary reagent that binds to the anti-V52 antibody. In certain embodiments, the V52 + The method for enriching cells includes using magnetic microparticles coated with an anti-V52 antibody. In certain embodiments, the magnetic microparticles coated with the anti-V52 antibody are coated with a secondary reagent that binds to the anti-V52 antibody. In certain embodiments, the V52 + The method of enriching cells comprises using magnetic nanoparticles coated with an anti-V52 antibody, hi certain embodiments, the anti-V52 antibody-coated magnetic nanoparticles are coated with a secondary reagent that binds to the anti-V52 antibody.

[0117] In certain embodiments, after enrichment of Vy9V52 T cells from a cell population comprising T cells, the percentage of Vy9V52 T cells in the cell population is increased compared to a non-enriched population. In some embodiments, the percentage of Vy9V52 T cells in the enriched cell population is greater than about 65%. In some embodiments, the percentage of Vy9V52 T cells in the enriched cell population is greater than about 70%. In some embodiments, the percentage of Vy9V52 T cells in the enriched cell population is greater than about 75%. In some embodiments, the percentage of Vy9V52 T cells in the enriched cell population is greater than about 80%. In some embodiments, the percentage of Vy9V52 T cells in the enriched cell population is greater than about 85%. In some embodiments, the percentage of Vy9V52 T cells in the enriched cell population is greater than about 90%. In some embodiments, the percentage of Vy9V52 T cells in the enriched cell population is greater than about 95%. In some embodiments, the percentage of Vy9V52 T cells in the enriched cell population is greater than about 99%.

[0118] 4.3. Isolated Vγ9Vδ2 T cell population In other embodiments, provided herein is an isolated population of Vy9V52 T cells produced by the methods provided in Section 4.2, below.

[0119] In some embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, or greater than 60%. In some embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is greater than 10%. In some embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is greater than 15%. In some embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is greater than 20%. In some embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is greater than 25%. In some embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is greater than 30%. In some embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is greater than 35%. In some embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is greater than 40%. In some embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is greater than 45%. In some embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is greater than 50%. In some embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is greater than 55%. In some embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is greater than 60%. In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is between 10% and 99%. In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is between 20% and 95%. In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is between 30% and 95%.In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is between 35% and 95%. In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is between 40% and 95%. In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is between 45% and 95%. In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is between 50% and 95%. In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is between 60% and 95%. In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is between 65% and 95%. In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is about 50%. In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is about 55%. In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is about 60%. In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is about 65%. In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is about 70%. In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is about 75%. In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is about 80%. In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is about 85%. In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is about 90%. In certain embodiments, the percentage of Vy9V52 T cells in the isolated Vy9V52 T cell population is about 95%.

[0120] In yet another aspect, provided herein are methods of using the Vy9V52 T cells provided herein in combination with multispecific antibodies, as described in more detail in sections 4.4, 4.7, 4.8, and 4.9 below.

[0121] In yet other aspects, the Vy9V52 T cells provided herein for allogeneic CAR T cell therapy, e.g., as described in more detail in Sections 4.5-4.9 below, are also provided. Methods for using the T cells are provided herein.

[0122] 4.4. Multispecific Antibodies for T Cell Redirection In other embodiments, provided herein are methods of using the Vy9V52 T cells provided herein in combination with a multispecific antibody such that the Vy9V52 T cells are directed to target cells.

[0123] In some embodiments, the multispecific antibody is trispecific. In some embodiments, the multispecific antibody is bispecific. In some embodiments, the multispecific antibody comprises a first binding domain that binds to an antigen expressed on (Vy9V52) T cells and a second binding domain that binds to an antigen expressed on an unhealthy cell.

[0124] Antigens expressed on Vy9V52 T cells are well known in the art. In some embodiments, the antigen expressed on Vy9V52 T cells is T cell receptor gamma variable 9. In some embodiments, the antigen expressed on Vy9V52 T cells is CD3.

[0125] In some embodiments, the unhealthy cells are cancer cells. In certain embodiments, the cancer cells are hematological cancer cells or solid tumor cancer cells. In some embodiments, the unhealthy cells are derived from a subject with an autoimmune or inflammatory disease. In some embodiments, the unhealthy cells are derived from a subject with a neurological disease.

[0126] In some embodiments, the antigen expressed on the unhealthy cell is a tumor antigen. Exemplary tumor antigens include glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, survivin, and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (GFAP), insulin-like growth factor (IGF ... These include, but are not limited to, IGF-I, IGF-II, IGF-I receptor, and mesothelin.

[0127] In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express numerous proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and gp100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER2 / Neu / ErbB-2. Yet another group of target antigens are oncofetal antigens, such as carcinoembryonic antigen (CEA).

[0128] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or tumor-associated antigen (TAA). TSAs are unique to tumor cells and are not present on other cells in the body. TAA-associated antigens are not unique to tumor cells, but instead are also expressed on normal cells under conditions that induce a state of immune tolerance to the antigen. Expression of an antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen. TAAs can be antigens that are expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or they can be antigens that are normally present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0129] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, and TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER2 / neu; unique tumor antigens resulting from chromosomal translocations; BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens, such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7.

[0130] Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMA, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0131] 4.5. METHODS FOR GENERATING Vγ9Vδ2 T CELLS EXPRESSING CHIMERIC ANTIGEN RECEPTORS 4.5.1.Vγ9Vδ2 T cells In other embodiments, provided herein are methods for producing chimeric antigen receptor (CAR) T cell products. In certain embodiments, the method comprises obtaining a cell population comprising Vy9V52 T cells and introducing a nucleic acid encoding a CAR into the Vy9V52 T cells. In some embodiments, the Vy9V52 T cells are produced according to the methods described in Section 4.2 above. In some embodiments, the Vy9V52 T cells are cells described in Section 4.3 above.

[0132] More specifically, in some embodiments, provided herein are methods for generating CAR T cells. In certain embodiments, the method comprises contacting a cell population comprising T cells with a culture system comprising IL-2, IL-15, and a bisphosphonate or mevalonate pathway intermediate, and culturing the cell population comprising T cells ex vivo in the culture system under hypoxic conditions for enhanced ex vivo activation and expansion of Vy9V52 T cells in the cell population comprising T cells.

[0133] T cells can be obtained from a number of sources. In some embodiments, the cell population comprising T cells is obtained from a cultured T cell line. In some embodiments, the cell population comprising T cells is collected, isolated, purified, or derived from a bodily fluid, tissue, or organ, including, but not limited to, peripheral blood, umbilical cord blood, bone marrow, lymph nodes, spleen, or other tissue or bodily fluid of a subject. In certain embodiments, the cell population comprising T cells is peripheral blood lymphocytes, T cell precursor cells (e.g., hematopoietic stem cells, lymphoid precursor cells, etc.), or a cell population containing the same. Immature T cells can be found in the thymus.

[0134] In certain embodiments, the cell population comprising T cells is peripheral blood mononuclear cells (PBMCs). In certain embodiments, the PBMCs are freshly obtained PBMCs. In certain embodiments, the PBMCs are frozen PBMCs. Various methods for collecting and preparing PBMCs are known in the art.

[0135] In certain embodiments, the cell population comprising T cells is derived from human tissue. In certain embodiments, the human tissue is fresh. In certain embodiments, the human tissue is frozen. Various methods for collecting and preparing human tissue are known in the art.

[0136] In certain embodiments, the cell population comprising T cells is tumor-infiltrating lymphocytes (TILs). In certain embodiments, the TILs are freshly obtained. In certain embodiments, the TILs are frozen. Various methods for collecting and preparing TILs are known in the art.

[0137] In some embodiments, the cell population comprising T cells is a mammalian cell. In certain embodiments, the mammalian cell is a human cell. In certain embodiments, the human cell is a genetically engineered cell. In certain embodiments, the human cell is a non-genetically engineered cell. In certain embodiments, the mammalian cell is a non-human cell. In certain embodiments, the non-human cell is a genetically engineered cell. In certain embodiments, the non-human cell is a non-genetically engineered cell.

[0138] In some embodiments, the cell population comprising T cells is obtained from a subject. In certain embodiments, the cell population comprising T cells is obtained from a healthy subject. In certain embodiments, the cell population comprising T cells is obtained from an unhealthy subject. In certain embodiments, the unhealthy subject has a solid tumor cancer. In certain embodiments, the unhealthy subject has a blood cancer. In certain embodiments, the unhealthy subject has both a solid tumor cancer and a blood cancer. In certain embodiments, the unhealthy subject has an autoimmune disease and an inflammatory disease. In certain embodiments, the unhealthy subject has a neurological disease.

[0139] The cell population comprising T cells is cultured ex vivo in a culture system for activation and expansion of Vy9V52 T cells.

[0140] In some embodiments, the activation and proliferation conditions include cytokines, including, but not limited to, lectins, hepatic growth factors, prostaglandins, fibroblast growth factors, prolactin, placental lactogen, OB protein, tumor necrosis factor-α, tumor necrosis factor-β, Müllerian inhibitory substance, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrins, thrombopoietin (TPO), nerve growth factor (NGF), platelet growth factors, TGF-α, TGF-β, insulin-like growth factor-I, insulin-like growth factor-II, erythropoietin (EPO), bone morphogenetic factors, interferon-α, interferon-β, insulin-like growth factor-II, and erythropoietin (EPO). These include interferon-lambda, macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF), interleukin-1 (IL-1), IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, LIF, kit-ligand, FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor, and LT (lymphotoxin).

[0141] In some embodiments, the activation and proliferation conditions include substances other than cytokines. In some embodiments, the activation and proliferation conditions include one or more anti-Vγ9 antibodies. In some embodiments, the activation and proliferation conditions include a bisphosphonate. In some embodiments, the activation and proliferation conditions include a mevalonate pathway intermediate. In certain embodiments, the bisphosphonate is selected from the group consisting of zoledronic acid, risedronate, ibandronic acid, alendronate, pamidronate, tiludronic acid, etidronic acid, and clodronate. In certain embodiments, the mevalonate pathway intermediate is selected from the group consisting of HMBPP, BrHPP, and isopentenyl pyrophosphate. In certain embodiments, the bisphosphonate is zoledronic acid. In certain embodiments, the bisphosphonate is risedronate. In certain embodiments, the bisphosphonate is ibandronic acid. In certain embodiments, the bisphosphonate is alendronate. In certain embodiments, the bisphosphonate is pamidronate. In certain embodiments, the bisphosphonate is tiludronic acid. In certain embodiments, the bisphosphonate is etidronic acid. In certain embodiments, the bisphosphonate is clodronate. In certain embodiments, the mevalonate pathway intermediate is HMBPP. In certain embodiments, the mevalonate pathway intermediate is BrHPP. In certain embodiments, the mevalonate pathway intermediate is isopentenyl pyrophosphate.

[0142] In some embodiments, the activation and proliferation conditions include both a cytokine and a bisphosphonate. In some embodiments, the activation and proliferation conditions include both a cytokine and a mevalonate pathway intermediate. In certain embodiments, the activation and proliferation conditions include IL-2, IL-15, and zoledronic acid.

[0143] In some embodiments, the IL-2 concentration in the culture system is 10 IU / mL to 1200 IU / mL. In some embodiments, the IL-2 concentration in the culture system is 50 IU / mL to 1200 IU / mL. In some embodiments, the IL-2 concentration in the culture system is 100 IU / mL to 1200 IU / mL. In some embodiments, the IL-2 concentration in the culture system is 100 IU / mL to 1100 IU / mL. In some embodiments, the IL-2 concentration in the culture system is 100 IU / mL to 1000 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 10 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 50 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 100 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 200 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 300 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 400 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 500 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 600 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 700 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 800 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 900 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 1000 IU / mL.

[0144] In some embodiments, the IL-2 concentration in the culture system is 1 IU / mL to 100 IU / mL, 1 IU / mL to 50 IU / mL, 1 IU / mL to 40 IU / mL, 1 IU / mL to 30 IU / mL, 1 IU / mL to 20 IU / mL, 5 IU / mL to 50 IU / mL, 5 IU / mL to 30 IU / mL, 5 IU / mL to 20 IU / mL, or 5 IU / mL to 15 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is about 10 IU / mL. In certain embodiments, the IL-2 concentration in the culture system is 10 IU / mL.

[0145] In some embodiments, the IL-2 concentration in the culture system remains the same throughout the culture process. In some embodiments, the IL-2 concentration in the culture system is adjusted during the culture process. In certain embodiments, the IL-2 concentration in the culture system is adjusted to gradually decrease during the culture process. In certain embodiments, the IL-2 concentration in the culture system is highest on days 0 and 1, lower on days 2, 3, and 4, and lowest on days 5 and beyond. In certain embodiments, the IL-2 concentration in the culture system is 1000 IU / mL or less on days 0 and 1, 800 IU / mL or less on days 2, 3, and 4, and 100 IU / mL or less on days 5 and beyond.

[0146] In some embodiments, the IL-15 concentration in the culture system is 5 ng / mL to 25 ng / mL. In some embodiments, the IL-15 concentration in the culture system is 5 ng / mL to 20 ng / mL. In some embodiments, the IL-15 concentration in the culture system is 10 ng / mL to 20 ng / mL. In certain embodiments, the IL-15 concentration in the culture system is 5 ng / mL. In certain embodiments, the IL-15 concentration in the culture system is 8 ng / mL. In certain embodiments, the IL-15 concentration in the culture system is 10 ng / mL. In certain embodiments, the IL-15 concentration in the culture system is 13 ng / mL. In certain embodiments, the IL-15 concentration in the culture system is 15 ng / mL. In certain embodiments, the IL-15 concentration in the culture system is 18 ng / mL. In certain embodiments, the IL-15 concentration in the culture system is 20 ng / mL. In certain embodiments, the IL-15 concentration in the culture system is 23 ng / mL. In one specific embodiment, the IL-15 concentration in the culture system is 25 ng / mL.

[0147] In some embodiments, the IL-15 concentration in the culture system is at least 50 ng / mL, at least 100 ng / mL, at least 150 ng / mL, at least 200 ng / mL, at least 250 ng / mL, at least 300 ng / mL, at least 400 ng / mL, at least 500 ng / mL, at most 50 ng / mL, at most 100 ng / mL, at most 150 ng / mL, at most 200 ng / mL, at most 250 ng / mL, at most 300 ng / mL, at most 400 ng / mL, or at most 500 ng / mL. In some embodiments, the IL-15 concentration in the culture system is 50 ng / mL to 500 ng / mL, 50 ng / mL to 400 ng / mL, 300 ng / mL to 250 ng / mL, 50 ng / mL to 200 ng / mL, 50 ng / mL to 150 ng / mL, 100 ng / mL to 500 ng / mL, 100 ng / mL to 400 ng / mL, 100 ng / mL to 300 ng / mL, 100 ng / mL to 250 ng / mL, 100 ng / mL to 200 ng / mL, 150 ng / mL to 500 ng / mL, 150 ng / mL to 400ng / mL, 150ng / mL~300ng / mL, 150ng / mL~250ng / mL, 150ng / mL~200ng / mL, 200ng / mL~500ng / mL, 200ng / mL~400ng / mL, 200ng / mL~300ng / m L, 250ng / mL~500ng / mL, 250ng / mL~400ng / mL, 250ng / mL~300ng / mL, 300ng / mL~500ng / mL, 300ng / mL~400ng / mL, or 400ng / mL~500ng / mL. In some embodiments, the IL-15 concentration in the culture system is 50 ng / mL or about 50 ng / mL, 100 ng / mL or about 100 ng / mL, 150 ng / mL or about 150 ng / mL, 200 ng / mL or about 200 ng / mL, 250 ng / mL or about 250 ng / mL, 300 ng / mL or about 300 ng / mL, 400 ng / mL or about 400 ng / mL, 500 ng / mL or about 500 ng / mL.

[0148] In some embodiments, the IL-15 concentration in the culture system is 50 ng / mL to 150 ng / mL, or 150 ng / mL to 250 ng / mL. In some embodiments, the IL-15 concentration in the culture system is at least 100 ng / mL. In some embodiments, the IL-15 concentration in the culture system is at least 200 ng / mL. In some embodiments, the IL-15 concentration in the culture system is up to 100 ng / mL. In some embodiments, the IL-15 concentration in the culture system is up to 200 ng / mL. In some embodiments, the IL-15 concentration in the culture system is 100 ng / mL or about 100 ng / mL. In some embodiments, the IL-15 concentration in the culture system is 200 ng / mL or about 200 ng / mL.

[0149] In some embodiments, the IL-15 concentration in the culture system remains the same throughout the culture process. In some embodiments, the IL-15 concentration in the culture system is adjusted during the culture process. In certain embodiments, the IL-15 concentration in the culture system is highest on days 2, 3, and 4 and lower on days 0, 1, 5, and thereafter. In certain embodiments, the IL-15 concentration in the culture system is 10 ng / mL or less on days 0 and 1, 20 ng / mL or less on days 2, 3, and 4, and 10 ng / mL or less on days 5 and thereafter.

[0150] In some embodiments, the concentration of zoledronic acid in the culture system is 100 nM to 1000 nM. In some embodiments, the concentration of zoledronic acid in the culture system is 200 nM to 500 nM. In some embodiments, the concentration of zoledronic acid in the culture system is 300 nM to 400 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 100 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 150 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 200 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 250 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 300 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 350 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 400 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 450 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 500 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 550 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 600 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 650 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 700 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 750 nM. In certain embodiments, the concentration of zoledronic acid in the culture system is 800 nM.

[0151] The T cell-containing cell population is activated and expanded ex vivo in a culture system under hypoxic conditions for enhanced ex vivo activation and expansion of Vy9V52 T cells in the T cell-containing cell population.

[0152] In some embodiments, the cell population comprising T cells is activated and cultured ex vivo in a culture system under hypoxic conditions for at least 3 days, at least 5 days, at least 7 days, at least 9 days, at least 11 days, at least 13 days, at least 15 days, at least 17 days, at least 19 days, or at least 21 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in a culture system under hypoxic conditions for at least 3 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in a culture system under hypoxic conditions for at least 5 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in a culture system under hypoxic conditions for at least 7 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in a culture system under hypoxic conditions for at least 9 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in a culture system under hypoxic conditions for at least 11 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for at least 13 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for at least 15 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for at least 17 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for at least 19 days. In some embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for at least 21 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 3-28 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 3-25 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo in a culture system under hypoxic conditions for 4 to 23 days.In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 5 to 21 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 6 to 19 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 7 to 17 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 8 to 15 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 9 to 14 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 10 to 14 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 11 to 14 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for 12-14 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 10 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 11 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 12 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 13 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 14 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 15 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 16 days, hi certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 17 days.In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 18 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 19 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 20 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo in culture under hypoxic conditions for about 21 days.

[0153] In some embodiments, the hypoxic oxygen concentration is less than 17%, 15%, less than 13%, less than 11%, less than 9%, less than 7%, less than 5%, less than 3%, less than 1%, or less than 0.5%. In some embodiments, the hypoxic oxygen concentration is less than 17%. In some embodiments, the hypoxic oxygen concentration is less than 15%. In some embodiments, the hypoxic oxygen concentration is less than 13%. In some embodiments, the hypoxic oxygen concentration is less than 11%. In some embodiments, the hypoxic oxygen concentration is less than 9%. In some embodiments, the hypoxic oxygen concentration is less than 7%. In some embodiments, the hypoxic oxygen concentration is less than 5%. In some embodiments, the hypoxic oxygen concentration is less than 3%. In some embodiments, the hypoxic oxygen concentration is less than 1%. In some embodiments, the hypoxic oxygen concentration is less than 0.5%. In certain embodiments, the hypoxic oxygen concentration is between 0.1% and 17%. In certain embodiments, the hypoxic oxygen concentration is 0.1% to 15%. In certain embodiments, the hypoxic oxygen concentration is 0.5% to 13%. In certain embodiments, the hypoxic oxygen concentration is 1% to 13%. In certain embodiments, the hypoxic oxygen concentration is 1% to 11%. In certain embodiments, the hypoxic oxygen concentration is 1% to 9%. In certain embodiments, the hypoxic oxygen concentration is 1% to 7%. In certain embodiments, the hypoxic oxygen concentration is 2% to 5%. In certain embodiments, the hypoxic oxygen concentration is about 15%. In certain embodiments, the hypoxic oxygen concentration is about 14%. In certain embodiments, the hypoxic oxygen concentration is about 13%. In certain embodiments, the hypoxic oxygen concentration is about 12%. In certain embodiments, the hypoxic oxygen concentration is about 11%. In certain embodiments, the hypoxic oxygen concentration is about 10%. In certain embodiments, the hypoxic oxygen concentration is about 9%. In certain embodiments, the hypoxic oxygen concentration is about 8%. In certain embodiments, the hypoxic oxygen concentration is about 7%. In certain embodiments, the hypoxic oxygen concentration is about 6%.In certain embodiments, the hypoxic oxygen concentration is about 5%. In certain embodiments, the hypoxic oxygen concentration is about 4%. In certain embodiments, the hypoxic oxygen concentration is about 3%. In certain embodiments, the hypoxic oxygen concentration is about 2%. In certain embodiments, the hypoxic oxygen concentration is about 1%.

[0154] In some embodiments, the cell population comprising T cells is further cultured ex vivo in a culture system under normoxic conditions to activate and expand Vy9V52 T cells in the cell population comprising T cells prior to being cultured ex vivo in a culture system under hypoxic conditions. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for at least 1 hour. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for at least 6 hours. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for at least 0.5 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for at least 1 day. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for at least 2 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for at least 3 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for at least 4 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for at least 5 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for 1 hour to 7 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for 6 hours to 7 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for 0.5 days to 7 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for 1 to 7 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for 1 to 6 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for 1 to 5 days. In certain embodiments, a cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for 1 to 4 days.In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for 1-3 days. In certain embodiments, the cell population comprising T cells is activated and expanded ex vivo under normoxic conditions for 1-2 days.

[0155] In some embodiments, the normoxic oxygen concentration is at least 18%. In some embodiments, the normoxic oxygen concentration is at least 19%. In some embodiments, the normoxic oxygen concentration is at least 20%. In certain embodiments, the normoxic oxygen concentration is 18%-22%. In certain embodiments, the normoxic oxygen concentration is 18%-21%. In certain embodiments, the normoxic oxygen concentration is 18%-20%. In certain embodiments, the normoxic oxygen concentration is 18.2% or about 18.2%. In certain embodiments, the normoxic oxygen concentration is 18.6% or about 18.6%. In certain embodiments, the normoxic oxygen concentration is 19% or about 19%. In certain embodiments, the normoxic oxygen concentration is 19.5% or about 19.5%. In certain embodiments, the normoxic oxygen concentration is 20% or about 20%. In certain embodiments, the normoxic oxygen concentration is at or about 20.5%. In certain embodiments, the normoxic oxygen concentration is at or about 21%.

[0156] In some embodiments, the amount of a particular type of cell is measured by methods known to those of skill in the art, hi some embodiments, the amount of a particular type of cell is measured by flow cytometry analysis.

[0157] In some embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, or more than 60%. In some embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to more than 10%. In some embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to more than 15%. In some embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to more than 20%. In some embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to more than 25%. In some embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to more than 30%. In some embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to greater than 35%. In some embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to greater than 40%. In some embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to greater than 45%. In some embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to greater than 50%. In some embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to greater than 55%. In some embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to greater than 60%. In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased from 10% to 99%. In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to between 20% and 95%. In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to between 30% and 95%. In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to between 35% and 95%.In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to between 40% and 95%. In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to between 45% and 95%. In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to between 50% and 95%. In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to between 60% and 95%. In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to between 65% and 95%. In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to about 50%. In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to about 55%. In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to about 60%. In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to about 65%. In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to about 70%. In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to about 75%. In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to about 80%. In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to about 85%. In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to about 90%. In certain embodiments, the total percentage of Vy9V52 T cells in a cell population comprising T cells is increased to about 95%.

[0158] In some embodiments, the total number of Vy9V52 T cells in the cell population comprising T cells is increased by at least 10-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 450-fold, at least 500-fold, at least 550-fold, or at least 600-fold compared to the total number of Vy9V52 T cells in the cell population comprising T cells prior to expansion. In some embodiments, the total number of Vy9V52 T cells in the cell population comprising T cells is increased by at least 10-fold. In some embodiments, the total number of Vy9V52 T cells in the cell population comprising T cells is increased by at least 30-fold. In some embodiments, the total number of Vy9V52 T cells in the cell population comprising T cells is increased by at least 50-fold. In some embodiments, the total number of Vy9V52 T cells in the cell population comprising T cells is increased by at least 100-fold. In some embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by at least 150-fold. In some embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by at least 200-fold. In some embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by at least 250-fold. In some embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by at least 300-fold. In some embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by at least 350-fold. In some embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by at least 400-fold. In some embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by at least 450-fold. In some embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by at least 500-fold. In some embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by at least 550-fold.In some embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by at least 600-fold. In certain embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by 10-900-fold. In certain embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by 10-800-fold. In certain embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by 30-700-fold. In certain embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by 30-650-fold. In certain embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by 50-600-fold. In certain embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by 100-600-fold. In certain embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by 150-600 fold. In certain embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by 200-600 fold. In certain embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by 250-600 fold. In certain embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by 300-600 fold. In certain embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by 350-600 fold. In certain embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by 400-600 fold. In certain embodiments, the total number of Vy9V52 T cells in a cell population comprising T cells is increased by 450-600 fold. In certain embodiments, the total number of Vγ9Vδ2 T cells in a cell population comprising T cells is increased by 500-600 fold.

[0159] In some embodiments, Vy9V52 T cells are isolated or enriched from a population of cells comprising T cells after culturing the population of cells comprising T cells ex vivo in a culture system under hypoxic conditions for enhanced ex vivo activation and expansion of Vy9V52 T cells in the population of cells comprising T cells.

[0160] In certain embodiments, Vγ9 + Vδ2 cells were first isolated and + In one particular embodiment, V52 cells are isolated second. + Cells were first isolated and Vγ9 + The cells are then isolated. + cells and Vδ2 + The cells are simultaneously isolated, in certain embodiments, Vy9V52 T cells are isolated or enriched as described in Section 6.1.5 below.

[0161] Vγ9 + Methods for isolating and enriching cells are known to those of skill in the art. + The method of enriching cells comprises positive selection. + Methods for enriching cells include negative selection.

[0162] In some embodiments, Vγ9 + Methods for enriching cells include negative selection, which involves incubating a cell mixture with a reagent that binds to unwanted cells. + Methods for enriching cells include density gradient centrifugation to remove unwanted cells, for example, using albumin, dextran, Ficoll, metrizamide, Percoll, and / or the like. + Methods for enriching cells include positive selection, which involves selecting or sorting for cells that have cell surface expression of Vγ9. + The method for enriching cells is Vγ9 + In some embodiments, the Vγ9 + The method for enriching cells includes using an anti-Vγ9 antibody that targets any of the Vγ9 chains. +The method for enriching cells includes an affinity column immobilized with a binding agent for Vγ9. In some embodiments, Vγ9 + Methods for enriching cells include a combination of two or more of the above methods.

[0163] In certain embodiments, Vγ9 + The method for enriching cells includes using magnetic beads coated with an anti-Vγ9 antibody. In certain embodiments, the magnetic beads coated with the anti-Vγ9 antibody are coated with a secondary reagent that binds to the anti-Vγ9 antibody. In certain embodiments, the Vγ9 + The method for enriching cells includes using magnetic microparticles coated with an anti-Vγ9 antibody. In certain embodiments, the magnetic microparticles coated with the anti-Vγ9 antibody are coated with a secondary reagent that binds to the anti-Vγ9 antibody. In certain embodiments, the γ9 + The method for enriching cells includes using magnetic nanoparticles coated with an anti-Vy9 antibody. In certain embodiments, the magnetic nanoparticles coated with the anti-Vy9 antibody are coated with a secondary reagent that binds to the anti-Vy9 antibody.

[0164] VVδ2 + Methods for isolating and enriching cells are known to those of skill in the art. + Methods for enriching cells include positive selection. + Methods for enriching cells include negative selection.

[0165] In some embodiments, V52 + Methods for enriching cells include negative selection, which involves incubating a cell mixture with a reagent that binds to undesired cells. + Methods for enriching cells include density gradient centrifugation to remove unwanted cells, for example, using albumin, dextran, Ficoll, metrizamide, Percoll, and / or the like. +Methods for enriching cells include positive selection, which involves sorting or selecting for cells that have cell surface expression of V52. + The method for enriching cells is Vδ2 + In some embodiments, the method further comprises FACS sorting of cells. + Methods for enriching cells include using an anti-V52 antibody that targets either of the V52 chains. + Methods for enriching cells include affinity columns immobilized with a binding agent for V52. In some embodiments, V52 + Methods for enriching cells include a combination of two or more of the above methods.

[0166] In certain embodiments, V52 + The method for enriching cells comprises using magnetic beads coated with an anti-V52 antibody. In certain embodiments, the magnetic beads coated with the anti-V52 antibody are coated with a secondary reagent that binds to the anti-V52 antibody. In certain embodiments, the Vγ2 + The method for enriching cells comprises using magnetic microparticles coated with an anti-Vγ2 antibody. In certain embodiments, the magnetic microparticles coated with the anti-Vδ2 antibody are coated with a secondary reagent that binds to the anti-Vδ2 antibody. In certain embodiments, the Vδ2 + The method of enriching cells comprises using magnetic nanoparticles coated with an anti-V52 antibody, hi certain embodiments, the anti-V52 antibody-coated magnetic nanoparticles are coated with a secondary reagent that binds to the anti-V52 antibody.

[0167] 4.5.2. Chimeric Antigen Receptors Provided herein are methods for generating CAR T cells, which involve introducing a nucleic acid encoding a chimeric antigen receptor (CAR) into a Vy9V52 T cell.

[0168] In some embodiments, a CAR provided herein comprises a polypeptide comprising: (a) an extracellular antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0169] signal peptide In certain embodiments, the CARs provided herein may include a signal peptide (also known as a signal sequence) at the N-terminus of the polypeptide. Generally, a signal peptide is a peptide sequence that targets a polypeptide to a desired site within a cell. In some embodiments, the signal peptide targets the effector molecule to the secretory pathway of a cell, allowing the effector molecule to be incorporated into and anchored to the lipid bilayer. It will be apparent to one of skill in the art that signal peptides comprising signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences are compatible for use in the CARs described herein. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and IgG1 heavy chain.

[0170] Extracellular antigen-binding domain The extracellular antigen-binding domain of the CAR described herein comprises one or more antigen-binding domains. In some embodiments, the extracellular antigen-binding domain of the CAR provided herein is monospecific. In other embodiments, the extracellular antigen-binding domain of the CAR provided herein is multispecific. In some embodiments, the extracellular antigen-binding domain comprises two or more antigen-binding domains that are directly fused to each other via a peptide bond or via a peptide linker.

[0171] In some embodiments, the extracellular antigen-binding domain comprises an antibody or a fragment thereof. For example, the binding domain can be derived from a monoclonal antibody (including agonist, antagonist, neutralizing, full-length or intact monoclonal antibodies), an antibody with polyepitopic or monoepitopic specificity, a polyclonal antibody or a univalent antibody, a multivalent antibody, a multispecific antibody formed from at least two intact antibodies (e.g., a bispecific antibody, so long as it exhibits the desired biological activity), a single-chain antibody, and fragments thereof (e.g., domain antibodies). The antibody can be human, humanized, chimeric, and / or affinity matured, and can be an antibody from another species, e.g., mouse, rabbit, llama, etc. In some embodiments, antibodies include polypeptide products of B cells within the immunoglobulin class of polypeptides, which are capable of binding to a specific molecular antigen and are composed of two identical paired polypeptide chains, each pair having one heavy chain (approximately 50-70 kDa) and one light chain (approximately 25 kDa), with the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids, and the carboxy-terminal portion of each chain containing a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2nd ed. 1995); and Kuby, Immunology (3rd ed. 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single-domain antibodies, such as those derived from camelid species (e.g., llamas and alpacas) or humanized variants thereof, intracellular antibodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above, which refer to portions of antibody heavy or light chain polypeptides that retain some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFv) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies.In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen-binding domains or molecules containing an antigen-binding site (e.g., one or more CDRs of an antibody) that bind to an antigen. Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989), Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995), Huston et al., 1993, Cell Biophysics 22:189-224, Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515, and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule. The antibody can be an agonist antibody or an antagonist antibody. The antibody may be neither an agonist nor an antagonist.

[0172] In a specific embodiment, the extracellular antigen-binding domain of the CAR of the invention comprises a single-chain Fv (sFv or scFv). ScFv is an antibody fragment comprising a VH antibody domain and a VL antibody domain connected in a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. See Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0173] In another specific embodiment, the extracellular antigen-binding domain of a CAR of the invention comprises one or more single domain antibodies (sdAbs). The sdAbs can be of the same or different origins and can be of the same or different sizes. Exemplary sdAbs include heavy chain-only antibodies (e.g., VHH or VHH). NAR ), a binding molecule naturally lacking a light chain, a single domain (V) derived from a conventional four-chain antibody H or V L Such sdAbs include, but are not limited to, humanized heavy chain-only antibodies, human single domain antibodies produced by transgenic mice or rats expressing human heavy chain segments, and engineered domains and single domain scaffolds other than those derived from antibodies. Any sdAb known in the art or developed by the present disclosure, such as the single domain antibodies described above in this disclosure, can be used to construct the CARs described herein. sdAbs can be derived from any species, including, but not limited to, mouse, rat, human, camel, llama, lamprey, fish, shark, goat, rabbit, and cow. Single domain antibodies contemplated herein also include naturally occurring single domain antibody molecules from species other than camelids and sharks.

[0174] In some embodiments, sdAbs are derived from naturally occurring single domain antigen binding molecules known as heavy chain antibodies that are devoid of light chains (also referred to herein as "heavy chain-only antibodies"). Such single domain molecules are disclosed, for example, in WO 94 / 04678 and Hamers-Casterman, C. et al., Nature 363:446-448 (1993). For clarity, variable domains derived from heavy chain molecules that naturally lack light chains are similar to the conventional V of four-chain immunoglobulins. HTo distinguish them from other VHH molecules, they are known herein as VHHs. Such VHH molecules can be derived from antibodies raised in Camelidae species, such as camel, llama, vicuna, dromedary, alpaca, and guanaco. Other non-Camelidae species can produce heavy chain molecules that naturally lack light chains, and such VHHs are within the scope of this disclosure. In addition, humanized versions and other modifications and variants of VHHs are also contemplated and within the scope of this disclosure. In some embodiments, sdAbs are derived from the variable region of immunoglobulins found in cartilaginous fish. For example, sdAbs can be derived from an immunoglobulin isotype known as a novel antigen receptor (NAR), which is found in shark serum. Methods for producing single domain molecules ("IgNARs") derived from the variable region of NARs are described in WO 03 / 014161 and Streltsov, Protein Sci. 14:2901-2909 (2005).

[0175] In some embodiments, naturally occurring VHH domains against a particular antigen or target can be obtained from a library (naive or immune) of Camelidae VHH sequences. Such methods may or may not involve screening such a library with the antigen or target, or at least one part, fragment, antigenic determinant, or epitope thereof, using one or more screening techniques known in the art. Such libraries and techniques are described, for example, in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020, and WO 03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from (naive or immune) VHH libraries can be used, such as VHH libraries obtained from (naive or immune) VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as described, for example, in WO 00 / 43507.

[0176] In some embodiments, sdAbs are recombinant, CDR-grafted, humanized, camelized, deimmunized, and / or generated in vitro (e.g., selected by phage display). In some embodiments, the amino acid sequence of the framework regions may be altered by "camelization" of specific amino acid residues within the framework regions. Camelization refers to the replacement or substitution of one or more amino acid residues in the amino acid sequence of a (naturally occurring) VH domain from a conventional four-chain antibody by one or more of the amino acid residues that occur at the corresponding position in the VHH domain of a heavy-chain antibody. This can be done by methods known in the art, as will be apparent to those skilled in the art. Such "camelizing" substitutions are preferably inserted at amino acid positions that form and / or are present at the VH-VL interface and / or the so-called camelid hallmark residues, as defined herein (see, e.g., WO 94 / 04678; Davies and Riechmann FEBS Letters 339:285-290 (1994); Davies and Riechmann, Protein Engineering 9(6):531-537 (1996); Riechmann, J. Mol. Biol. 259:957-969 (1996); and Riechmann and Muyldermans, J. Immunol. Meth. 231:25-38 (1999)).

[0177] In some embodiments, the sdAb is a human single domain antibody produced by transgenic mice or rats expressing human heavy chain segments. See, e.g., U.S. Patent Application Publication No. 20090307787, U.S. Patent No. 8,754,287, U.S. Patent Application Publication Nos. 20150289489, 20100122358, and WO 2004049794.

[0178] In some embodiments, single domain antibodies are generated from traditional four-chain antibodies (see, e.g., EP 0 368 684; Ward et al., Nature, 341(6242):544-6 (1989); Holt et al., Trends Biotechnol., 21(11):484-490 (2003); WO 06 / 030220 and WO 06 / 003388).

[0179] In some embodiments, the extracellular antigen-binding domain comprises a humanized antibody or fragment thereof. A humanized antibody may comprise human framework regions and human constant region sequences.

[0180] Humanized antibodies can be produced by a variety of techniques, including CDR grafting (EP 239,400; WO 91 / 09967; U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering, or resurfacing (EP 592,106, 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1994, PNAS 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and, for example, U.S. Pat. Nos. 6,407,213, 5,766,886, WO 93 / 17105, Tan et al., J. Immunol. 169:1119 25 (2002), Caldas et al., Protein Eng. 13 (5):353-60 (2000), Morea et al., Methods 20 (3):267 79 (2000), Baca et al., J. Biol. Chem. 272 ​​(16):10678-84 (1997), Roguska et al., Protein Eng. 9 (10):895 904 (1996), Couto et al., Cancer Res. 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res. 55(8):1717-22 (1995), Sandhu JS, Gene 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol. 235(3):959-73 (1994). See also U.S. Patent Application Publication No. 2005 / 0042664(A1) (February 24, 2005), each of which is incorporated herein by reference in its entirety.

[0181] Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are typically taken from an "import" variable domain. Humanization can be performed, for example, by substituting hypervariable region sequences for the corresponding sequences of a human antibody according to the methods of Jones et al., 1986, Nature 321:522-25; Riechmann, et al., Nature, 1988, 332:323-27; and Verhoeyen, et al., Science, 1988, 239:1534-36).

[0182] In some cases, humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six CDRs of a parent non-human antibody (e.g., a rodent) are grafted onto a human antibody framework. For example, Padlan et al. determined that only about one-third of the CDR residues actually contact the antigen, and called these "specificity-determining residues" or SDRs (Padlan et al., 1995, FASEB J. 9:13339). In the SDR grafting technique, only the SDR residues are grafted onto a human antibody framework (see, for example, Kashmiri et al., 2005, Methods 36:2534).

[0183] The selection of human variable domains, both light and heavy, used to create a humanized antibody can be important to reduce antigenicity. For example, according to the so-called "best-fit" method, the sequence of the variable domain of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent human sequence can be selected as the human framework for the humanized antibody (Sims et al., 1993, J. Immunol. 151:2296-308, and Chothia et al., 1987, J. Mol. Biol. 196:90117). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:428589, and Presta et al., 1993, J. Immunol. 151:2623-32). In some cases, the framework is derived from the consensus sequences of the most abundant human subclasses, VL6 subgroup I (VL6I) ​​and VH subgroup III (VHIII). In other methods, human germline genes are used as the source of the framework regions.

[0184] In an alternative paradigm based on CDR comparison, called superhumanization, the homology of FRs is irrelevant. This method involves comparing non-human sequences with a functional human germline gene repertoire. Genes encoding the same or closely related canonical structures as the mouse sequences are then selected. Next, among the genes that share the canonical structures with non-human antibodies, the genes with the highest homology within the CDRs are selected as FR donors. Finally, non-human CDRs are grafted onto these frameworks (see, for example, Tan et al., 2002, J. Immunol. 169:1119-25).

[0185] Furthermore, it is generally desirable for antibodies to be humanized with retention of affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformations of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, 2000, Protein Eng. 13:819-24), Modeller (Sali and Blundell, 1993, J. Mol. Biol. 234:779-815), and Swiss PDB Viewer (Guex and Peitsch, 1997, Electrophoresis 18:2714-23). Inspection of these displays permits analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, for example, analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is achieved. In general, the hypervariable region residues are directly and most substantially involved in antigen binding.

[0186] Another method for antibody humanization is based on a metric for antibody humanity called Human String Content (HSC). This method compares the mouse sequence with the human germline gene repertoire and scores the differences as HSC. The target sequence is then humanized by maximizing its HSC, rather than using an overall identity measure to generate diverse humanized variants (Lazar et al., 2007, Mol. Immunol. 44:1986-98).

[0187] In addition to the methods described above, empirical methods can be used to generate and select humanized antibodies. These methods involve generating large libraries of humanized variants and selecting the best clones using enrichment or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosomal, and yeast display libraries, as well as by bacterial colony screening (see, e.g., Hoogenboom, 2005, Nat. Biotechnol. 23:1105-16; Dufner et al., 2006, Trends Biotechnol. 24:523-29; Feldhaus et al., 2003, Nat. Biotechnol. 21:163-70; and Schlapschy et al., 2004, Protein Eng. Des. Sel. 17:847-60).

[0188] In the FR library approach, a collection of multiple residue variants is introduced at specific positions in the FR, and the library is then screened to select the FR that best supports the grafted CDR. The substituted residues can include some or all of the "vernier" residues identified as potentially contributing to CDR structure (see, e.g., Foote and Winter, 1992, J. Mol. Biol. 224:48799), or those from the more limited set of target residues identified by Baca et al. (1997, J. Biol. Chem. 272:10678-84).

[0189] In FR shuffling, entire FRs are combined with non-human CDRs rather than creating a combinatorial library of selected residue variants (see, e.g., Dall'Acqua et al., Methods, 2005, 36:43-60). The library can be screened for binding in a two-step process, first humanizing the VL, followed by humanizing the VH. Alternatively, a one-step FR shuffling process can be used. Such a process has been shown to be more efficient than two-step screening, as the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., 2007, Mol. Immunol. 44:3049-60).

[0190] The "humaneering" method is based on the experimental identification of essential minimum specificity determinants (MSDs) and the sequential substitution of non-human fragments into a human FR library and evaluation of binding. Humaneering begins with the CDR3 regions of the non-human VH and VL chains, and gradually substitutes other regions of the non-human antibody, including CDR1 and CDR2 of both VH and VL, with human FRs. This approach typically identifies multiple subclasses of antibodies that retain the epitope but have distinct CDRs in the human V segments. Humaneering allows the isolation of antibodies that are 91-96% homologous to human germline antibodies (see, e.g., Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007).

[0191] "Human engineering" methods involve modifying non-human antibodies or antibody fragments, such as murine or chimeric antibodies or antibody fragments, by making specific changes to the antibody's amino acid sequence to generate modified antibodies that reduce immunogenicity in humans while retaining the desired binding characteristics of the original non-human antibody. Generally, the techniques involve classifying amino acid residues in non-human (e.g., murine) antibodies as "low risk," "moderate risk," or "high risk" residues. Classification is performed using a global risk / benefit calculation that assesses the predicted benefit of making a particular substitution (e.g., for immunogenicity in humans) against the risk that the substitution will affect the folding of the resulting antibody. Specific human amino acid residues to be substituted at a given position (e.g., low risk or moderate risk) in a non-human (e.g., murine) antibody sequence can be selected by aligning amino acid sequences from the variable regions of the non-human antibody with the corresponding regions of specific or consensus human antibody sequences. Amino acid residues at low or moderate risk positions in the non-human sequence can be substituted with the corresponding residue in the human antibody sequence depending on the alignment. Techniques for producing human engineered proteins are described in detail in Studnicka et al., 1994, Protein Engineering 7:80514; U.S. Patent Nos. 5,766,886; 5,770,196; 5,821,123; and 5,869,619, and WO 93 / 11794.

[0192] Composite human antibodies can be produced, for example, using Composite Human Antibody™ technology (Antitope Ltd., Cambridge, United Kingdom). To produce a composite human antibody, fragments of multiple human antibody variable region sequences are engineered to minimize the immunogenicity of the resulting antibody by avoiding T-cell epitopes. Such antibodies may contain human constant region sequences, such as a human light chain constant region and / or a human heavy chain constant region.

[0193] A deimmunized antibody is an antibody from which T cell epitopes have been removed. Methods for producing deimmunized antibodies have been described. See, for example, Jones et al., Methods Mol Biol. 2009;525:40523, xiv, and De Groot et al., Cell. Immunol. 244:148-153 (2006). A deimmunized antibody comprises a T cell epitope-depleted variable region and a human constant region. Briefly, the VH and VL of an antibody are cloned, and then T cell epitopes are identified by examining overlapping peptides derived from the VH and VL of the antibody in a T cell proliferation assay. T cell epitopes are identified by in silico methods to identify peptides that bind to human MHC class II. Mutations are introduced into the VH and VL to abolish binding to human MHC class II. The VH and VL are then used to generate a deimmunized antibody.

[0194] In certain embodiments, the extracellular antigen-binding domain comprises multiple binding domains. In some embodiments, the extracellular antigen-binding domain comprises a multispecific antibody or fragment thereof. In other embodiments, the extracellular antigen-binding domain comprises a multivalent antibody or fragment thereof. The term "specificity" refers to the antigen-binding protein's selective recognition of a specific epitope of an antigen. As used herein, the term "multispecific" denotes that the antigen-binding protein has two or more antigen-binding sites, at least two of which bind different antigens. As used herein, the term "valency" denotes the presence of a specific number of binding sites in the antigen-binding protein. A full-length antibody has two binding sites and is bivalent. Thus, the terms "trivalent," "tetravalent," "pentavalent," and "hexavalent" refer to the presence of two, three, four, five, and six binding sites in the antigen-binding protein, respectively.

[0195] Multispecific antibodies, such as bispecific antibodies, are antibodies that have binding specificities for at least two different antigens.Methods for producing multispecific antibodies are known in the art, for example, by co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (see, for example, Milstein and Cuello, 1983, Nature 305:537-40).For further details on the production of multispecific antibodies (e.g., bispecific antibodies), see, for example, Bispecific Antibodies (Kontermann ed., 2011).

[0196] Antibodies of the present disclosure may be multivalent antibodies with two or more antigen-binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acids encoding the antibody polypeptide chains. In certain embodiments, the multivalent antibody comprises (or consists of), for example, three to about eight antigen-binding sites. In one such embodiment, the multivalent antibody comprises (or consists of) four antigen-binding sites. The multivalent antibody comprises at least one polypeptide chain (e.g., two polypeptide chains), wherein the polypeptide chain comprises two or more variable domains. For example, the polypeptide chain may comprise VD1-(X1)n-VD2-(X2)n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain may comprise a VH-CH1-flexible linker-VH-CH1-Fc region chain; or a VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein may further comprise at least two (e.g., four) light chain variable domain polypeptides. The multivalent antibody herein may comprise, for example, from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated herein comprise a light chain variable domain and optionally further comprise a CL domain.

[0197] When multiple binding domains are present in the extracellular antigen-binding domain of the CAR of the present invention, the various domains may be fused to each other via peptide linkers. In some embodiments, the domains are fused directly to each other without any peptide linkers. The peptide linkers may be the same or different. Each peptide linker may have the same or different length and / or sequence depending on the structural and / or functional characteristics of the various domains. Each peptide linker may be independently selected and optimized. The length, flexibility, and / or other properties of the peptide linker used in the CAR may have some effect on properties including, but not limited to, affinity, specificity, or avidity for one or more particular antigens or epitopes. In some embodiments, the peptide linker contains flexible residues (such as glycine and serine) to allow adjacent protein domains to move freely relative to each other. For example, a glycine-serine doublet may be a suitable peptide linker.

[0198] The peptide linker may have a naturally occurring or non-naturally occurring sequence. For example, a sequence from the hinge region of a heavy chain-only antibody can be used as a linker. See, e.g., WO 1996 / 34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n , (GGGS) n , and (GGGGS) nand the like, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Other linkers known in the art, for example, as described in International Publication Nos. 2016014789, 2015158671, 2016102965, U.S. Patent Application Publication No. 20150299317, WO 2018067992, U.S. Patent No. 7,741,465, Colcher et al., J. Nat. Cancer Inst. 82:1191-1197 (1990), and Bird et al., Science 242:423-426 (1988), the disclosures of each of which are incorporated herein by reference, can also be included in the CARs provided herein.

[0199] In some embodiments, the extracellular antigen-binding domain provided in the CAR of the present invention recognizes an antigen that acts as a cell surface marker on target cells associated with a particular disease state. The antigen targeted by the CAR can be an antigen on a single diseased cell or can be an antigen expressed on different cells, each contributing to the disease. The antigen targeted by the CAR can be directly or indirectly involved in the disease. In some embodiments, the extracellular antigen binding domain binds to an antigen expressed on an unhealthy cell. In some embodiments, the unhealthy cell is a cancer cell. In certain embodiments, the cancer cell is a blood cancer cell or a solid tumor cancer cell. In some embodiments, the unhealthy cell is derived from a subject with an autoimmune disease or inflammatory disease. In some embodiments, the unhealthy cell is derived from a subject with a neurological disease.

[0200] In some embodiments, the extracellular antigen-binding domain binds to a cancer antigen. Exemplary tumor antigens include glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, survivin, and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (GFAP), and telomerase inhibitors. These include, but are not limited to, IGF-I, IGF-II, IGF-I receptor, and mesothelin.

[0201] In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express numerous proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and gp100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER2 / Neu / ErbB-2. Yet another group of target antigens are oncofetal antigens, such as carcinoembryonic antigen (CEA).

[0202] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or tumor-associated antigen (TAA). TSAs are unique to tumor cells and are not present on other cells in the body. TAA-associated antigens are not unique to tumor cells, but instead are also expressed on normal cells under conditions that induce a state of immune tolerance to the antigen. Expression of an antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen. TAAs can be antigens that are expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or they can be antigens that are normally present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0203] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, and TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER2 / neu; unique tumor antigens resulting from chromosomal translocations; BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens, such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7.

[0204] Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMA, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0205] Hinge Area In some embodiments, the CAR provided herein comprises a hinge domain located between the extracellular antigen-binding domain and the transmembrane domain. A hinge domain is generally an amino acid segment found between two domains of a protein, and can allow the protein to be flexible and one or both domains to move relative to each other. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen-binding domain relative to the transmembrane domain of the effector molecule can be used.

[0206] Hinge domains of antibodies (e.g., IgG, IgA, IgM, IgE, or IgD antibodies) are also suitable for use in the pH-dependent chimeric receptor systems described herein. In some embodiments, the hinge domain is a hinge domain that connects the constant domains CH1 and CH2 of an antibody. In some embodiments, the hinge domain is an antibody hinge domain and comprises an antibody hinge domain and one or more antibody constant regions. In some embodiments, the hinge domain comprises an antibody hinge domain and an antibody CH3 constant region. In some embodiments, the hinge domain comprises an antibody hinge domain and an antibody CH2 and CH3 constant region. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises an IgG1 antibody hinge region and a CH2 and CH3 constant region. In some embodiments, the hinge region comprises an IgG1 antibody hinge region and a CH3 constant region.

[0207] Non-naturally occurring peptides can also be used as hinge domains in the chimeric receptors described herein. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain of an Fc receptor is a peptide linker, such as a (GxS)n linker, where x and n can independently be integers between 3 and 12, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

[0208] The hinge domain can contain any one of about 10 to 100 amino acids, e.g., about 15 to 75 amino acids, 20 to 50 amino acids, or 30 to 60 amino acids. In some embodiments, the hinge domain is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length.

[0209] In some embodiments, the hinge domain is the hinge domain of a naturally occurring protein. The hinge domain of any protein known in the art to contain a hinge domain is suitable for use in the chimeric receptors described herein. In some embodiments, the hinge domain is at least a portion of the hinge domain of a naturally occurring protein, conferring flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the hinge domain is a portion of the hinge domain of CD8α, e.g., a fragment containing about 15 to 100 (e.g., 20, 25, 30, 35, or 40) contiguous amino acids of the hinge domain of CD8α.

[0210] Transmembrane domain The CAR of the present disclosure comprises a transmembrane domain that can be directly or indirectly fused to an extracellular antigen-binding domain. The transmembrane domain can be derived from either natural or synthetic sources. As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. A transmembrane domain suitable for use in the CAR described herein can be obtained from a naturally occurring protein. Alternatively, the transmembrane domain can be a synthetic, non-naturally occurring protein segment (e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane).

[0211] Transmembrane domains are classified based on their three-dimensional structure. For example, transmembrane domains can form an α-helix, a complex of two or more α-helices, a β-barrel, or any other stable structure that can span a cellular phospholipid bilayer. Furthermore, transmembrane domains can also, or alternatively, be classified based on their transmembrane domain topology (e.g., the number of times the transmembrane domain passes through the membrane and the orientation of the protein). For example, single-pass membrane proteins cross the cellular membrane once, while multi-pass membrane proteins cross the cellular membrane at least twice (e.g., 2, 3, 4, 5, 6, 7, or more times). Membrane proteins can be defined as type I, type II, or type III depending on their termini relative to the inside and outside of the cell and the topology of the transmembrane segments. Type I membrane proteins have a single transmembrane region and are oriented such that the N-terminus of the protein is present on the extracellular side of the cellular lipid bilayer and the C-terminus of the protein is present on the cytoplasmic side. Type II membrane proteins also have a single transmembrane region, but are oriented such that the C-terminus of the protein is on the extracellular side of the cell's lipid bilayer and the N-terminus of the protein is on the cytoplasmic side. Type III membrane proteins have multiple transmembrane segments and can be further subclassified based on the number of transmembrane segments and the location of the N- and C-termini.

[0212] In some embodiments, the transmembrane domain of a CAR described herein is derived from a type I single-pass membrane protein. In some embodiments, transmembrane domains from multi-pass membrane proteins may also be adapted for use in the CARs described herein. Multi-pass membrane proteins may comprise complex (at least 2, 3, 4, 5, 6, 7, or more) alpha helical or beta sheet structures. In some embodiments, the N- and C-termini of a multi-pass membrane protein are on opposite sides of a lipid bilayer (e.g., the N-terminus of the protein is on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is on the extracellular side).

[0213] The transmembrane domains used in the CARs described herein can also comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment has a length of about 15-100 amino acids. In some embodiments, the protein segment has a length of at least about 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids. Examples of synthetic transmembrane domains are known in the art and are described, for example, in U.S. Pat. No. 7,052,906 and WO 2000 / 032776, the relevant disclosures of which are incorporated herein by reference.

[0214] The transmembrane domains provided herein may comprise a transmembrane region and a cytoplasmic region located at the C-terminus of the transmembrane domain. The cytoplasmic region of the transmembrane domain may comprise three or more amino acids, and in some embodiments, helps orient the transmembrane domain within the lipid bilayer. In some embodiments, one or more cysteine ​​residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine ​​residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain comprises a positively charged amino acid. In some embodiments, the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.

[0215] In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane domain of a CAR provided herein comprises an artificial hydrophobic sequence. For example, a triplet of phenylalanine, tryptophan, and valine may be present at the C-terminus of the transmembrane domain. In some embodiments, the transmembrane region comprises mostly hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the tissue region is hydrophobic. In some embodiments, the transmembrane region comprises a poly-leucine-alanine sequence. The hydropathy, i.e., the hydrophobic or hydrophilic characteristics, of a protein or protein segment can be assessed by any method known in the art (e.g., Kyte and Doolittle hydropathy analysis).

[0216] In some embodiments, the transmembrane domain of the CAR is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDI la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL-2Rβ, IL-2Rγ, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI ld, ITGAE, CD103, ITGAL, CDI la, LFA-1, ITGAM, CD11b, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT and comprising a transmembrane domain selected from AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0217] Intracellular signaling domains The intracellular signaling domain of the CAR provided herein is involved in activating at least one of the normal effector functions of immune effector cells expressing the CAR. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity, including cytokine secretion. Thus, the term "cytoplasmic signaling domain" refers to a portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Typically, the entire cytoplasmic signaling domain can be used, but in many cases, it is not necessary to use the entire chain. To the extent that a cytoplasmic signaling domain can be used, such a truncated portion can be used in place of the intact chain, as long as it transmits the effector function signal. Thus, the term cytoplasmic signaling domain is meant to include any truncated portion of the cytoplasmic signaling domain sufficient to transmit the effector function signal.

[0218] In some embodiments, the intracellular signaling domain comprises a major intracellular signaling domain of an immune effector cell. In some embodiments, the CAR comprises an intracellular signaling domain consisting essentially of a major intracellular signaling domain of an immune effector cell. A "major intracellular signaling domain" refers to a cytoplasmic signaling sequence that acts stimulatory to induce immune effector function. In some embodiments, the major intracellular signaling domain contains a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. As used herein, "ITAM" is a conserved protein motif typically present in the tail portion of signaling molecules expressed in many immune cells. The motif may comprise two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, where each x is independently any amino acid, generating the conserved motif YxxL / Ix(6-8)YxxL / I. ITAMs within signaling molecules are important for intracellular signaling, mediated at least in part by phosphorylation of tyrosine residues within the ITAM following TCR or CAR binding. ITAMs may also serve as docking sites for other proteins involved in signaling pathways. Exemplary ITAM-containing major cytoplasmic signaling sequences include those derived from CD3 zeta, FcRγ (FCER1G), FcRβ (FCER1B), CD3γ, CD3Δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0219] In some embodiments, the major intracellular signaling domain is derived from CD3 zeta. In some embodiments, the intracellular signaling domain consists of the cytoplasmic signaling domain of CD3 zeta. In some embodiments, the major intracellular signaling domain is the cytoplasmic signaling domain of wild-type CD3 zeta.

[0220] Costimulatory signaling domain In some embodiments, the CAR comprises at least one costimulatory signaling domain. As used herein, the term "costimulatory signaling domain" refers to at least a portion of a protein that mediates intracellular signal transduction to induce an immune response, such as an effector function. Many immune effector cells require costimulation in addition to stimulating antigen-specific signals to promote cell proliferation, differentiation, and survival, as well as to activate cell effector functions.

[0221] The costimulatory signaling domain of a CAR described herein can be a cytoplasmic signaling domain derived from a costimulatory protein that transmits a signal and regulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils. A "costimulatory signaling domain" can be the cytoplasmic portion of a costimulatory molecule, or one or more domains thereof. The term "costimulatory molecule" refers to a cognate binding partner on an immune cell (such as a T cell) that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the immune cell, for example, but not limited to, proliferation and survival.

[0222] In some embodiments, the intracellular signaling domain comprises a single costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (such as about any of 2, 3, 4, or more) costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more identical costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains from different costimulatory proteins, such as any two or more costimulatory proteins described herein. In some embodiments, the intracellular signaling domain comprises a major intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3 zeta) and one or more costimulatory signaling domains. In some embodiments, the one or more costimulatory signaling domains and the major intracellular signaling domain (such as the cytoplasmic signaling domain of CD3 zeta) are fused to each other via any peptide linker. The major intracellular signaling domain and one or more costimulatory signaling domains can be arranged in any suitable order. In some embodiments, the one or more costimulatory signaling domains are positioned between the transmembrane domain and the major intracellular signaling domain (such as the cytoplasmic signaling domain of CD3 zeta). Multiple costimulatory signaling domains can provide additive or synergistic stimulatory effects.

[0223] Activation of a costimulatory signaling domain in a host cell (e.g., an immune cell) can induce the cell to increase or decrease cytokine production and secretion, phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. The costimulatory signaling domain of any costimulatory molecule can be adapted for use in the CARs described herein. The type of costimulatory signaling domain is selected based on factors such as the type of immune effector cell on which the effector molecule will be expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC effect, proliferation, cytokine release, and cytotoxicity). Examples of costimulatory signaling domains for use in CARs include members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6); members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BlyS / TNFSF13B, BAFF R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF 8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-α / TNF-β, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α, and TNF RII / TNFRSF1B);SLAM family members (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150); and any other costimulatory molecules, e.g., CD7, CD53, CD82 / Kai-1, CD The cytoplasmic signaling domain may be a cytoplasmic signaling domain of a costimulatory protein, including, but not limited to, 90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA class I, HLADR, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C. In some embodiments, the one or more costimulatory signaling domains are selected from the group consisting of ligands that bind to CD27, CD28, CD137, OX40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. In some embodiments, a costimulatory signaling domain for use in a CAR comprises an intracellular signaling domain derived from a cytokine receptor;

[0224] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure comprises a costimulatory signaling domain derived from CD137 (i.e., 4-1BB). In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of CD3 zeta and the costimulatory signaling domain of CD137.

[0225] In some embodiments, the costimulatory signaling domain is a variant of any of the costimulatory signaling domains described herein, such that the costimulatory signaling domain can modulate the immune response of an immune cell. In some embodiments, the costimulatory signaling domain comprises up to 10 (e.g., 1, 2, 3, 4, 5, or 8) amino acid residue variants compared to the wild-type counterpart. Such costimulatory signaling domains comprising one or more amino acid variants may be referred to as variants. Mutation of amino acid residues in the costimulatory signaling domain may result in increased signaling and enhanced stimulation of an immune response compared to a costimulatory signaling domain that does not comprise the mutation. Mutation of amino acid residues in the costimulatory signaling domain may result in decreased signaling and reduced stimulation of an immune response compared to a costimulatory signaling domain that does not comprise the mutation.

[0226] Polynucleotides In certain embodiments, the present disclosure provides a polynucleotide encoding a CAR described herein. The polynucleotide of the present disclosure can be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, which can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or non-coding (antisense) strand. In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0227] The present disclosure further relates to variants of the polynucleotides described herein, where the variants encode, for example, fragments, analogs, and / or derivatives of an antibody or CAR of the present disclosure. In certain embodiments, the present disclosure provides polynucleotides, including polynucleotides comprising a nucleotide sequence that is at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98%, or 99% identical to a polynucleotide encoding a CAR of the present disclosure. As used herein, the phrase "a polynucleotide comprising a nucleotide sequence at least, e.g., 95% "identical" to a reference nucleotide sequence" is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to five point mutations for every 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide comprising a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides of the reference sequence may be deleted or replaced with alternative nucleotides, or up to 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence. These variations in the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or anywhere between these terminal positions, and may be dispersed individually among the nucleotides of the reference sequence or in one or more contiguous groups within the reference sequence.

[0228] Polynucleotide variants can contain alterations in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain alterations that result in silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, polynucleotide variants contain silent substitutions that do not result in changes to the amino acid sequence of a polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants can be generated for a variety of reasons, such as to optimize codon expression for a particular host (i.e., changing codons in human mRNA to those preferred by a bacterial host, such as E. coli). In some embodiments, polynucleotide variants contain at least one silent mutation in a non-coding or coding region of the sequence.

[0229] In some embodiments, polynucleotide variants are produced to modulate or alter expression (or expression levels) of an encoded polypeptide. In some embodiments, polynucleotide variants are produced to increase expression of an encoded polypeptide. In some embodiments, polynucleotide variants are produced to decrease expression of an encoded polypeptide. In some embodiments, polynucleotide variants increase expression of an encoded polypeptide compared to the parent polynucleotide sequence. In some embodiments, polynucleotide variants decrease expression of an encoded polypeptide compared to the parent polynucleotide sequence.

[0230] Vectors Also provided are vectors comprising the nucleic acids described herein. In one embodiment, the nucleic acids can be incorporated into recombinant expression vectors.

[0231] The present disclosure provides vectors for cloning and expressing any one of the CARs described herein. In some embodiments, the vector is suitable for replication and integration in eukaryotic cells, such as mammalian cells. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex viral vectors, and derivatives thereof. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.

[0232] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to genetically engineered mammalian cells in vitro or ex vivo. Many different retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Numerous adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying immunomodulatory agent (such as immune checkpoint inhibitor) coding sequences and / or self-inactivating lentiviral vectors carrying chimeric antigen receptors can be packaged using protocols known in the art. The resulting lentiviral vectors can be used to transduce mammalian cells (such as primary human T cells) using methods known in the art. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of the transgene and its propagation in progeny cells. Lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.

[0233] In some embodiments, the vector comprises any one of the nucleic acids encoding a CAR described herein. The nucleic acid can be cloned into the vector using any molecular cloning method known in the art, such as, for example, using a restriction endonuclease site and one or more selectable markers. In some embodiments, the nucleic acid is operably linked to a promoter. A variety of promoters have been investigated for gene expression in mammalian cells, and any promoter known in the art can be used in the present disclosure. Promoters can be broadly classified as constitutive promoters or regulatable promoters (e.g., inducible promoters).

[0234] In some embodiments, the nucleic acid encoding the CAR is operably linked to a constitutive promoter. A constitutive promoter allows a heterologous gene (also called a transgene) to be constitutively expressed in a host cell. Exemplary constitutive promoters contemplated herein include, but are not limited to, the cytomegalovirus (CMV) promoter, human elongation factor-1α (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerokinase promoter (PGK), simian virus 40 early promoter (SV40), and chicken β-actin promoter (CAGG) associated with the CMV early enhancer. The efficiency of such constitutive promoters to promote transgene expression has been extensively compared in numerous studies. For example, Michael C. Milone et al. compared the efficiency of CMV, hEF1α, UbiC, and PGK to promote chimeric antigen receptor expression in primary human T cells and concluded that the hEF1α promoter not only drives the highest level of transgene expression but also optimally maintains it in CD4 and CD8 human T cells (Molecular Therapy, 17(8):1453-1464(2009)). In some embodiments, the nucleic acid encoding the CAR is operably linked to the hEF1α promoter.

[0235] In some embodiments, the nucleic acid encoding the CAR is operably linked to an inducible promoter. Inducible promoters belong to the category of regulatable promoters. Inducible promoters can be induced by one or more conditions (such as physical conditions), the microenvironment of the engineered immune effector cells, or the physiological state of the engineered immune effector cells, an inducer (i.e., an inducer), or a combination thereof.

[0236] In some embodiments, the inducing conditions do not induce expression of an endogenous gene in the genetically engineered mammalian cell and / or in a subject receiving the pharmaceutical composition, hi some embodiments, the inducing conditions are selected from the group consisting of an inducer, irradiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox conditions, the tumor environment, and an activation state of the genetically engineered mammalian cell.

[0237] In some embodiments, the vector also contains a selectable marker gene or reporter gene to select cells expressing the CAR from a population of host cells transfected via the lentiviral vector. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. For example, the vector can contain transcription and translation terminators, initiation sequences, and promoters useful for controlling the expression of the nucleic acid sequence.

[0238] 4.6. CAR-expressing Vγ9Vδ2 T cells In yet another aspect, provided herein is a CAR T cell product produced according to the methods provided herein (e.g., as described in Section 5.4, above).

[0239] More specifically, in some embodiments, the CAR-expressing CAR T cells are Vy9V52 T cells.

[0240] In some embodiments, the CAR in the CAR T cells of the invention comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CAR further comprises one or more additional regions / domains, such as a signal peptide, a hinge region, a costimulatory signaling domain, a linker, etc., each of which is as described above in Section 4.5.2.

[0241] Specifically, in some embodiments, the CARs provided herein may further comprise a signal peptide at the N-terminus of the polypeptide. In some embodiments, the signal peptide targets the effector molecule to the secretory pathway of the cell and enables incorporation and anchoring of the effector molecule into the lipid bilayer. Signal peptides, including signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences, suitable for use in the CARs described herein will be apparent to those of skill in the art. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and IgG1 heavy chain.

[0242] The extracellular antigen-binding domain of a CAR described herein comprises one or more antigen-binding domains. In some embodiments, the extracellular antigen-binding domain comprises an antibody or a fragment thereof. In certain embodiments, the extracellular antigen-binding domain of a CAR of the invention comprises a single-chain Fv (sFv or scFv). In some embodiments, the extracellular antigen-binding domain comprises a humanized antibody or a fragment thereof.

[0243] In certain embodiments, the extracellular antigen-binding domain comprises multiple binding domains. In some embodiments, the extracellular antigen-binding domain comprises a multispecific antibody or fragment thereof. In other embodiments, the extracellular antigen-binding domain comprises a multivalent antibody or fragment thereof. When multiple binding domains are present in the extracellular antigen-binding domain of the CAR of the invention, the various domains may be fused to each other via peptide linkers. In some embodiments, the domains are fused directly to each other without any peptide linker. The peptide linkers may be the same or different. Each peptide linker may have the same or different length and / or sequence depending on the structural and / or functional characteristics of the various domains. Each peptide linker may be independently selected and optimized.

[0244] In some embodiments, the extracellular antigen-binding domain provided in the CAR of the present invention recognizes an antigen that acts as a cell surface marker on target cells associated with a particular disease state. The antigen targeted by the CAR can be an antigen on a single diseased cell or can be an antigen expressed on different cells, each contributing to the disease. The antigen targeted by the CAR can be directly or indirectly involved in the disease. In some embodiments, the extracellular antigen binding domain binds to an antigen expressed on an unhealthy cell. In some embodiments, the unhealthy cell is a cancer cell. In certain embodiments, the cancer cell is a blood cancer cell or a solid tumor cancer cell. In some embodiments, the unhealthy cell is derived from a subject with an autoimmune disease or inflammatory disease. In some embodiments, the unhealthy cell is derived from a subject with a neurological disease.

[0245] In some embodiments, the extracellular antigen-binding domain binds to a cancer antigen. Exemplary tumor antigens include glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, survivin, and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (GFAP), and telomerase inhibitors. These include, but are not limited to, IGF-I, IGF-II, IGF-I receptor, and mesothelin.

[0246] In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express numerous proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and gp100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER2 / Neu / ErbB-2. Yet another group of target antigens are oncofetal antigens, such as carcinoembryonic antigen (CEA).

[0247] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or tumor-associated antigen (TAA). TSAs are unique to tumor cells and are not present on other cells in the body. TAA-associated antigens are not unique to tumor cells, but are instead expressed on normal cells under conditions that cannot induce a state of immune tolerance to the antigen. Expression of an antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen. TAAs can be antigens that are expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or they can be antigens that are normally present at very low levels on normal cells but are expressed at much higher levels on tumor cells.

[0248] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, and TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER2 / neu; unique tumor antigens resulting from chromosomal translocations; BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens, such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7.

[0249] Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMA, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0250] In some embodiments, a CAR provided herein comprises a hinge domain located between the extracellular antigen-binding domain and the transmembrane domain. In some embodiments, the hinge domain is that of a naturally occurring protein. The hinge domain of any protein known in the art to contain a hinge domain is suitable for use in the chimeric receptors described herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein, conferring flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the hinge domain is a portion of the hinge domain of CD8α, for example, a fragment containing about 15 to 100 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8α.

[0251] The CARs of the present disclosure comprise a transmembrane domain that can be fused directly or indirectly to an extracellular antigen-binding domain. The transmembrane domain can be derived from either natural or synthetic sources. Transmembrane domains suitable for use in the CARs described herein can be obtained from naturally occurring proteins. Alternatively, the transmembrane domain can be a synthetic, non-naturally occurring protein segment (e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane). In some embodiments, the transmembrane domain is derived from a type I, type II, or type III membrane protein. In some embodiments, the transmembrane domain of a CAR described herein is derived from a type I single-pass membrane protein. In some embodiments, transmembrane domains from multi-pass membrane proteins can also be suitable for use in the CARs described herein. The transmembrane domain used in the CARs described herein can also comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some embodiments, the protein segment has a length of about 15-100 amino acids. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment has a length of at least about 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids.

[0252] The transmembrane domains provided herein may comprise a transmembrane region and a cytoplasmic region located C-terminal to the transmembrane domain. In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues.

[0253] In some embodiments, the transmembrane domain of the CAR is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDI la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL-2Rβ, IL-2Rγ, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CDI la, LFA-1, ITGAM, CD11b, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT and comprising a transmembrane domain selected from AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0254] The intracellular signaling domain within a CAR provided herein is responsible for activating at least one of the normal effector functions of an immune effector cell expressing the CAR. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, a CAR comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of an immune effector cell. In some embodiments, the primary intracellular signaling domain contains a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Exemplary ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3 zeta, FcRγ (FCER1G), FcRβ (FCER1B), CD3γ, CD3Δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0255] In some embodiments, the major intracellular signaling domain is derived from CD3 zeta. In some embodiments, the intracellular signaling domain consists of the cytoplasmic signaling domain of CD3 zeta. In some embodiments, the major intracellular signaling domain is the cytoplasmic signaling domain of wild-type CD3 zeta.

[0256] In some embodiments, the CAR comprises at least one costimulatory signaling domain. The costimulatory signaling domain of the chimeric receptors described herein can be a cytoplasmic signaling domain derived from a costimulatory protein that transmits a signal and modulates a response mediated by an immune cell. In some embodiments, the intracellular signaling domain comprises a single costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (such as about any of two, three, four, or more) costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more identical costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains derived from different costimulatory proteins, such as any two or more costimulatory proteins described herein. In some embodiments, the intracellular signaling domain comprises a major intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3 zeta) and one or more costimulatory signaling domains. In some embodiments, the one or more costimulatory signaling domains and the major intracellular signaling domain (such as the cytoplasmic signaling domain of CD3 zeta) are fused to each other via any peptide linker. The major intracellular signaling domain and one or more costimulatory signaling domains can be arranged in any suitable order. In some embodiments, one or more costimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (such as the cytoplasmic signaling domain of CD3 zeta). Multiple costimulatory signaling domains can provide additive or synergistic stimulatory effects.

[0257] The costimulatory signaling domain of any costimulatory molecule may be adapted for use in the CARs described herein. Examples of costimulatory signaling domains for use in CARs include members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6); members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BlyS / TNFSF13B, BAFF R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF 8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-α / TNF-β, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α, and TNF RII / TNFRSF1B); members of the SLAM family (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150);and the cytoplasmic signaling domain of any other costimulatory protein, including, but not limited to, CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA class I, HLADR, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C. In some embodiments, the one or more costimulatory signaling domains are selected from the group consisting of ligands that bind to CD27, CD28, CD137, OX40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. In some embodiments, a costimulatory signaling domain for use in a CAR comprises an intracellular signaling domain derived from a cytokine receptor;

[0258] In some embodiments, the intracellular signaling domain in a CAR of the present disclosure comprises a costimulatory signaling domain derived from CD137 (i.e., 4-1BB). In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of CD3 zeta and the costimulatory signaling domain of CD137.

[0259] Pharmaceutical Compositions In one aspect, the present disclosure further provides a pharmaceutical composition comprising the activated and expanded Vy9V52 T cells or engineered Vy9V52 T cells of the present disclosure. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the activated and expanded Vy9V52 T cells or engineered Vy9V52 T cells of the present disclosure and a pharmaceutically acceptable excipient.

[0260] In specific embodiments, the term "excipient" can refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), carrier, or vehicle. Pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerin, propylene, glycol, water, ethanol, and the like. If desired, the compositions can further contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Such compositions will contain a prophylactically or therapeutically effective amount of the active ingredients provided herein, such as in purified form, in combination with a suitable amount of excipients to provide a form for proper administration to a patient. The formulation should suit the mode of administration.

[0261] In some embodiments, the choice of excipient will be determined in part by the particular cells and / or by the method of administration, and thus a variety of suitable formulations exist.

[0262] Typically, acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants (such as ascorbic acid, methionine, vitamin E, sodium metabisulfite, and the like); preservatives, tonicity agents, stabilizers, metal complexes (e.g., Zn-protein complexes); chelating agents (such as EDTA and / or nonionic surfactants).

[0263] Buffers can be used to control the pH within a range that optimizes therapeutic efficacy, especially when stability is pH-dependent. Buffers suitable for use in the present disclosure include both organic and inorganic acids and their salts, such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, and acetate. Additionally, buffers can include histidine and trimethylamine salts (such as Tris).

[0264] Preservatives may be added to retard microbial growth. Suitable preservatives for use in the present disclosure include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chlorides, bromides, iodides), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens (such as methyl or propyl paraben); catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0265] Tonicity adjusting agents, sometimes known as "stabilizers," may be present to adjust or maintain the tonicity of the liquid in the composition. When used with large charged biomolecules (such as proteins and antibodies), tonicity adjusting agents are often called "stabilizers" because they can interact with the charged groups of amino acid side chains, thereby reducing the possibility of inter- and intra-molecular interactions. Exemplary tonicity adjusting agents include polyhydric sugar alcohols, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0266] Further exemplary excipients include (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to container walls. Such excipients include polyhydric sugar alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myo-inisitose, myo-inositol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polysaccharides, and the like. Examples of suitable reducing agents include ethylene glycol; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides (e.g., raffinose); and polysaccharides such as dextrin or dextran.

[0267] A non-ionic surfactant or detergent (also known as a "wetting agent") may be present to aid in solubilizing the therapeutic agent and to protect the therapeutic protein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stresses without causing denaturation of the active therapeutic protein or antibody. Suitable non-ionic surfactants include, for example, polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0268] For pharmaceutical compositions to be used for in vivo administration, pharmaceutical compositions are preferably sterile.Pharmaceutical compositions can be sterilized by filtering through a sterile filtration membrane.The pharmaceutical compositions herein can generally be placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0269] The route of administration will be in accordance with known and accepted methods, such as, for example, injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical administration, inhalation, or by sustained or extended release means, as single or multiple boluses, or infusion over an extended period of time, in an appropriate manner.

[0270] In another embodiment, the pharmaceutical composition can be provided as a controlled-release or sustained-release system. In one embodiment, a pump can be used to achieve controlled or sustained release (see, e.g., Sefton, Crit. Ref. Biomed. Eng. 14:201-40 (1987); Buchwald et al., Surgery 88:507-16 (1980); and Saudek et al., N. Engl. J. Med. 321:569-74 (1989)). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of prophylactic or therapeutic agents (e.g., fusion proteins described herein) or compositions provided herein (see, e.g., Medical Applications of Controlled Release (Langer and Wise, eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126 (1983); Levy et al., Science 228:190-92 (1985); During et al., Ann. Neurol. 25:351-56 (1989); Howard et al., J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126 (1983)). al., J. Neurosurg. 71:105-12 (1989); U.S. Patent Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, and 5,128,326; WO 99 / 15154 and WO 99 / 20253).Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymers used in sustained-release formulations are inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In yet another embodiment, controlled-release or sustained-release systems can be placed in close proximity to specific target tissues, such as the nasal cavity or lungs, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984)). Controlled-release systems are discussed, for example, by Langer, Science 249:1527-33 (1990). Any technique known to those of skill in the art can be used to prepare sustained release formulations comprising one or more of the agents described herein (see, e.g., U.S. Pat. No. 4,526,938; WO 91 / 05548 and WO 96 / 20698; Ning, et al., Radiotherapy & Oncology, 39:179-89 (1996); Song et al., PDA J. of Pharma. Sci. & Tech. 50:372-97 (1995); Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-54 (1997); and Lam et al., Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-60 (1997)).

[0271] The pharmaceutical compositions described herein may also contain more than one active compound or agent, as needed for the particular condition being treated. Alternatively, or in addition, the composition may include a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressant, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts effective for the intended purpose.

[0272] The active ingredient can also be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, prepared, for example, by coacervation techniques or by interfacial polymerization, respectively, in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences 18 th It is disclosed in the edition.

[0273] Various compositions and delivery systems are known and can be used with the therapeutic agents provided herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, construction of a nucleic acid as part of a recombinant cell, retrovirus or other vector capable of expressing a single domain antibody or therapeutic molecule provided herein.

[0274] In some embodiments, the pharmaceutical compositions provided herein contain binding molecules and / or cells in an amount effective for treating or preventing a disease or disorder, e.g., a therapeutically or prophylactically effective amount. In some embodiments, the therapeutic or prophylactic effect is monitored by periodic evaluation of the treated subject. In the case of repeated administration over several days or longer, depending on the condition, treatment is repeated until the desired suppression of disease symptoms occurs. However, other drug administration methods may be useful and can be determined.

[0275] 4.8. Methods and Uses In other embodiments, provided herein are methods and uses for using activated and expanded Vy9V52 T cells, such as those described in sections 4.3 and 4.4 above.

[0276] In yet another aspect, provided herein are methods and uses of engineered Vy9V52 T cells expressing recombinant receptors, such as those described in section 4.6 above, including CAR T cells, wherein the CAR T cells are Vy9V52 T cells.

[0277] In some embodiments, provided herein are methods for treating a disease or disorder in a subject, the method comprising administering to the subject (i) a therapeutically effective amount of activated and expanded Vy9V52 T cells, or a pharmaceutical composition comprising activated and expanded Vy9V52 T cells, and (ii) a therapeutically effective amount of one or more multispecific antibodies, such that the activated and expanded Vy9V52 T cells are directed to target cells. In certain embodiments, provided herein are methods for treating a disease or disorder in a subject, the method comprising administering to the subject (i) a therapeutically effective amount of activated and expanded Vy9V52 T cells, or a pharmaceutical composition comprising activated and expanded Vy9V52 T cells, and (ii) a therapeutically effective amount of a Vy9xTAA bispecific antibody. In certain embodiments, provided herein are methods for treating a disease or disorder in a subject, the method comprising administering to the subject (i) a therapeutically effective amount of activated and expanded Vy9V52 T cells or a pharmaceutical composition comprising activated and expanded Vy9V52 T cells, and (ii) a therapeutically effective amount of a CD3xTAA bispecific antibody. In certain embodiments, provided herein are methods for treating a disease or disorder in a subject, the method comprising administering to the subject (i) a therapeutically effective amount of activated and expanded Vy9V52 T cells or a pharmaceutical composition comprising activated and expanded Vy9V52 T cells, and (ii) a therapeutically effective amount of a Vy9xTAA and CD3xTAA bispecific antibody.

[0278] In some embodiments, provided herein are methods for treating a disease or disorder in a subject comprising administering to the subject a therapeutically effective amount of CAR T cells, wherein the CAR T cells are produced by a method comprising: (i) obtaining a cell population comprising Vy9V52 T cells; and (ii) introducing into the cell population a nucleic acid encoding a chimeric antigen receptor (CAR).

[0279] In some embodiments, the activated and expanded Vy9V52 T cells or engineered Vy9V52 T cells provided herein are useful as allogeneic CAR T cell therapy. In some embodiments, the activated and expanded Vy9V52 T cells or engineered Vy9V52 T cells provided herein have safety features not present in conventional autologous T cell therapies, such as no or low cytokine storm, no stimulation of regulatory T cells, reduced autologous tissue damage, reduced autoimmune induction, reduced graft versus host disease, etc.

[0280] Such methods and uses include, for example, therapeutic methods and uses that involve administering cells or compositions containing the cells to a subject having a disease or disorder. In some embodiments, the cells are administered in an amount effective to treat the disease or disorder. Uses include use of the cells in such methods and treatments, and in the preparation of medicaments for carrying out such treatments. In some embodiments, the methods are carried out by administering cells or compositions containing the cells to a subject having or suspected of having a disease or condition. In some embodiments, the methods thereby treat the disease or disorder in the subject.

[0281] In some embodiments, the treatments provided herein result in a complete or partial improvement or reduction of a disease or disorder, or its associated symptoms, adverse effects or outcomes, or phenotype. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, improvement or palliation of the disease state, and remission or improved prognosis. These terms include, but do not imply, complete cure of the disease or complete elimination of any or all symptoms or effects on outcomes.

[0282] As used herein, in some embodiments, the treatments provided herein delay the onset of a disease or disorder, e.g., postpone, prevent, slow, retard, stabilize, inhibit, and / or postpone the onset of a disease (such as cancer). This delay can be of varying duration, depending on the disease history and / or the individual receiving treatment. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease or disorder. For example, late-stage cancer, such as the development of metastases, can be delayed. In other embodiments, the methods or uses provided herein prevent a disease or disorder.

[0283] In some embodiments, the T cell therapy of the present invention is used to treat solid tumor cancers. In some embodiments, the T cell therapy of the present invention is used to treat hematological cancers. In some embodiments, the T cell therapy of the present invention is used to treat autoimmune and inflammatory diseases. In some embodiments, the T cell therapy of the present invention is used to treat neurological diseases.

[0284] In some embodiments, the methods include adoptive cell therapy, in which genetically engineered cells are administered to a subject. Such administration can promote cellular activation (e.g., T cell activation) so that cells of the disease or disorder are targeted for destruction.

[0285] In some embodiments, the methods involve administering cells or compositions containing the cells to a subject, tissue, or cell having, e.g., at risk for, or suspected of having a disease or disorder. In some embodiments, the cells, populations, and compositions are administered to a subject having the particular disease or disorder being treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells or compositions are administered to a subject, e.g., having or at risk for the disease or disorder. In some embodiments, the methods thereby treat, e.g., ameliorate, one or more symptoms of the disease or disorder.

[0286] Methods for administering cells for adoptive cell therapy are known, as described, for example, in U.S. Patent Application Publication No. 2003 / 0170238; U.S. Patent No. 4,690,915; Rosenberg, Nat Rev Clin Oncol. 8(10):577-85 (2011); Themeli et al., Nat Biotechnol. 31(10):928-933 (2013); Tsukahara et al., Biochem Biophys Res Commun 438(1):84-9 (2013); and Davila et al., PloS ONE 8(4):e61338 (2013). These methods can be used in connection with the methods and compositions provided herein.

[0287] In some embodiments, cell therapy (e.g., adoptive T cell therapy) is performed via autologous transplantation, where cells are isolated and / or otherwise prepared from the subject receiving cell therapy or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from the subject in need of treatment, and the cells, after isolation and processing, are administered to the same subject. In other embodiments, cell therapy (e.g., adoptive T cell therapy) is performed via allogeneic transplantation, where cells are isolated and / or otherwise prepared from a subject other than the subject receiving or ultimately receiving cell therapy (e.g., a first subject). In such embodiments, the cells are then administered to a different subject of the same species, e.g., a second subject. In some embodiments, the first subject and the second subject are genetically identical. In some embodiments, the first subject and the second subject are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0288] In some embodiments, the subject to which the cells, cell populations, or compositions are administered is a primate, such as a human. The subject may be male or female and of any suitable age, including infants, juveniles, adolescents, adults, and geriatric subjects. In some examples, the subject is a validated animal model for disease, adoptive cell therapy, and / or for assessing toxicity outcomes.

[0289] The compositions provided herein can be administered by any suitable means, for example, by injection, for example, intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjunctival injection, subconjunctival injection, sub-Tenon injection, retrobulbar injection, periocular injection, or posterior juxtascleral delivery. In some embodiments, they are administered parenterally, intrapulmonaryly, and intranasally, and, if desired for local therapy, by intralesional administration. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0290] The amount of a prophylactic or therapeutic agent provided herein that is effective in the prevention and / or treatment of a disease or condition can be determined by standard clinical techniques. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. The appropriate dose of binding molecule or cell for disease prevention or treatment can depend on the type of disease or disorder being treated, the type of binding molecule, the severity and course of the disease or disorder, whether the agent is administered prophylactically or therapeutically, previous treatments, the patient's medical history and response to the agent, and the discretion of the attending physician. In some embodiments, the compositions, molecules, and cells are suitably administered to the patient at one time or over a series of treatments. Multiple doses can be administered intermittently. An initial higher loading dose, followed by one or more lower doses, can be administered.

[0291] In the context of genetically engineered cells, in some embodiments, a subject may be administered a range of about one million to about 100 billion cells and / or that amount of cells per kilogram of body weight. In some embodiments, where the pharmaceutical composition comprises any one of the genetically engineered immune cells described herein, the pharmaceutical composition is administered at a dose of at least about 10, 10, 10, 10, 10, or 10 cells per kg of body weight of the individual. Doses may vary depending on the disease or disorder, and / or the patient, and / or other treatment-specific attributes.

[0292] In some embodiments, the pharmaceutical composition is administered a single time. In some embodiments, the pharmaceutical composition is administered multiple times (such as two, three, four, five, six, or more times). In some embodiments, the pharmaceutical composition is administered one or more times during a dosing cycle. The dosing cycle can be, for example, one week, two weeks, three weeks, four weeks, five weeks, or more, or one month, two months, three months, four months, five months, or more. The optimal dosage and treatment regimen for a particular patient can be determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0293] In some embodiments, the compositions provided herein are administered as part of a combination therapy, e.g., simultaneously with another therapeutic intervention, such as another antibody or engineered cell or receptor or agent (such as a cytotoxic or therapeutic agent), or sequentially in any order.

[0294] In some embodiments, the compositions provided herein are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some embodiments, the cells are co-administered with another therapy close enough in time so that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the compositions provided herein are administered before one or more additional therapeutic agents. In some embodiments, the compositions provided herein are administered after one or more additional therapeutic agents.

[0295] In certain embodiments, once the cells are administered to a mammal (e.g., a human), the biological activity of the genetically engineered cell population is measured by any of a number of known methods. Parameters assessed include specific binding of genetically engineered or natural T cells or other immune cells to an antigen in vivo (e.g., by imaging) or ex vivo (e.g., by ELISA or flow cytometry). In certain embodiments, the ability of the genetically modified cells to destroy target cells can be measured using any suitable method known in the art, such as the cytotoxicity assays described in Kochenderfer et al., J. Immunotherapy, 32(7):689-702 (2009) and Herman et al., J. Immunological Methods, 285(1):25-40 (2004). In certain embodiments, the biological activity of the cells can also be measured by assaying the expression and / or secretion of certain cytokines, such as CD107a, IFNγ, IL-2, and TNF. In some aspects, biological activity is measured by assessing a clinical outcome, such as reduction in tumor burden or tumor burden.

[0296] In yet another aspect, provided herein are methods for establishing in vivo engraftment of Vy9V52 T cells disclosed herein in a recipient subject. In some embodiments, the method comprises (i) obtaining a cell population comprising Vy9V52 T cells disclosed herein, and (ii) adoptively transferring the cell population into the recipient subject. In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer is obtained using the methods for ex vivo activation and expansion of Vy9V52 T cells described herein.

[0297] In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer is produced by ex vivo activation and expression of a cell population comprising T cells obtained from the recipient subject. In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer is produced by ex vivo activation and expression of a cell population comprising T cells obtained from a subject other than the recipient subject. In some embodiments, the subject from which the cell population comprising T cells is obtained is the donor subject. In some embodiments, the donor subject and recipient subject are of the same species. In some embodiments, the donor subject and recipient subject from the same species are genetically related. In some embodiments, the donor subject and recipient subject are of different species. In some embodiments, the recipient subject is a human in need thereof. In some embodiments, the recipient subject is a model animal. In some embodiments, the donor subject is a human. In some embodiments, the donor subject is a human individual and the recipient subject is a different human individual. In some embodiments, the donor subject is a human individual and the recipient subject is the same human individual. In some embodiments, the donor subject is a human individual and the recipient subject is a non-human mammal. In some embodiments, the non-human animal is a model animal. In some embodiments, the non-human individual is a mouse, pig, monkey, chimpanzee, cow, or sheep. In some embodiments, the recipient subject is a patient suffering from a disease or condition. In some embodiments, the recipient subject has cancer. In some embodiments, the recipient subject has a hematological cancer. In some embodiments, the recipient subject has a solid tumor.

[0298] In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, or more than 60% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise more than 10% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise more than 15% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise more than 20% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise more than 25% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise more than 30% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise more than 35% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise more than 40% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise more than 45% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise more than 50% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise more than 55% Vy9V52 T cells in the cell population. In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises more than 60% Vy9V52 T cells in the cell population.

[0299] In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise 10% to 99%, 20% to 95%, 30% to 95%, 35% to 95%, 40% to 95%, 45% to 95%, 50% to 95%, 55% to 95%, 60% to 95%, or 65% to 95% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise 10% to 99% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise 20% to 95% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise 30% to 95% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise 35%-95% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise 40%-95% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise 45%-95% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise 50%-95% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise 55%-95% Vy9V52 T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise 60%-95% Vy9V52 T cells in the cell population, hi some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise 65%-95% Vy9V52 T cells in the cell population.

[0300] In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer are enriched for Vy9V52 T cells. In some embodiments, enrichment of Vy9V52 T cells uses any method for enriching Vy9V52 T cells described herein. In some embodiments, enriched cell populations comprising Vy9V52 T cells for adoptive transfer comprise more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 99% Vy9V52 T cells in the cell population. In some embodiments, enriched cell populations comprising Vy9V52 T cells for adoptive transfer comprise more than 65% Vy9V52 T cells in the cell population. In some embodiments, enriched cell populations comprising Vy9V52 T cells for adoptive transfer comprise more than 70% Vy9V52 T cells in the cell population. In some embodiments, the enriched cell population comprising Vy9V52 T cells for adoptive transfer comprises more than 75% Vy9V52 T cells in the cell population. In some embodiments, the enriched cell population comprising Vy9V52 T cells for adoptive transfer comprises more than 80% Vy9V52 T cells in the cell population. In some embodiments, the enriched cell population comprising Vy9V52 T cells for adoptive transfer comprises more than 85% Vy9V52 T cells in the cell population. In some embodiments, the enriched cell population comprising Vy9V52 T cells for adoptive transfer comprises more than 90% Vy9V52 T cells in the cell population. In some embodiments, the enriched cell population comprising Vy9V52 T cells for adoptive transfer comprises more than 95% Vy9V52 T cells in the cell population. In some embodiments, the enriched cell population comprising Vy9V52 T cells for adoptive transfer comprises greater than 99% Vy9V52 T cells in the cell population.

[0301] In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer is a purified Vy9V52 T cell population. In some embodiments, the purified Vy9V52 T cell population for adoptive transfer comprises more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 99% Vy9V52 T cells in the cell population. In some embodiments, the purified Vy9V52 T cell population for adoptive transfer comprises more than 65% Vy9V52 T cells in the cell population. In some embodiments, the purified Vy9V52 T cell population for adoptive transfer comprises more than 65% Vy9V52 T cells in the cell population. In some embodiments, the purified Vy9V52 T cell population for adoptive transfer comprises more than 70% Vy9V52 T cells in the cell population. In some embodiments, the purified Vy9V52 T cell population for adoptive transfer comprises more than 75% Vy9V52 T cells in the cell population. In some embodiments, the purified Vy9V52 T cell population for adoptive transfer comprises more than 80% Vy9V52 T cells in the cell population. In some embodiments, the purified Vy9V52 T cell population for adoptive transfer comprises more than 85% Vy9V52 T cells in the cell population. In some embodiments, the purified Vy9V52 T cell population for adoptive transfer comprises more than 90% Vy9V52 T cells in the cell population. In some embodiments, the purified Vy9V52 T cell population for adoptive transfer comprises more than 95% Vy9V52 T cells in the cell population. In some embodiments, the purified Vy9V52 T cell population for adoptive transfer comprises more than 99% Vy9V52 T cells in the cell population. In some embodiments, the purified Vy9V52 T cell population for adoptive transfer comprises 100% Vy9V52 T cells in the cell population.

[0302] In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1% or no αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 95% αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 90% αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 85% αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 80% αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 75% αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 70% αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 65% αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 60% αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 55% αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 50% αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 45% αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 40% αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 35% αβ T cells in the cell population.In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 30% αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 25% αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 20% αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 15% αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 10% αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 5% αβ T cells in the cell population. In some embodiments, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 1% αβ T cells in the cell population. In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer lacks αβ T cells in the cell population.

[0303] In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises at least about or about 5×10 6 cells, at least about or about 1 x 10 7 cells, at least about or about 10 x 10 7 cells, at least about or about 20 x 10 7 cells, at least about or about 30 x 10 7 cells, at least about or about 40 x 10 7 cells, at least about or about 50 x 10 7 cells, at least about or about 60 x 10 7 cells, at least about or about 70 x 10 7 cells, at least about or about 80 x 10 7 cells, at least approximately 90 x 10 7 cells, at least about or about 1 x 10 8 cells, at least about or about 10 x 10 8 cells, at least about or about 20 x 10 8 cells, at least about or about 30 x 10 8cells, at least about or about 40 x 10 8 cells, at least about or about 50 x 10 8 cells, at least about or about 60 x 10 8 cells, at least about or about 70 x 10 8 cells, at least about or about 80 x 10 8 cells, at least approximately 90 x 10 8 cells, or at least about or about 1 x 10 9 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 5x10 6 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 1 x 10 7 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 10 x 10 7 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 20 x 10 7 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 30 x 10 7 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 40 x 10 7 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 50 x 10 7 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 60 x 10 7 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 70 x 10 7 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 80 x 10 7 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises approximately 90 x 10 7 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 1 x 10 8In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 10 x 10 8 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 20 x 10 8 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 30 x 10 8 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 40 x 10 8 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 50 x 10 8 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 60 x 10 8 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 70 x 10 8 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 80 x 10 8 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises approximately 90 x 10 8 In some embodiments, the cell population comprising Vy9V52 T cells for adoptive transfer comprises about 1 x 10 9In any embodiment described herein, a cell population comprising Vy9V52 T cells for adoptive transfer comprises more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, or more than 60% Vy9V52 T cells in the cell population. In any embodiment described herein, a cell population comprising Vy9V52 T cells for adoptive transfer comprises 10% to 99%, 20% to 95%, 30% to 95%, 35% to 95%, 40% to 95%, 45% to 95%, 50% to 95%, 55% to 95%, 60% to 95%, or 65% to 95% Vy9V52 T cells in the cell population. In any of the embodiments described herein, the cell population comprising Vy9V52 T cells for adoptive transfer is enriched for Vy9V52 T cells. In some embodiments, the enriched cell population comprising Vy9V52 T cells for adoptive transfer comprises more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 99% Vy9V52 T cells in the cell population. In any of the embodiments described herein, the cell population comprising Vy9V52 T cells for adoptive transfer is a purified Vy9V52 T cell population. In some embodiments, the purified Vy9V52 T cell population for adoptive transfer comprises more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 99% Vy9V52 T cells in the cell population. In some embodiments, purified Vy9V52 T cell populations for adoptive transfer comprise 100% Vy9V52 T cells. In any of the embodiments described herein, cell populations comprising Vy9V52 T cells for adoptive transfer comprise less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1% or no αβ T cells in the cell population.

[0304] In some embodiments, enrichment of Vy9V52 T cells in the cell population for adoptive transfer is achieved by any of the methods for enriching Vy9V52 T cells described herein. In some embodiments, enrichment of Vy9V52 T cells in the cell population for adoptive transfer is achieved at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days before adoptive transfer of the cell population comprising Vy9V52 T cells into the recipient subject. In some embodiments, enrichment of Vy9V52 T cells in the cell population for adoptive transfer is achieved at least 1 day before adoptive transfer of the cell population comprising Vy9V52 T cells into the recipient subject. In some embodiments, enrichment of Vy9V52 T cells in the cell population for adoptive transfer is achieved at least 2 days before adoptive transfer of the cell population comprising Vy9V52 T cells into the recipient subject. In some embodiments, the enrichment of Vy9V52 T cells in the cell population for adoptive transfer is obtained at least 3 days before adoptive transfer of the cell population comprising Vy9V52 T cells into the recipient subject. In some embodiments, the enrichment of Vy9V52 T cells in the cell population for adoptive transfer is obtained at least 4 days before adoptive transfer of the cell population comprising Vy9V52 T cells into the recipient subject. In some embodiments, the enrichment of Vy9V52 T cells in the cell population for adoptive transfer is obtained at least 5 days before adoptive transfer of the cell population comprising Vy9V52 T cells into the recipient subject. In some embodiments, the enrichment of Vy9V52 T cells in the cell population for adoptive transfer is obtained at least 6 days before adoptive transfer of the cell population comprising Vy9V52 T cells into the recipient subject. In some embodiments, the enrichment of Vy9V52 T cells in the cell population for adoptive transfer is obtained at least 7 days before adoptive transfer of the cell population comprising Vy9V52 T cells into the recipient subject.

[0305] In some embodiments, adoptively transferring the cell population comprising Vy9V52 T cells comprises administering the cell population to the recipient subject. In some embodiments, the method further comprises (iii) administering an effective amount of a second composition concurrently and / or consecutively with the adoptive transfer. In some embodiments, the method further comprises administering an effective amount of the second composition concurrently with the adoptive transfer. In some embodiments, the method further comprises administering an effective amount of the second composition consecutively with the adoptive transfer. In some embodiments, the method further comprises administering an effective amount of the second composition both concurrently and consecutively with the adoptive transfer.

[0306] In some embodiments, administration of the cell population comprising Vy9V52 T cells to the recipient subject is by intravenous administration. In some embodiments, administration of the cell population comprising Vy9V52 T cells to the recipient subject is by intraperitoneal administration. In some embodiments, administration of the second composition to the recipient subject is by intravenous administration. In some embodiments, administration of the second composition to the recipient subject is by intraperitoneal administration. In some embodiments, administration of the cell population comprising Vy9V52 T cells to the recipient subject is by intravenous administration and administration of the second composition to the recipient subject is by intravenous administration. In some embodiments, administration of the cell population comprising Vy9V52 T cells to the recipient subject is by intravenous administration and administration of the second composition to the recipient subject is by intraperitoneal administration. In some embodiments, administration of the cell population comprising Vy9V52 T cells to the recipient subject is by intraperitoneal administration and administration of the second composition to the recipient subject is by intravenous administration. In some embodiments, administering the population of cells comprising Vy9V52 T cells to the recipient subject is by intraperitoneal administration, and administering the second composition to the recipient subject is by intraperitoneal administration.

[0307] In some embodiments, the second composition administered simultaneously with and / or sequentially with the adoptive transfer of a cell population comprising Vy9V52 T cells into the recipient subject is a composition comprising IL-2, IL-15, a bisphosphonate, or a mevalonate pathway intermediate, or a combination thereof. In some embodiments, the second composition comprises an effective amount of IL-2. In some embodiments, the second composition comprises an effective amount of IL-15. In some embodiments, the second composition comprises an effective amount of a bisphosphonate. In some embodiments, the second composition comprises an effective amount of a mevalonate pathway intermediate. In some embodiments, the second composition comprises effective amounts of IL-2 and IL-15. In some embodiments, the second composition comprises effective amounts of IL-2 and a bisphosphonate. In some embodiments, the second composition comprises effective amounts of IL-2 and a mevalonate pathway intermediate. In some embodiments, the second composition comprises effective amounts of IL-15 and a bisphosphonate. In some embodiments, the second composition comprises effective amounts of IL-15 and a mevalonate pathway intermediate. In some embodiments, the second composition comprises effective amounts of IL-2, a bisphosphonate, and a mevalonate pathway intermediate. In some embodiments, the second composition comprises effective amounts of IL-15, a bisphosphonate, and a mevalonate pathway intermediate.

[0308] In some embodiments, the bisphosphonate is selected from the group consisting of zoledronic acid, risedronic acid, ibandronic acid, alendronic acid, pamidronic acid, tiludronic acid, etidronic acid, and clodronic acid. In some embodiments, the bisphosphonate is zoledronic acid. In some embodiments, the bisphosphonate is risedronic acid. In some embodiments, the bisphosphonate is ibandronic acid. In some embodiments, the bisphosphonate is alendronic acid. In some embodiments, the bisphosphonate is pamidronic acid. In some embodiments, the bisphosphonate is tiludronic acid. In some embodiments, the bisphosphonate is etidronic acid. In some embodiments, the bisphosphonate is clodronic acid.

[0309] In some embodiments, the mevalonate pathway intermediate is selected from the group consisting of HMBPP, BrHPP, and isopentenyl pyrophosphate. In some embodiments, the mevalonate pathway intermediate is HMBPP. In some embodiments, the mevalonate pathway intermediate is BrHPP. In some embodiments, the mevalonate pathway intermediate is isopentenyl pyrophosphate.

[0310] In some embodiments, IL-2 is about 1 x 10 3 IU / kg body weight ~ approx. 1×10 5 In some embodiments, IL-2 is administered at a dose of about 2×10 IU / kg body weight. 3 In some embodiments, IL-2 is administered at a dose of about 3×10 IU / kg body weight. 3 In some embodiments, IL-2 is administered at a dose of about 4×10 IU / kg body weight. 3 In some embodiments, IL-2 is administered at a dose of about 5×10 IU / kg body weight. 3 In some embodiments, IL-2 is administered at a dose of about 6×10 IU / kg body weight. 3 In some embodiments, IL-2 is administered at a dose of about 7×10 IU / kg body weight. 3 In some embodiments, IL-2 is administered at a dose of about 8×10 IU / kg body weight. 3 In some embodiments, IL-2 is administered at a dose of about 9×10 IU / kg body weight. 3 In some embodiments, IL-2 is administered at a dose of about 1×10 IU / kg body weight. 4 In some embodiments, IL-2 is administered at a dose of about 2×10 IU / kg body weight. 4 In some embodiments, IL-2 is administered at a dose of about 3×10 IU / kg body weight. 4 In some embodiments, IL-2 is administered at a dose of about 4×10 IU / kg body weight. 4 In some embodiments, IL-2 is administered at a dose of about 5×10 IU / kg body weight.4 In some embodiments, IL-2 is administered at a dose of about 6×10 IU / kg body weight. 4 In some embodiments, IL-2 is administered at a dose of about 7×10 IU / kg body weight. 4 In some embodiments, IL-2 is administered at a dose of about 8×10 IU / kg body weight. 4 In some embodiments, IL-2 is administered at a dose of about 9×10 IU / kg body weight. 4 In some embodiments, IL-2 is administered at a dose of about 1×10 IU / kg body weight. 5 It is administered at a dose of 1 IU / kg body weight.

[0311] In some embodiments, the bisphosphonate is administered at a dosage of about 10 μg / kg body weight to about 100 mg / kg body weight. In some embodiments, the bisphosphonate is administered...

Claims

1. 1. A method for ex vivo activation and expansion of Vγ9Vδ2 T cells, comprising: a) contacting a cell population comprising T cells with a culture system comprising IL-2, IL-15, and a bisphosphonate or mevalonate pathway intermediate; b) culturing said cell population ex vivo in said culture system under hypoxic conditions to activate and expand Vγ9Vδ2 T cells.

2. The method of claim 1 , wherein the method further comprises obtaining the cell population from a subject.

3. The method of claim 2 , wherein the subject is healthy.

4. The method of claim 2 , wherein the subject is in poor health.

5. The method of claim 1 , wherein the cell population is a mammalian cell population.

6. The method of claim 5 , wherein the mammalian cell is a human cell.

7. The method of claim 6 , wherein the human cells are genetically engineered cells.

8. The method of claim 6, wherein the human cells are not genetically engineered cells.

9. 2. The method of claim 1, wherein the cell population is a peripheral blood mononuclear cell (PBMC) population.

10. 10. The method of claim 9, wherein the PBMCs are freshly obtained PBMCs.

11. 10. The method of claim 9, wherein the PBMCs are frozen PBMCs.

12. The method of claim 1 , wherein the cell population is derived from human tissue.

13. The method of claim 12, wherein the human tissue is fresh.

14. The method of claim 12, wherein the human tissue is frozen.

15. The method of claim 1 , wherein the cell population comprises tumor infiltrating lymphocytes (TILs).

16. The method of claim 15, wherein the TIL is a freshly obtained TIL.

17. The method of claim 15, wherein the TIL is a frozen TIL.

18. 10. The method of claim 1, wherein the cell population is cultured in the culture system under the hypoxic conditions for at least 3 days.

19. 10. The method of claim 1, wherein the hypoxic oxygen concentration is less than 15%.

20. 20. The method of claim 19, wherein the hypoxic oxygen concentration is about 5%.

21. 2. The method of claim 1, further comprising culturing the cell population in the culture system under normoxic conditions to activate and expand Vγ9Vδ2 T cells.

22. 22. The method of claim 21, wherein the cell population is cultured under said normoxic conditions for at least 1 hour before being cultured under said hypoxic conditions.

23. 22. The method of claim 21, wherein the oxygen concentration in the normoxic state is about 18.2% or 18.6%.

24. The method of claim 1, wherein the IL-2 concentration in the culture system is 10 IU / mL to 1200 IU / mL.

25. The method of claim 24, wherein the IL-2 concentration in the culture system is adjusted to decrease during culture.

26. 26. The method of claim 25, wherein the IL-2 concentration in the culture system is 1000 IU / mL or less on days 0 and 1, 800 IU / mL or less on days 2, 3, and 4, and 100 IU / mL or less on days 5 and thereafter.

27. 2. The method of claim 1, wherein the IL-15 concentration in the culture system is 5 ng / mL to 25 ng / mL, or 50 ng / mL to 300 ng / mL, or about 100 ng / mL, or about 200 ng / mL.

28. The method of claim 27, wherein the IL-15 concentration in the culture system is adjusted during culture.

29. The method of claim 28, wherein the IL-15 concentration in the culture system is 10 ng / mL or less on days 0 and 1, 20 ng / mL or less on days 2, 3, and 4, and 10 ng / mL or less on days 5 and thereafter.

30. 2. The method of claim 1, wherein the bisphosphonate is selected from the group consisting of zoledronic acid, risedronic acid, ibandronic acid, alendronic acid, pamidronic acid, tiludronic acid, etidronic acid, and clodronic acid.

31. 2. The method of claim 1, wherein the mevalonate pathway intermediate is selected from the group consisting of HMBPP, BrHPP, and isopentenyl pyrophosphate.

32. 2. The method of claim 1, wherein the method increases the total percentage of Vy9V52 T cells in the cell population to greater than 10%.

33. 2. The method of claim 1, wherein the method increases the total number of Vy9V52 T cells in the cell population by at least 10-fold compared to the total number of Vy9V52 T cells in the cell population before the contacting.

34. 2. The method of claim 1, further comprising enriching said Vγ9Vδ2 T cells in said cell population.

35. 35. The method of claim 34, wherein said enrichment results in a percentage of vγ9Vδ2 T cells in said cell population of greater than 95%.

36. 1. A method for ex vivo activation and expansion of Vγ9Vδ2 T cells, comprising: a) contacting a cell population comprising T cells with a culture system comprising IL-2, IL-15, and zoledronic acid; b) culturing the cell population ex vivo in the culture system under hypoxic conditions for about 14 days to activate and expand Vγ9Vδ2 T cells; (i) the IL-2 concentration in the culture system is (1) 1000 IU / mL or less on days 0 and 1, 800 IU / mL or less on days 2, 3, and 4, and 100 IU / mL or less on days 5 and thereafter, or (2) 10 or about 10; and (ii) the IL-2 concentration in the culture system is (1) 10 ng / mL or less on days 0 and 1, and 2 (iii) the oxygen concentration in the hypoxic state is about 5%; and optionally, (iv) the concentration of zoledronic acid in the culture system is 350 nM or about 350 nM.

37. 1. A method for ex vivo activation and expansion of Vγ9Vδ2 T cells, comprising: a) contacting a cell population comprising T cells with a culture system comprising IL-2, IL-15, and zoledronic acid; b) culturing the cell population under normoxic conditions for at least 1 hour to activate and expand Vγ9Vδ2 T cells before culturing under hypoxic conditions; c) culturing the cell population under said hypoxic conditions for about 14 days to further activate and expand Vγ9Vδ2 T cells; (i) the IL-2 concentration in the culture system is (1) 1000 IU / mL or less on days 0 and 1, 800 IU / mL or less on days 2, 3, and 4, and 100 IU / mL or less on days 5 and thereafter, or (2) 10 or about 10; and (ii) the IL-15 concentration in the culture system is (1) 10 ng / mL or less on days 0 and 1, and 20 ng / mL or less on days 2, 3, and 4. (iii) the oxygen concentration in the hypoxic state is about 5%; (iv) the oxygen concentration in the normoxic state is 18.2% or 18.6%, or about 18.2% or about 18.6%; and optionally, (iv) the concentration of zoledronic acid in the culture system is about 350 nM.

38. 2. An isolated Vγ9Vδ2 T cell population produced by the method of claim 1.

39. 1. An isolated cell population, wherein the percentage of Vγ9Vδ2 T cells in said isolated cell population is greater than 10%.

40. 40. A pharmaceutical composition comprising the isolated Vy9V52 T cell population of claim 38 or the isolated cell population of claim 39, and optionally a pharmaceutically acceptable excipient.

41. 1. A process for generating chimeric antigen receptor (CAR) T cells, comprising: (i) performing the functions to obtain the isolated Vy9V52 T cell population of claim 38 or the isolated cell population of claim 39; (ii) performing a function of expressing a CAR in the Vγ9Vδ2 T cell; Optionally, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain, further optionally, the extracellular domain binds to an antigen expressed on an unhealthy cell, further optionally, the unhealthy cell is a cancer cell, further optionally, the cancer cell is a blood cancer cell or a solid tumor cancer cell.

42. 40. A method for generating chimeric antigen receptor (CAR) T cells, comprising: (i) obtaining the isolated Vy9V52 T cell population of claim 38 or the isolated cell population of claim 39; and (ii) introducing a nucleic acid encoding a CAR into the Vy9V52 T cells; Optionally, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain, further optionally, the extracellular domain binds to an antigen expressed on an unhealthy cell, further optionally, the unhealthy cell is a cancer cell, further optionally, the cancer cell is a blood cancer cell or a solid tumor cancer cell.

43. 43. A CAR T cell produced by the method of claim 42.

44. 44. A pharmaceutical composition comprising the CAR T cells of claim 43 and optionally a pharmaceutically acceptable excipient.

Images