Method for producing gamma delta T cells

Abstract

The present application is directed to methods for producing populations of cells comprising γδ T cells. The application is also directed to, for example, compositions useful in such production methods, the produced populations of cells, and methods of their use. In some embodiments, the population of cells comprising γδ T cells has a γδ T cell purity of at least about 85%. In some embodiments, the population of cells comprising γδ T cells has a γδ T cell purity of at least about 95%. In some embodiments, the population of cells comprising γδ T cells comprises vδ2 γδ T cells and, optionally, vδ1 γδ T cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 439,556, filed January 17, 2023, the contents of which are hereby incorporated by reference in their entirety.

[0002] The present application is directed to methods for producing populations of cells comprising γδ T cells. The present application is also directed to, for example, compositions and methods useful in such production methods, the populations of cells produced, and methods of their use. [Background technology]

[0003] Adoptive cell therapy involves isolating and expanding human immune cells, with or without cell engineering, and administering these cells to patients for the treatment of cancer and other diseases. One promising form of adoptive cell therapy is CAR (chimeric antigen receptor) T-cell therapy. In this therapy, αβ T cells are engineered to express CAR molecules that recognize tumor-associated antigens (TAAs) expressed on the surface of cancer cells, thereby activating and killing TAA-expressing cancer cells. Several CAR T-cell therapies have been approved for the treatment of hematologic malignancies, and others are in development for solid tumors.

[0004] To avoid graft-versus-host disease (GVHD) caused by mismatched αβ T cells, all currently approved CAR-T therapies are autologous cell therapies. That is, CAR-T cells are generated using the patient's own T cells. Autologous cell therapies have limitations, including the inability to obtain sufficient numbers of high-quality T cells from heavily pretreated cancer patients, the long turnover time (e.g., several weeks) required for CAR-T cell production, and the risk of CAR-T cell production failure from a particular patient. Given these limitations, allogeneic cell therapies, which can be prepared using various sources, such as healthy donor cells, stem cell bank cells, or cell lines, and can be used as ready-to-use treatments, are highly desirable.

[0005] Innate immune cell therapy has the potential to overcome some of these limitations. Innate immune cells (e.g., NK, NKT, γδ T, and myeloid cells) lack HLA restriction and can regulate alloresponses, thereby avoiding GVHD during adoptive transfer of allogeneic innate immune cells (see, for example, Khatwani et al. Front Immunol, 13, 2022). Notably, NK cells are easily procured from peripheral blood, present at significantly higher levels than γδ T cells, and are easier to generate than γδ T cells. As a result, the majority of innovations in innate immune cell therapy have focused on NK cell therapy.

[0006] γδ T cells are innate-like T lymphocytes and comprise a small fraction (0.5%–5%) of human peripheral blood mononuclear cells. Despite their low cell numbers in healthy individuals, previous studies have demonstrated that these cells possess potent anti-cancer and anti-infective capabilities. Unlike conventional αβ T cells, γδ T cells inhibit tumor growth and eliminate pathogen-infected cells in an MHC-unrestricted manner. This feature makes γδ T cells an attractive option for allogeneic immune cell therapy with minimal risk of GVHD. γδ T cells are characterized by expressing a unique TCR composed of gamma and delta chains, as opposed to the alpha and beta chains of αβ T cells. In humans, there are two major subsets of γδ T cells, distinguished by the delta chain. vδ1 cells reside primarily in peripheral tissues, while vδ2 cells are more abundant in peripheral blood. The antigens recognized by the vδ1 TCR remain largely unknown, whereas vδ2 cells recognize non-peptide phosphoantigens that are intermediates in the mevalonate metabolic pathway. Despite growing interest in developing γδ T cell-based cell therapies, there are currently obstacles that limit the realization of such therapies. One key issue is how to obtain the large numbers of pure γδ T cells (e.g., 1 × 10) required for adoptive cell therapy from limited cell numbers in the starting material (e.g., human peripheral blood) while maintaining γδ T cell function and cytotoxicity. 8Current techniques use aminobisphosphonates, antibodies, and artificial antigen-presenting cells (feeder cells) to promote the expansion of γδ T cells (e.g., Dokouhaki et al., Cancer Lett, 297, 2010; Van Acker et al., J Hematol Oncol, 9, 2016; Li et al., J Immunother, 33, 2010; Salot et al., J Immunol Methods, 326, 2007; Kondo et al., Cytotherapy, 10, 2008; Deniger et al., Cancer Res, 20, 2014; Xiao et al., Cytotherapy, 20, 2018; Zhou et al., Cell Mol Immunol, 9, 2012; and Makkouk et al., J Immunother Cancer, 9, 2021. However, the use of certain manufacturing components introduces additional manufacturing considerations and complexities. For example, the use of feeder cells requires additional QA / QC steps to verify the feeder cell content in the final product and raises safety concerns, such as the potential introduction of any residual feeder cells (e.g., K562 cancer cells) into human patients. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Khatwani et al. Front Immunol, 13, 2022 [Non-patent document 2] Dokouhaki et al., Cancer Lett, 297, 2010 [Non-patent document 3] Van Acker et al., J Hematol Oncol, 9, 2016; Li et al., J Immunother, 33, 2010 [Non-patent document 4] Salot et al., J Immunol Methods, 326, 2007 [Non-patent document 5] Kondo et al., Cytotherapy, 10, 2008 [Non-patent document 6] Deniger et al., Cancer Res, 20, 2014 [Non-Patent Document 7] Xiao et al., Cytotherapy, 20, 2018 [Non-patent document 8] Zhou et al., Cell Mol Immunol, 9, 2012 [Non-Patent Document 9] Makkouk et al., J Immunother Cancer, 9, 2021 Summary of the Invention

[0008] In some aspects, provided herein are methods of producing a population of cells comprising γδ T cells, the method comprising culturing an isolated pool of mixed immune cells depleted of αβ T cells, or a derivative thereof, in a culture medium comprising: (a) a basal medium, (b) a cytokine, and (c) one or more of: (i) a metabolic polypeptide, (ii) an iron source, (iii) an antioxidant enzyme cofactor, (iv) a lipid precursor, and (v) a carrier protein; and harvesting the population of cells comprising γδ T cells.

[0009] In some embodiments, the population of cells comprising γδ T cells has a γδ T cell purity of at least about 85%. In some embodiments, the population of cells comprising γδ T cells has a γδ T cell purity of at least about 95%.

[0010] In some embodiments, the population of cells comprising γδ T cells comprises vδ2 γδ T cells, and optionally vδ1 γδ T cells.

[0011] In some embodiments, the population of cells comprising γδ T cells has a vδ2 γδ T cell purity relative to total T cells (such as γδ T cells) in the population of at least about 70%. In some embodiments, the population of cells comprising γδ T cells has a vδ2 γδ T cell purity relative to total T cells (such as γδ T cells) in the population of at least about 95%.

[0012] In some embodiments, the population of cells comprising γδ T cells comprises less than about 15% of NK cells, hi some embodiments, the population of cells comprising γδ T cells comprises less than about 2.5% of NK cells.

[0013] In some embodiments, the basal culture medium is NK MACS medium.

[0014] In some embodiments, the cytokine is one or more of IL-15, IL-2, IL-7, SCF, TPO, FLT-3L, BMP4, VEGF, or bFGF. In some embodiments, the cytokine is IL-15. In some embodiments, the cytokine is added to the culture medium in an amount of about 100 pg / mL to about 100 ng / mL.

[0015] In some embodiments, the metabolic polypeptide is insulin or an analog thereof. In some embodiments, the insulin is human insulin. In some embodiments, the metabolic polypeptide is added to the culture medium in an amount of about 1 mg / L to about 50 mg / L.

[0016] In some embodiments, the iron source is transferrin. In some embodiments, the transferrin is human transferrin. In some embodiments, the iron source is added to the culture medium in an amount of about 1 mg / L to about 50 mg / L.

[0017] In some embodiments, the antioxidant enzyme cofactor is a selenium compound. In some embodiments, the selenium compound is sodium selenite. In some embodiments, the antioxidant enzyme cofactor is added to the culture medium in an amount of about 0.001 mg / L to about 0.1 mg / L.

[0018] In some embodiments, the lipid precursor is ethanolamine. In some embodiments, the lipid precursor is added to the culture medium in an amount of about 1 mg / L to about 10 mg / L.

[0019] In some embodiments, the carrier protein is albumin. In some embodiments, the albumin is human albumin. In some embodiments, the human albumin is human serum albumin. In some embodiments, the carrier protein is added to the culture medium in an amount of about 100 mg / L to about 5,000 mg / L.

[0020] In some embodiments, the culture medium comprises two or more of: (i) a metabolic polypeptide; (ii) an iron source; (iii) an antioxidant enzyme cofactor; (iv) a lipid precursor; and (v) a carrier protein.

[0021] In some embodiments, the culture medium comprises three or more of: (i) a metabolic polypeptide, (ii) an iron source, (iii) an antioxidant enzyme cofactor, (iv) a lipid precursor, and (v) a carrier protein.

[0022] In some embodiments, the culture medium comprises four or more of: (i) a metabolic polypeptide, (ii) an iron source, (iii) an antioxidant enzyme cofactor, (iv) a lipid precursor, and (v) a carrier protein.

[0023] In some embodiments, the culture medium comprises (i) a metabolic polypeptide, (ii) an iron source, (iii) an antioxidant enzyme cofactor, (iv) a lipid precursor, and (v) a carrier protein.

[0024] In some embodiments, the culture medium comprises ITSEA.

[0025] In some embodiments, the culture medium further comprises serum. In some embodiments, the serum is AB serotype (AB) serum. In some embodiments, the AB serum is human AB serum. In some embodiments, the serum is added to the culture medium in an amount of about 0.1% (v / v) to about 10% (v / v).

[0026] In some embodiments, the culturing is for about 0 to about 14 days.

[0027] In some embodiments, the method further comprises cryopreserving the harvested population of cells comprising γδ T cells.

[0028] In some embodiments, the isolated pool of αβ T cell-depleted mixed immune cells is derived from peripheral blood mononuclear cells (PBMCs). In some embodiments, the isolated pool of αβ T cell-depleted mixed immune cells is derived from a stem cell bank or cell line.

[0029] In some embodiments, the method further comprises performing a step of depleting αβ T cells on the isolated pool of mixed immune cells. In some embodiments, the step of depleting αβ T cells comprises antibody-based depletion. In some embodiments, the step of depleting Aβ T cells comprises (a) exposing the isolated pool of mixed immune cells to an anti-TCR α / β antibody, and (b) separating the antibody-conjugated αβ TCR+ T cells from other cells in the isolated pool of mixed immune cells to obtain a population of αβ T cell-depleted cells. In some embodiments, the separation is performed by a cell sorting technique.

[0030] In some embodiments, the method further comprises cryopreserving cells obtained from the step of depleting αβ T cells from the isolated pool of mixed immune cells prior to culturing the isolated pool of mixed immune cells or a derivative thereof in culture medium.

[0031] In some embodiments, the method further comprises isolating the pool of mixed immune cells from a donor. In some embodiments, the donor is not the intended recipient. In some embodiments, the donor is the intended recipient. In some embodiments, the isolating comprises leukapheresis. In some embodiments, the isolation is performed on blood from the donor. In some embodiments, the donor is a human individual. In some embodiments, the method further comprises withdrawing blood from the donor.

[0032] In some embodiments, the method further comprises performing a step of transducing γδ T cells. In some embodiments, the culturing step is divided by a step of transducing the cells in culture to obtain transduced γδ T cells. In some embodiments, the culturing step comprises a first culturing step from about day 0 to about day 4, and transducing is performed around day 4. In some embodiments, the culturing step comprises a second culturing step from about day 0 to about day 10. In some embodiments, the transduction comprises introducing one or more transgenes. In some embodiments, the one or more transgenes comprise a CAR. In some embodiments, the CAR specifically recognizes globohexaosylceramide, AKR1C3, SSEA-4, or TROP-2.

[0033] In some embodiments, the method further comprises cryopreserving the produced population of cells comprising γδ T cells after the harvesting step.

[0034] In some embodiments, the method is a feeder-cell-free method for producing a population of cells comprising γδ T cells.

[0035] In some embodiments, the method is an antibody-free method for producing a population of cells comprising γδ T cells. For example, in some embodiments, the method does not involve the use of an antibody in culturing the pool of mixed immune cells or derivatives thereof after αβ T cell depletion. In some embodiments, the method is an antibody-free method for γδ TCR stimulation for producing a population of cells comprising γδ T cells.

[0036] In some embodiments, the method does not include using an aminobisphosphonate in culturing the pool of mixed immune cells or derivatives thereof, hi some embodiments, the aminobisphosphonate is zoledronic acid or pamidronic acid, or a mixture thereof.

[0037] In another aspect, provided herein is a population of cells comprising γδ T cells produced using any of the methods described herein. In some embodiments, the population comprises at least about 1×10 8 In some embodiments, the population comprises one or more transgenes. In some embodiments, the one or more transgenes comprise a CAR.

[0038] In another aspect, provided herein are methods of treating a disease in an individual. The method comprises obtaining an isolated pool of mixed immune cells, or a derivative thereof, that are depleted of αβ T cells from a donor individual, producing a population of cells that comprises γδ T cells using any method described herein, and administering the population of cells that comprises γδ T cells to the individual. In some embodiments, the disease is cancer. In some embodiments, the donor individual is not the individual. In some embodiments, the donor individual is the individual.

[0039] Those skilled in the art will appreciate that modifications to the embodiments and details described herein may be made without departing from the scope of the present disclosure. Additionally, while various advantages, aspects, and objectives have been described with respect to various implementations, the scope of the present disclosure should not be limited with respect to such advantages, aspects, and objectives.

[0040] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. [Brief explanation of the drawings]

[0041] [Figure 1A] The percentage of surviving γδ T cells after cell expansion (n=4) is shown. [Figure 1B] The percentages of γδ T cell lineages, i.e., vδ1, vδ2, and vδ1- / vδ2- γδ T cells, in the final cell population on day 14 (n=2) are shown. Briefly, peripheral blood apheresis cells from healthy humans were first depleted of αβ T cells and then expanded in vitro for 14 days. Cell viability after expansion was assessed by AOPI staining assay. The lineage of γδ T cells was determined by FACS.

[0042] [Figure 2A] The degree of proliferation of γδ T cells by IL-15 alone, ITSEA alone, and the combination of IL-15 and ITSEA is shown. [Figure 2B] The extent of NK cell proliferation under the indicated conditions is shown. [Figure 2C] The extent of proliferation of non-γδ T cells / non-NK cells under the indicated conditions is shown.

[0043] [Figure 3A] The percentage of γδ T cell purity in the final cell population on day 14 is shown. [Figure 3B] The percentage of NK cell purity in the final cell population on day 14 is shown. [Figure 3C] The percentage of non-γδ T cell / non-NK cell purity in the final cell population on day 14 is shown.

[0044] [Figure 4A] Figure 1 shows the percentage of specific lysis of SKOV3 cancer cells by γδ T cells expanded in vitro over 14 days. [Figure 4B] The percentage of specific lysis of A549 cancer cells by γδ T cells expanded in vitro over 14 days is shown. Briefly, luciferase-expressing tumor cells (i.e., SKOV3 and A549) pretreated with or without zoledronic acid (Zol) were co-cultured with γδ T cells at an E / T ratio of 4 / 1 in the presence of rhIL-15 during culture. Cytotoxicity (specific lysis rate) was determined on days 1 and 3 based on the disappearance of the luciferase signal (n = 3 technical replicates). rhIL-15: recombinant human IL-15; Zol: zoledronic acid; E / T: effector cell to target cell ratio.

[0045] [Figure 5A] Figure 1 shows the percentage of specific lysis of SKOV3 cancer cells grown in a 3D model by γδ T cells expanded in vitro over 14 days. [Figure 5B] Percent specific lysis of A549 cancer cells grown in a 3D model by in vitro expanded γδ T cells over 14 days is shown. Briefly, fluorescently tagged tumor cells were cultured in 3D culture and allowed to form tumor-like masses. After 2 days, γδ T cells were added with rhIL-15 at E / T:4 / 1. Fluorescence intensity, indicative of tumor mass, was measured after 3 and 6 days (n=3 technical replicates). TC: tumor cells; rhIL-15: recombinant human IL-15; E / T: effector to target cell ratio.

[0046] [Figure 6A] A schematic diagram of the anti-TROP-2 CAR construct transduced into αβ T cell-depleted cells before further expansion is shown. [Figure 6B] Transduction efficiency of non-transduced γδ T cells and anti-TROP-2 CAR-transduced γδ T cells is shown. [Figure 6C]Fold expansion of non-transduced γδ T cells and anti-TROP-2 CAR-transduced γδ T cells is shown. [Figure 6D] Against a TROP-2 expressing cell line (i.e., SKOV3), γδ CAR-T cells show increased antitumor cytotoxic activity. n=3 technical replicates per condition. [Figure 6E] For cell lines that do not express TROP-2 (i.e., A549), no significant changes were observed over untransduced γδ T cells. n=3 technical replicates per condition. DETAILED DESCRIPTION OF THE INVENTION

[0047] In some aspects, the present application provides methods for producing populations of cells comprising γδ T cells. The methods include culturing using the culture medium described herein. In other aspects, provided herein are compositions useful for such production methods, produced populations of cells, and methods of use thereof. The disclosure of the present application is based on the inventors' unique perspective and findings regarding methods for rapidly expanding γδ T cells in vitro using a novel feeder cell-free and antibody-free platform that selectively and potently expands unmodified or genetically engineered human γδ T cells. In certain embodiments, the resulting γδ T cells are primarily composed of the vδ2 subtype of γδ T cells and exhibit potent anti-tumor activity. In certain embodiments, the disclosure provided herein eliminates extra manufacturing steps for feeder cells and antibodies, thus eliminating QA / QC steps to verify feeder cell content in the final product and eliminating safety concerns regarding the introduction of residual feeder cells (e.g., cancer cells) into humans, such as human patients. As demonstrated herein, the produced cell populations containing γδ T cells do not require any further enrichment or isolation after cell expansion, and due to the lack of αβ T cells, are sufficiently pure to minimize the risk of graft-versus-host disease (GvHD) when administered to patients as allogeneic cell therapy. Thus, as described in further detail herein, the populations of cells, including γδ T cells, obtained by the methods taught herein can be used safely, efficiently, and effectively in prophylactic, therapeutic, experimental, and commercial applications.

[0048] More specifically, for example, after extensive investigation, it was unexpectedly discovered that the methods described herein for expanding γδ T cells in vitro produce populations of cells containing significantly expanded γδ T cells compared to (for example) feeder-cell-free methods. It was also found that in the populations of cells thus produced containing γδ T cells, NK cells did not significantly expand and remained at low levels, such as less than 5% of the cells in the population. For example, the method described in Example 1 below produced an average expansion of γδ T cells of over 2000-fold, as shown in Figure 2A. The robustness of the expansion of γδ T cells and the purity of the expansion method described herein were unexpected given the prevalence in the art of methods that promote γδ T cell expansion without simultaneously promoting NK cell expansion, e.g., using feeder cells and / or γδ T cell-activating antibodies. Thus, the novel methods described herein provide similar or superior efficacy using more efficient protocols than those currently described in the art, while also providing a safer product.

[0049] Thus, in some aspects, provided herein are methods for producing a population of cells comprising γδ T cells. The methods include culturing an isolated pool of αβ T cell-depleted mixed immune cells, or a derivative thereof, in a culture medium comprising (a) a basal medium, (b) a cytokine, and (c) one or more of: (i) a metabolic polypeptide, (ii) an iron source, (iii) an antioxidant enzyme cofactor, (iv) a lipid precursor, and (v) a carrier protein; and, optionally, harvesting the population of cells comprising γδ T cells. In some embodiments, the basal medium is NK MACS medium (Miltenyi Biotec). In some embodiments, the pool of αβ T cell-depleted mixed immune cells is peripheral blood mononuclear cells (PBMCs) isolated after αβ T cell depletion. In some embodiments, the population of cells comprises γδ T cells having at least about 95% γδ T cell purity. In some embodiments, the population of cells comprises γδ T cells, including vδ1 γδ T cells and vδ2 γδ T cells. In some embodiments, the population of cells comprising γδ T cells comprises less than about 5% NK cells. In some embodiments, the cytokine is IL-15. In some embodiments, the metabolic polypeptide is insulin or an analog thereof. In some embodiments, the iron source is transferrin. In some embodiments, the antioxidant enzyme cofactor is a selenium compound (e.g., sodium selenite). In some embodiments, the lipid precursor is ethanolamine. In some embodiments, the carrier protein is albumin (e.g., human albumin, including human serum albumin). In some embodiments, the culturing is for about 0 to about 14 days. In some embodiments, the method is a feeder-cell-free method for producing a population of cells comprising γδ T cells. In some embodiments, the method is an antibody-free method for producing a population of cells comprising γδ T cells. For example, in some embodiments, the method does not involve the use of an antibody in culturing the pool of mixed immune cells or derivatives thereof after αβ T cell depletion. In some embodiments, the method is a γδ TCR-stimulating antibody-free method for producing a population of cells comprising γδ T cells.In some embodiments, the method does not include the use of an aminobisphosphonate in culturing the PBMCs or derivatives thereof.

[0050] In another aspect, provided herein are methods for producing a population of cells comprising γδ T cells, wherein the γδ T cells have been engineered to contain a transgene, the method comprising culturing an isolated pool of αβ T cell-depleted mixed immune cells, or a derivative thereof, in a culture medium comprising: (a) a basal medium; (b) a cytokine; and (c) one or more of: (i) a metabolic polypeptide, (ii) an iron source, (iii) an antioxidant enzyme cofactor, (iv) a lipid precursor, and (v) a carrier protein; transducing the population of cells comprising γδ T cells (or their intermediates / precursors) to deliver the transgene to the γδ T cells; and optionally harvesting the population of cells comprising γδ T cells. In some embodiments, the method further comprises performing a step of depleting αβ T cells (e.g., antibody-based depletion) on the isolated pool of αβ T cell-depleted mixed immune cells. In some embodiments, the population of cells comprises γδ T cells having a γδ T cell purity of at least about 95%. In some embodiments, the population of cells comprises γδ T cells, primarily comprising vδ2 γδ T cells and optionally vδ1 γδ T cells. In some embodiments, the population of cells comprising γδ T cells comprises less than about 5% NK cells. In some embodiments, the cytokine is IL-15. In some embodiments, the metabolic polypeptide is insulin or an analog thereof. In some embodiments, the iron source is transferrin. In some embodiments, the antioxidant enzyme cofactor is a selenium compound (e.g., sodium selenite). In some embodiments, the lipid precursor is ethanolamine. In some embodiments, the carrier protein is albumin (e.g., human albumin, including human serum albumin). In some embodiments, the transgene is a CAR. In some embodiments, the culturing is from about 0 days to about 14 days. In some embodiments, the method is a feeder-cell-free method for producing a population of cells comprising γδ T cells. In some embodiments, the method is an antibody-free method for producing a population of cells comprising γδ T cells.For example, in some embodiments, the method does not include the use of an antibody in culturing the pool of mixed immune cells or derivatives thereof after αβ T cell depletion. In some embodiments, the method is a γδ TCR-stimulating, antibody-free method for producing a population of cells comprising γδ T cells. In some embodiments, the method does not include the use of an aminobisphosphonate in culturing the pool of mixed immune cells depleted of αβ T cells or derivatives thereof.

[0051] In another aspect, provided herein is a population of cells comprising γδ T cells produced using the methods described herein.

[0052] In another aspect, provided herein are methods of treating a disease in an individual. The method comprises obtaining an isolated pool of mixed immune cells, or a derivative thereof, that have been depleted of αβ T cells from a donor individual, producing a population of cells comprising γδ T cells using the methods described herein, and administering the population of cells comprising γδ T cells to the individual. In some embodiments, the disease is cancer. In some embodiments, the donor individual is not the individual receiving the population of cells comprising γδ T cells (i.e., the method of treatment is allogeneic treatment). In some embodiments, the donor individual is the individual receiving the population of cells comprising γδ T cells (i.e., the method of treatment is autologous treatment).

[0053] Also provided are kits for producing the populations of cells comprising γδ T cells described herein, pharmaceutical compositions and kits comprising the populations of cells comprising γδ T cells described herein, and methods of their use to treat or otherwise ameliorate diseases such as cancer.

[0054] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. For example, some aspects of the present disclosure are presented in a modular format, and such presentation should not be construed as limiting the possible combinations of approaches taught herein.

[0055] I. Definition For the purposes of interpreting this specification, the following definitions shall apply, and where appropriate, terms used in the singular shall include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth herein shall control.

[0056] As used herein, the terms "polypeptide" and "protein" may be used interchangeably to refer to polymers comprising amino acid residues and are not limited to a minimum length. Such polymers may contain natural or non-natural amino acid residues, or combinations thereof, and include, but are not limited to, peptides, polypeptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Full-length polypeptides or proteins, as well as fragments thereof, are encompassed by this definition. The terms also include modified species thereof, such as post-translational modifications of one or more residues, such as methylation, phosphorylation, glycosylation, sialylation, or acetylation.

[0057] As used herein, "treatment" or "treating" refers to an approach to obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing one or more symptoms attributable to the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease, preventing or slowing the onset or recurrence of the disease, slowing or slowing the progression of the disease, ameliorating the state of the disease, providing remission (partial or complete) of the disease, reducing the dose of one or more other agents required to treat the disease, slowing the progression of the disease, improving quality of life, and / or prolonging survival. Also encompassed by "treatment" is the alleviation of the pathological consequences of the disease. The methods of the present application contemplate any one or more of these aspects of treatment.

[0058] The terms "individual," "subject," and "patient" are used interchangeably herein to describe mammals, including humans. In some embodiments, the individual is human. In some embodiments, the individual is afflicted with cancer. In some embodiments, the individual is in need of treatment.

[0059] The term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.), humanized antibodies, chimeric antibodies, full-length antibodies and antigen-binding fragments thereof, single-chain Fvs, nanobodies, and Fc-fusion proteins, so long as they exhibit the desired antigen-binding activity. Antibodies and / or antibody fragments may be derived from murine antibodies, rabbit antibodies, chicken antibodies, human antibodies, fully humanized antibodies, camelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.

[0060] Those skilled in the art will understand that the term "cell" includes the primary subject cell and its progeny.

[0061] A "pharmaceutically acceptable carrier" refers to one or more ingredients contained in a pharmaceutical formulation, other than the active ingredient, that are non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, cryoprotectants, tonicity agents, preservatives, and combinations thereof. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicological and manufacturing testing and / or are included in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration or other state / federal governments, or are listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more specifically, humans.

[0062] As used herein, the terms "comprising," "having," "containing," and "including," as well as other similar forms and grammatical equivalents thereof, are intended to be equivalent in meaning and to be open-ended in that the item or items preceding any one of these words do not imply an exhaustive listing of such items or items or are limited to only the listed item or items. For example, an article "comprising" components A, B, and C may consist of (i.e., contain only) components A, B, and C, or may include not only components A, B, and C but also one or more other components. As such, "comprising" and similar forms and grammatical equivalents thereof are intended and understood to include disclosure of embodiments that "consist essentially of" or "consist of."

[0063] Where a range of values ​​is provided, unless the context clearly dictates otherwise, each intervening value between the upper and lower limit of that range, and at any other stated or intervening value within that stated range, to the tenth of the unit of the lower limit, is encompassed within the scope of the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0064] Reference herein to "about" a value or parameter includes (and describes) a variation that is directed to that value or parameter itself. For example, a reference to "about X" includes the description of "X."

[0065] As used in this specification, including the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0066] II. Methods for Producing a Population of Cells Comprising γδ T Cells In some aspects, provided herein are methods for producing a population of cells comprising γδ T cells, comprising culturing a population of PBMCs or a derivative thereof. In some embodiments, the methods include steps related to cell depletion (e.g., αβ T cell depletion), obtaining an isolated pool of mixed immune cells (e.g., a population comprising PBMCs), transduction, and cryopreservation. In some embodiments, the culturing involves the use of a culture medium comprising: (a) a basal medium; (b) a cytokine (e.g., IL-15); and (c) one or more (including all) of: (i) a metabolic polypeptide (e.g., insulin); (ii) an iron source (e.g., transferrin); (iii) an antioxidant enzyme cofactor (e.g., a selenium compound); (iv) a lipid precursor (e.g., ethanolamine); and (v) a carrier protein (e.g., albumin). In some embodiments, the methods are feeder-cell-free methods for producing a population of cells comprising γδ T cells. In some embodiments, the methods are antibody-free methods (e.g., γδ TCR-stimulating antibody-free methods) for producing a population of cells comprising γδ T cells. In some embodiments, the method does not include the use of an aminobisphosphonate in culturing an isolated pool of mixed immune cells (such as PBMCs) or derivatives thereof.

[0067] In some embodiments, a population of cells comprising γδ T cells produced using the methods provided herein has a γδ T cell purity of at least about 85%, examples of which include any of at least about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%, or about 100%. In some embodiments, a population of cells comprising γδ T cells produced using the methods provided herein comprises vδ2 γδ T cells. In some embodiments, a population of cells comprising γδ T cells produced using the methods provided herein comprises vδ2 γδ T cells and vδ1 γδ T cells. In some embodiments, a population of cells comprising γδ T cells produced using the methods provided herein has a vδ2 γδ T cell purity relative to, e.g., total T cells (such as total γδ T cells) in the population of at least about 70%, examples of which include any of at least about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%, or about 100%. In some embodiments, a population of cells comprising γδ T cells produced using the methods provided herein comprises less than about 15% of NK cells, examples of which include any of: less than about 14% of NK cells, less than about 13% of NK cells, less than about 12% of NK cells, less than about 11% of NK cells, less than about 10% of NK cells, less than about 9% of NK cells, less than about 8% of NK cells, less than about 7% of NK cells, less than about 6% of NK cells, less than about 5% of NK cells, less than about 4.5% of NK cells, less than about 4% of NK cells, less than about 3.5% of NK cells, less than about 3% of NK cells, less than about 2.5% of NK cells, less than about 2% of NK cells, less than about 1.5% of NK cells, less than about 1% of NK cells, or less than about 0.5% of NK cells.

[0068] Certain aspects of the methods taught herein are discussed in more detail in a modular manner below. One of skill in the art will readily appreciate how aspects of the present specification can be combined to provide any method of producing a population of cells, including γδ T cells, encompassed by the teachings provided herein. Discussing such methods (including their components and steps) in a modular manner does not limit the scope of the description encompassed herein.

[0069] A. Culture Methods of producing a population of cells comprising γδ T cells include culturing an isolated pool of mixed immune cells that have been depleted of αβ T cells (such as a population of cells comprising PBMCs that have been depleted of αβ T cells) or a derivative thereof in a culture medium. In some embodiments, as used herein, an isolated pool of mixed immune cells or a derivative thereof refers to a population of cells obtained from any source, such as an individual, or a population of cells that has been treated, such as by an αβ depletion process, but the derivative does not include a harvested population of cells that comprises γδ T cells produced using the methods described herein.

[0070] Numerous anatomical sources of mixed immune cell populations have been identified in humans, including immune cell collection methods, such as circulating peripheral blood mononuclear cells, skin- or mucosal-resident immune cells, bone marrow immune cells, immune cells circulating in lymph nodes and lymph nodes, tissue-resident immune cells from organ biopsies, immune cells in tumors or tumor microenvironments, immune cells in cerebrospinal fluid (see, e.g., Schroder et al. (2018), Front Neurol 9:1081), and peritoneal-resident immune cells (see, e.g., Shen et al. (2015), Stem Cell Res 15(2):299-304; and Ruiz-Alcaraz et al. (2020), Immunol Cell Biol, 98(2):114-126). In some embodiments, the isolated pool of αβ T cell-depleted mixed immune cells may be obtained from any source known in the art, including, but not limited to, PBMCs, cell banks (e.g., stem cell banks, blood cell banks, cell line banks), induced pluripotent stem cells, bone marrow aspirates, peritoneal immunoisolates (e.g., peritoneal dialysis), immunoisolates from lymph and / or lymph node collections, mucosal immunoisolates, or skin biopsy immunoisolates. In some embodiments, the isolated pool of αβ T cell-depleted mixed immune cells comprises αβ T cell-depleted PBMCs. In some embodiments, the isolated pool of αβ T cell-depleted mixed immune cells is a freshly isolated cell population. In some embodiments, the isolated pool of αβ T cell-depleted mixed immune cells is a cryopreserved cell population.

[0071] In some embodiments, the culturing step includes culturing the isolated pool of αβ T cell-depleted mixed immune cells, or a derivative thereof, in culture medium for a period of time until harvesting a desired population of cells, including γδ T cells. In certain aspects of the descriptions provided herein, the culturing includes time-based characteristics, such as the time the isolated pool of αβ T cell-depleted mixed immune cells, or a derivative thereof, is cultured in culture medium. In such embodiments, an initial culture using culture medium used for expanding γδ T cells, as described below, can be used as a starting point, e.g., day 0. In some embodiments, the culturing includes changing the culture medium, such as removing the existing medium and replacing it with fresh culture medium. For example, in some embodiments, the culture medium is changed every 2-10 days, e.g., every 4-7 days. In some embodiments, the culturing includes adding a culture medium component, such as a cytokine (e.g., IL-15), without changing the culture medium. For example, in some embodiments, cytokines are added to the culture medium every 1-4 days, e.g., every 2-3 days, of the culture process.

[0072] In some embodiments, the culture proceeds for about 30 days or less, examples of which include about 28 days or less, about 21 days or less, or about 14 days or less. In some embodiments, the culture period is based on obtaining a resultant population of cells comprising γδ T cells, examples of which include those assessed with one or more markers. In some embodiments, the culture period is based on obtaining the number of cells in the population of cells and / or the number of γδ T cells in the population of cells. In some embodiments, the culture period is based on obtaining a fold expansion, examples of which include a 500-fold, 1000-fold, 1500-fold, or 2000-fold expansion of γδ T cells in the population of cells comprising γδ T cells compared to the starting PBMCs or derivatives thereof.

[0073] Culture techniques, including those for expanding γδ T cells, are well known in the art. See, e.g., Segeritz et al., Basic Science Methods for Clinical Researchers, 2017; and Fundamental Techniques in Cell Culture, 3rd Edition, 2016, the contents of which are incorporated herein by reference in their entireties. Typical culture conditions often involve culturing cells at 37°C in a standard atmosphere containing 5% CO2, with relative humidity maintained at approximately 100%. Those skilled in the art will readily appreciate that specific conditions (e.g., temperature, percent CO2, humidity, oxygen, pH, etc. for culture) can be adapted to further provide culture conditions suitable for expanding γδ T cells. In certain aspects, such techniques may be adapted to produce products suitable for human use, an example of which is the administration of a population of cells comprising γδ T cells. In some embodiments, the culture techniques are Good Manufacturing Practice (GMP).

[0074] i. Culture media and their components Various culture media taught herein are useful in the taught methods of producing a population of cells comprising γδ T cells. The culture media provided herein comprise (a) a basal medium; (b) a cytokine; and (c) one or more (including all) of: (i) a metabolic polypeptide, (ii) an iron source, (iii) an antioxidant enzyme cofactor, (iv) a lipid precursor, and (v) a carrier protein. In some embodiments, the culture medium further comprises serum. In some embodiments, the methods of producing a population of cells comprising γδ T cells use a single type of culture medium. In some embodiments, the methods of producing a population of cells comprising γδ T cells may use two or more different culture media; for example, the methods include culturing in a first culture medium according to the disclosure provided herein and culturing in a second culture medium according to the disclosure provided herein, wherein the first culture medium and the second culture medium are different.

[0075] The culture medium described herein comprises a basal culture medium. In some embodiments, the basal culture medium is selected from the group consisting of NK MACS, TexMACS, RPMI-1640, AIM-V, OpTmizer, DMEM, IMDM, and X-VIVO. In some embodiments, the basal culture medium comprises or is NK MACS medium. In some embodiments, the cytokine is a mitogenic cytokine. In some embodiments, the cytokine is one or more of IL-15, IL-2, IL-7, IL-12, IL-18, IL-21, SCF, TPO, FLT-3L, BMP4, VEGF, or bFGF. In some embodiments, the cytokine is IL-15. In some embodiments, cytokines are added to the culture medium in an amount of about 100 pg / mL to about 500 ng / mL, examples of which include about 1 ng / mL to about 100 ng / mL, or about 1 ng / mL to about 20 ng / mL, or about 5 ng / mL to about 15 ng / mL, or about 5 ng / mL to about 500 ng / mL, or about 1 ng / mL to about 500 ng / mL. In some embodiments, cytokines are added to the culture medium in an amount of any of about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, or about 20 ng / mL. In some embodiments, cytokines are added to the culture medium every 1-4 days during culture, e.g., every 2 days or every 3 days, examples of which include addition in the amounts described herein for each instance of cytokine added to the culture medium.

[0076] In some embodiments, the metabolic polypeptide is a hormone, such as a human hormone. In some embodiments, the metabolic polypeptide is selected from the group consisting of insulin, proinsulin, and insulin-like growth factor. In some embodiments, the metabolic polypeptide is insulin or an analog thereof. In some embodiments, the insulin is human insulin. In some embodiments, the metabolic polypeptide (e.g., insulin) is added to the culture medium in an amount of about 1 mg / L to about 50 mg / L, examples of which include any of about 1 mg / L to about 18 mg / L, or about 15 mg / L to about 25 mg / L, or about 18 mg / L to about 22 mg / L, or about 20 mg / L to about 40 mg / L, or about 35 mg / L to about 50 mg / L. In some embodiments, a metabolic polypeptide (e.g., insulin) is added to the culture medium in an amount of any of about 15 mg / L, about 16 mg / L, about 17 mg / L, about 18 mg / L, about 19 mg / L, about 20 mg / L, about 21 mg / L, about 22 mg / L, about 23 mg / L, about 24 mg / L, or about 25 mg / L.

[0077] In some embodiments, the iron source increases cellular uptake of non-heme iron, such as through transferrin or non-transferrin-based cellular mechanisms. In some embodiments, the iron source is transferrin. In some embodiments, the transferrin is human transferrin. In some embodiments, the iron source is a component of non-transferrin-bound iron (NTBI) uptake, such as DCYTB, DMT1, ZIP8, and ZIP14, or increases the expression or activity of one or more such components. In some embodiments, the iron source (e.g., transferrin) is added to the culture medium in an amount of about 1 mg / L to about 50 mg / L, examples of which include about 5 mg / L to about 45 mg / L. In some embodiments, an iron source (e.g., transferrin) is added to the culture medium in an amount of any of about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, about 10 mg / L, about 11 mg / L, about 12 mg / L, about 13 mg / L, about 14 mg / L, about 15 mg / L, about 16 mg / L, about 17 mg / L, about 18 mg / L, about 19 mg / L, about 20 mg / L, about 25 mg / L, about 30 mg / L, about 35 mg / L, about 40 mg / L, about 45 mg / L, about, or about 50 mg / L.

[0078] In some embodiments, the antioxidant enzyme cofactor (e.g., a cofactor for glutathione peroxidase) is a selenium compound. In some embodiments, the selenium compound is selenite (a salt containing selenium). In some embodiments, the selenium compound is sodium selenite or disodium selenite. In some embodiments, the antioxidant enzyme cofactor (e.g., sodium selenite) is added to the culture medium in an amount of about 0.001 mg / L to about 0.1 mg / L. In some embodiments, the antioxidant enzyme cofactor (e.g., sodium selenite) is added to the culture medium in an amount of any of about 0.001 mg / L, about 0.005 mg / L, about 0.01 mg / L, about 0.05 mg / L, or about 0.1 mg / L. In some embodiments, an antioxidant enzyme cofactor (e.g., sodium selenite) is added to the culture medium in an amount of any of about 0.01 mg / L, about 0.011 mg / L, about 0.012 mg / L, about 0.013 mg / L, about 0.014 mg / L, about 0.015 mg / L, about 0.016 mg / L, about 0.017 mg / L, about 0.018 mg / L, about 0.019 mg / L, or about 0.02 mg / L.

[0079] In some embodiments, the lipid precursor is ethanolamine or a glyceride (e.g., a diglyceride). In some embodiments, the lipid precursor is ethanolamine. In some embodiments, the lipid precursor (e.g., ethanolamine) is added to the culture medium in an amount of about 1 mg / L to about 10 mg / L. In some embodiments, the lipid precursor (e.g., ethanolamine) is added to the culture medium in an amount of about 1 mg / L, about 2 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, or about 10 mg / L.

[0080] In some embodiments, the carrier protein is a globular protein. In some embodiments, the carrier protein is selected from the group consisting of albumin, lysozyme, and myoglobin. In some embodiments, the carrier protein is albumin. In some embodiments, the albumin is human albumin. In some embodiments, the human albumin is human serum albumin. In some embodiments, the carrier protein (e.g., albumin) is added to the culture medium in an amount of about 100 mg / L to about 5,000 mg / L. In some embodiments, a carrier protein (e.g., albumin) is added to the culture medium in an amount of any of about 100 mg / L, about 200 mg / L, about 300 mg / L, about 400 mg / L, about 500 mg / L, about 600 mg / L, about 700 mg / L, about 800 mg / L, about 900 mg / L, about 1000 mg / L, about 1500 mg / L, about 2000 mg / L, about 2500 mg / L, about 3000 mg / L, about 3500 mg / L, about 4000 mg / L, about 4500 mg / L, or about 5000 mg / L.

[0081] In some embodiments, the culture medium comprises two or more, three or more, four or more, or all of the following components (e.g., added to a basal medium (e.g., NK MACS): (i) a metabolic polypeptide (e.g., insulin), (ii) an iron source (e.g., transferrin), (iii) an antioxidant enzyme cofactor (e.g., a selenium compound), (iv) a lipid precursor (e.g., ethanolamine), or (v) a carrier protein (e.g., albumin). In some embodiments, the culture medium comprises ITSEA (e.g., added to a basal medium): In some embodiments, the metabolic polypeptide (e.g., insulin) is added in an amount of about 15 mg / L to about 25 mg / L, examples of which include any of about 16 mg / L, about 17 mg / L, about 18 mg / L, about 19 mg / L, about 20 mg / L, about 21 mg / L, about 22 mg / L, about 23 mg / L, or about 24 mg / L. In some embodiments, the iron source (e.g., transferrin) is added in an amount of about 5 mg / L to about 15 mg / L, examples of which include about 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, or 14 mg / L. In some embodiments, the antioxidant enzyme cofactor (e.g., selenium compound) is added in an amount of about 0.01 mg / L to about 0.02 mg / L, examples of which include about 0.011 mg / L, 0.012 mg / L, 0.013 mg / L, 0.014 mg / L, 0.015 mg / L, 0.016 mg / L, 0.017 mg / L, 0.018 mg / L, or 0.019 mg / L. In some embodiments, the lipid precursor (e.g., ethanolamine) is added in an amount of about 1 mg / L to about 10 mg / L, examples of which include any of about 2 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, or about 9 mg / L.In some embodiments, the carrier protein (e.g., albumin) is added in an amount of about 100 mg / L to about 1000 mg / L, examples of which include any of about 200 mg / L, about 300 mg / L, about 400 mg / L, about 500 mg / L, about 600 mg / L, about 700 mg / L, about 800 mg / L, or about 900 mg / L.

[0082] In some embodiments, the culture medium (e.g., comprising the following components added to basal medium (e.g., NK MACS)) comprises a cytokine (e.g., IL-15) and two or more, three or more, four or more, or all of: (i) a metabolic polypeptide (e.g., insulin), (ii) an iron source (e.g., transferrin), (iii) an antioxidant enzyme cofactor (e.g., a selenium compound), (iv) a lipid precursor (e.g., ethanolamine), or (v) a carrier protein (e.g., albumin). In some embodiments, the culture medium (e.g., comprising the following components added to basal medium) comprises a cytokine (e.g., IL-15) and ITSEA. In some embodiments, the cytokine (e.g., IL-15) is added in an amount of about 1 ng / mL to about 500 ng / mL, examples of which include any of about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, or 19 ng / mL. In some embodiments, the cytokine is added to the culture medium every 1 to 4 days during culture, e.g., every 2 days or every 3 days, examples of which include addition in the amounts described herein for each instance of cytokine added to the culture medium. In some embodiments, a metabolic polypeptide (e.g., insulin) is added in an amount of about 15 mg / L to about 25 mg / L, examples of which include any of about 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, 20 mg / L, 21 mg / L, 22 mg / L, 23 mg / L, or 24 mg / L. In some embodiments, an iron source (e.g., transferrin) is added in an amount of about 5 mg / L to about 15 mg / L, examples of which include any of about 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, or 14 mg / L.In some embodiments, antioxidant enzyme cofactors (e.g., selenium compounds) are added in amounts of about 0.01 mg / L to about 0.02 mg / L, examples of which include about 0.011 mg / L, about 0.012 mg / L, 0.013 mg / L, about 0.014 mg / L, about 0.015 mg / L, about 0.016 mg / L, about 0.017 mg / L, about 0.018 mg / L, or about 0.019 mg / L. In some embodiments, lipid precursors (e.g., ethanolamine) are added in amounts of about 1 mg / L to about 10 mg / L, examples of which include about 2 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, or about 9 mg / L. In some embodiments, the carrier protein (e.g., albumin) is added in an amount of about 100 mg / L to about 1000 mg / L, examples of which include any of about 200 mg / L, about 300 mg / L, about 400 mg / L, about 500 mg / L, about 600 mg / L, about 700 mg / L, about 800 mg / L, or about 900 mg / L.

[0083] In some embodiments, the culture medium further comprises serum. In some embodiments, the serum is AB serotype (AB) serum. In some embodiments, the serum is human AB serum. In some embodiments, the human AB serum is collected from a human male with AB blood type. In some embodiments, the human AB serum has reduced reactivity with γδ T cells grown in the culture medium. In some embodiments, serum is added to the culture medium in an amount of about 0.1% (v / v) to about 10% (v / v), examples of which include any of about 0.5% (v / v), about 1% (v / v), about 1.5% (v / v), about 2% (v / v), about 2.5% (v / v), about 3% (v / v), about 3.5% (v / v), about 4% (v / v), about 4.5% (v / v), about 5% (v / v), about 5.5% (v / v), about 6% (v / v), about 6.5% (v / v), about 7% (v / v), about 7.5% (v / v), about 8% (v / v), about 8.5% (v / v), about 9% (v / v), about 9.5% (v / v), or about 10% (v / v).

[0084] In some embodiments, the culture medium comprises (e.g., the following components added to basal medium (e.g., NK MACS)): a cytokine (e.g., IL-15), serum (e.g., AB serum), and two or more, three or more, four or more, or all of: (i) a metabolic polypeptide (e.g., insulin), (ii) an iron source (e.g., transferrin), (iii) an antioxidant enzyme cofactor (e.g., a selenium compound), (iv) a lipid precursor (e.g., ethanolamine), or (v) a carrier protein (e.g., albumin). In some embodiments, the culture medium comprises (e.g., the following components added to basal medium): a cytokine (e.g., IL-15), and ITSEA. In some embodiments, the cytokine (e.g., IL-15) is added in an amount of about 1 ng / mL to about 50 ng / mL, examples of which include any of about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, or about 19 ng / mL. In some embodiments, the cytokine is added to the culture medium every 1 to 4 days during culture, e.g., every 2 days or every 3 days, examples of which include addition in the amount described herein for each instance of cytokine added to the culture medium. In some embodiments, a metabolic polypeptide (e.g., insulin) is added in an amount of about 15 mg / L to about 25 mg / L, examples of which include any of about 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, 20 mg / L, 21 mg / L, 22 mg / L, 23 mg / L, or 24 mg / L. In some embodiments, an iron source (e.g., transferrin) is added in an amount of about 5 mg / L to about 15 mg / L, examples of which include any of about 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, or 14 mg / L.In some embodiments, antioxidant enzyme cofactors (e.g., selenium compounds) are added in amounts of about 0.01 mg / L to about 0.02 mg / L, examples of which include about 0.011 mg / L, about 0.012 mg / L, 0.013 mg / L, about 0.014 mg / L, about 0.015 mg / L, about 0.016 mg / L, about 0.017 mg / L, about 0.018 mg / L, or about 0.019 mg / L. In some embodiments, lipid precursors (e.g., ethanolamine) are added in amounts of about 1 mg / L to about 10 mg / L, examples of which include about 2 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, or about 9 mg / L. In some embodiments, the carrier protein (e.g., albumin) is added in an amount of about 100 mg / L to about 1000 mg / L, examples of which include any of about 200 mg / L, about 300 mg / L, about 400 mg / L, about 500 mg / L, about 600 mg / L, about 700 mg / L, about 800 mg / L, or about 900 mg / L. In some embodiments, serum (e.g., AB serum) is added to the culture medium in an amount of about 0.5% (v / v) to about 5% (v / v), examples of which include any of about 0.5% (v / v), about 1% (v / v), about 1.5% (v / v), about 2% (v / v), about 2.5% (v / v), about 3% (v / v), about 3.5% (v / v), about 4% (v / v), about 4.5% (v / v), or about 5% (v / v).

[0085] B. Collection In certain aspects, the method of producing a population of cells comprising γδ T cells comprises harvesting the population of cells comprising γδ T cells after culturing. In some embodiments, the cells are harvested at other stages of the methods provided herein (such as the transduction stage), and the harvesting techniques described herein are also applicable for performing harvesting techniques at such stages. In some embodiments, no harvesting step is performed to obtain a population of cells comprising γδ T cells. One of skill in the art will readily understand whether a harvesting step is necessary in the methods provided herein based, for example, on the downstream use of the population of cells comprising γδ T cells.

[0086] Harvesting techniques, including culture techniques for expanding γδ T cells, are well known in the art. See, e.g., Segeritz et al., Basic Science Methods for Clinical Researchers, 2017; and Fundamental Techniques in Cell Culture, 3rd Edition, 2016, the contents of which are incorporated herein by reference in their entireties. In some embodiments, harvesting comprises obtaining a population of cells comprising γδ T cells in solution, e.g., suspending the population of cells comprising γδ T cells. In some embodiments, harvesting comprises concentrating the population of cells comprising γδ T cells, e.g., by using centrifugation. In some embodiments, harvesting comprises removing at least about 50%, 60%, 70%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the culture medium from a population of cells comprising γδ T cells, such as the culture medium used during culturing. In some embodiments, harvesting comprises resuspending the cells in a solution (e.g., DMSO) for cryopreservation. In some embodiments, harvesting comprises resuspending the cells in a solution (e.g., a pharmaceutically acceptable excipient) for administration to a subject (e.g., a human patient) in need thereof.

[0087] In some embodiments, harvesting is performed, e.g., performed at one time, based on achieving one or more characteristics of the population of produced cells comprising γδ T cells, including one or more characteristics of γδ T cells. In some embodiments, the one or more characteristics include the number of cells (e.g., the number of produced γδ T cells), the extent of expansion (e.g., the extent of expansion of γδ T cells), or the phenotype (e.g., based on lineage, activation, exhaustion, memory, and / or antigen presentation). Additional details regarding the characteristics, including the phenotype of the population of cells comprising γδ T cells, are provided elsewhere in this disclosure. In some embodiments, harvesting is performed on about day 7 to about day 28, including from about day 7 to about day 18, or from about day 10 to about day 14. In some embodiments, harvesting is performed on any of the following days: about the 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, or 24th day.

[0088] C. Steps to be performed before culturing In certain embodiments, the methods provided herein include one or more additional step(s) performed prior to culturing the isolated pool of mixed immune cells (such as an isolated pool of PBMCs) or a derivative thereof that has been depleted of αβ T cells in culture medium. In some embodiments, the method further includes a step of depleting αβ T cells. In some embodiments, the method further includes a step of obtaining (e.g., isolating) the pool of mixed immune cells or the pool of mixed immune cells that has been depleted of αβ T cells.

[0089] i.αβ T cell depletion In certain embodiments, the method comprises performing a step of depleting αβ T cells on the isolated pool of mixed immune cells to obtain a population of cells depleted of αβ T cells, hi some embodiments, αβ T cells are removed from the pool of mixed immune cells by negative selection or use of a suitable isolation technique.

[0090] In some embodiments, the step of depleting αβ T cells comprises antibody-based depletion. In some embodiments, the step of depleting αβ T cells comprises (a) exposing a pool of mixed immune cells to an anti-TCR α / β antibody, and (b) separating the antibody-conjugated αβ TCR+ T cells from other cells in the pool of mixed immune cells to obtain a population of αβ T cell-depleted cells. In some embodiments, the separation is performed by a cell sorting technique, including, but not limited to, magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS).

[0091] In some embodiments, the population of cells resulting from αβ T cell depletion (an isolated pool of mixed immune cells depleted of αβ T cells) is cryopreserved for a period of time before carrying out the culturing steps taught herein. Cryopreservation techniques are described in more detail elsewhere in this disclosure.

[0092] ii. Isolate / obtain a pool of mixed immune cells In certain embodiments, the method includes obtaining an isolated pool of mixed immune cells, including an isolated pool of mixed immune cells that have been depleted of αβ T cells. In some embodiments, the pool of mixed immune cells that have been depleted of αβ T cells is an isolated pool of PBMCs. As described herein, the isolated pool of PBMCs may include additional cells, e.g., at least about 85% PBMC purity. In some embodiments, the one or more precursors (e.g., before αβ T cell depletion) include PBMCs, examples of which include obtaining isolated PBMCs from an individual.

[0093] In some embodiments, the method includes isolating a pool of mixed immune cells, such as PBMCs, from a donor (such as a healthy human donor or a human donor including a human patient with a disease or disorder). In some embodiments, the isolated pool of mixed immune cells, such as PBMCs, from a donor (such as a human donor) is from a single collection from the donor (e.g., a single blood draw or cell collection). In some embodiments, the isolated pool of mixed immune cells, such as PBMCs, from a donor (such as a human donor) is from multiple collections from the donor. In some embodiments, the isolated pool of mixed immune cells, such as PBMCs, is pooled from one or more donors (such as human donors) (e.g., multiple collections from a single donor), or is one or more collections from multiple donors. In some embodiments, the pooled immune cells from one or more sources (such as one or more collections or one or more donors) are subjected to αβ T cell depletion. In some embodiments, the sample collected from the donor (such as a human donor) includes peripheral blood cells from apheresis, buffy coat, whole blood, LRSCs, leukopaque, or the like. In some embodiments, an isolated pool of mixed immune cells (such as PBMCs) is isolated from tumor-infiltrating or tissue-resident immune cells, natural or induced pluripotent / stem / progenitor cells, ascites, or other human body fluids or bone marrow cells.

[0094] In some embodiments, the isolating comprises leukapheresis. In some embodiments, the isolating is performed on blood from a donor (e.g., a human donor). In some embodiments, the method further comprises withdrawing blood from the donor (e.g., a human donor).

[0095] In some embodiments, the donor is a human individual. In some embodiments, the donor is a healthy individual (e.g., an individual not having the disease or condition to be treated with the manufactured cell population comprising γδ T cells). In some embodiments, the donor is an individual suspected of having or diagnosed with a disease, disorder, and / or medical condition. In some embodiments, the donor is the intended recipient of the manufactured cell population comprising γδ T cells. In some embodiments, the donor is not the intended recipient of the manufactured cell population comprising γδ T cells. One of skill in the art will readily recognize that when the donor and recipient are different individuals, the therapy is characterized as allogeneic, and when the donor and recipient are the same individual, the therapy is characterized as autologous.

[0096] D. Transduction In certain aspects, the methods provided herein include transducing the cells of the population with a transgene. In some embodiments, the transgene is a chimeric antigen receptor (CAR).

[0097] Various techniques are known for transducing cells to modify gene expression. See, for example, Bilal et al., Immunol Cell Biol, 93, 2015 (the contents of which are incorporated herein by reference in their entirety). Generally speaking, transduction involves introducing a factor, such as an exogenous factor (via viral delivery, e.g., a virus or viral vector) that modifies cellular gene expression. In some embodiments, transduction involves delivering a gene or modified gene (including a transgene) via a retrovirus. Examples of viruses or viral vectors used for viral-mediated transgene delivery include adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes simplex virus (HSV), measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, and picornavirus. Other techniques for modifying cells are also known and may be used in the methods taught herein. For example, non-viral approaches are known, including delivery of substances via plasmids, nanoparticles, lipoplexes, liposomes, lipid conjugates, or exosomes. In some embodiments, a gene (e.g., a transgene) is delivered to a cell. In some embodiments, the gene (e.g., a transgene) is further modified. In some embodiments, the modified gene (e.g., a modified transgene) is a CAR.

[0098] In some embodiments, cells are modified by delivery of a nucleic acid editing system, examples of which include CRISPR / Cas9, TALENS, ZFNs, etc.

[0099] In some embodiments, transduction and associated cell harvesting and culture methods include static techniques, examples of which include static bag or plate transduction. In some embodiments, transduction and associated cell harvesting and culture methods include spinoculation techniques, examples of which include Sepax spinoculation and plate or bag spinoculation. See, e.g., Remley et al., J Translational Medicine, 19, 2021. In some embodiments, the bag or plate is coated. In some embodiments, the coating is retronectin.

[0100] In some embodiments, the method includes two or more culture steps, and the transduction step occurs after the first culture step and before the second culture step. In such embodiments, the first culture step is from about day 0 to about day 4. In some embodiments, the transduction step is performed around day 4. In some embodiments, the second culture step is from about day 0 to about day 10.

[0101] The present disclosure encompasses numerous cell modifications, which may be directed by the use of the population of cells produced. For example, in some embodiments, transduction involves introducing a CAR. In some embodiments, the CAR specifically recognizes globohexaosylceramide, AKR1C3, SSEA-4, or TROP-2.

[0102] E. Freezing storage In some embodiments, the methods provided herein include one or more cryopreservation steps, examples of which include cryopreserving cells at different stages of the method. In some embodiments, cryopreservation is performed on an isolated pool of mixed immune cells, or an isolated pool of mixed immune cells that has been depleted of αβ T cells (such as PBMCs) or a derivative thereof (e.g., a population of PBMCs that has been depleted of αβ T cells). In some embodiments, cryopreservation is performed on a harvested population of cells that comprises γδ T cells. In some embodiments, cryopreservation is performed on a population of cells that comprises γδ T cells after the harvested population of cells that comprises γδ T cells has been subjected to a transduction step.

[0103] Cryopreservation techniques are well known in the art. In some embodiments, cryopreservation techniques involve combining cells (such as an αβ T cell-depleted PBMC population or a population of cells comprising γδ T cells produced using the methods described herein) with freezing medium and placing the resulting composition in a cryopreservation container, examples of which include a liquid nitrogen freezer (−195° C.) or an ultra-low temperature freezer (−65° C., −80° C., or −120° C.) for storage for up to or at least about any of 1 day, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 3 years, or about 5 years. The freezing medium contains dimethyl sulfoxide (DMSO), and / or sodium chloride (NaCl), and / or dextrose, and / or dextran sulfate, and / or hydroxyethyl starch (HES), along with a physiological pH buffer, and is capable of maintaining a pH of about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5, about 7.5 to about 8.0, or about 6.5 to about 7.5. In some embodiments, the freezing medium comprises CryoStor CS10.

[0104] Cryopreserved cells may retain functionality and may be thawed for later use, depending on the stage in the method at which they were cryopreserved. For example, in some embodiments, a population of mixed immune cells depleted of αβ T cells may be cryopreserved and then thawed to continue culturing as taught herein to produce a population of cells comprising γδ T cells. In some embodiments, the produced population of cells comprising γδ T cells may be cryopreserved and later thawed for methods of use, e.g., methods of treatment, described herein.

[0105] In some embodiments, cryopreservation involves flushing the cell plate and / or bag (such as a GREX plate and / or bag) with culture medium and transferring the cells in the culture medium (such as a population of cells containing γδ T cells) to a container such as a 50 mL conical tube. In some embodiments, the resulting cell population is counted. In some embodiments, the container is centrifuged to pellet the cells, followed by removal of the supernatant. In some embodiments, the resulting cell pellet is resuspended in CryoStor CS10 freezing medium at 4°C. In some embodiments, the resuspended cells are placed in a cell freezing container and stored at -80°C for a period of time (such as overnight) before being transferred to long-term storage (such as in liquid nitrogen).

[0106] F. Exemplary Methods In some embodiments, a method for producing a population of cells comprising γδ T cells is provided. The method includes culturing an isolated pool of mixed immune cells (e.g., PBMCs depleted of αβ T cells) or a derivative thereof that has been depleted of αβ T cells in a culture medium comprising: (a) a basal medium; (b) IL-15; and (c) the following: (i) insulin or a derivative thereof, (ii) transferrin, (iii) sodium selenite, (iv) ethanolamine, and (v) albumin or a derivative thereof; and, optionally, harvesting the population of cells comprising γδ T cells. In some embodiments, the IL-15 is human IL-15, an example of which is recombinant human IL-15. In some embodiments, the recombinant human IL-15 is added to the culture medium in an amount of about 10 ng / mL. In some embodiments, the insulin is human insulin. In some embodiments, the human insulin is added to the culture medium in an amount of about 20 mg / L. In some embodiments, the transferrin is human transferrin. In some embodiments, human transferrin is added to the culture medium in an amount of about 11 mg / L. In some embodiments, sodium selenite is added to the culture medium in an amount of about 0.0134 mg / L. In some embodiments, ethanolamine is added to the culture medium in an amount of about 4 mg / L. In some embodiments, the albumin is human albumin. In some embodiments, human albumin is added to the culture medium in an amount of about 400 mg / L. In some embodiments, the culture medium comprises ITSEA. In some embodiments, the culture medium further comprises serum. In some embodiments, the serum is human AB serum. In some embodiments, human serum is added to the culture medium in an amount of about 1% (v / v). In some embodiments, the basal culture medium is NK MACS medium. In some embodiments, the culturing is performed for about 14 days. In some embodiments, the produced population of cells comprises γδ T cells having a γδ T cell purity of at least about 85% (e.g., at least about 95%). In some embodiments, the population of cells produced comprises γδ T cells, including vδ2 γδ T cells, and optionally vδ1 γδ T cells.In some embodiments, the population of cells comprising γδ T cells has a vδ2 γδ T cell purity relative to the total T cells (e.g., γδ T cells) in the population of at least about 70%. In some embodiments, the manufactured population of cells comprising γδ T cells has a vδ2 γδ T cell purity relative to the total T cells (e.g., γδ T cells) in the population of at least about 95%. In some embodiments, the manufactured population of cells comprising γδ T cells comprises less than about 5% NK cells (e.g., less than about 2.5% NK cells). In some embodiments, the manufactured population of cells comprising γδ T cells comprises γδ T cells comprising a CAR. In some embodiments, the CAR specifically recognizes globohexaosylceramide, AKR1C3, SSEA-4, or TROP-2. In some embodiments, the method further comprises cryopreserving the manufactured population of cells comprising γδ T cells after the harvesting step.

[0107] In some embodiments, a method for producing a population of cells comprising γδ T cells is provided. The method includes culturing an isolated pool of mixed immune cells (e.g., PBMCs depleted of αβ T cells) or a derivative thereof that has been depleted of αβ T cells in a culture medium comprising: (a) a basal culture medium; (b) IL-15; and (c) the following: (i) insulin or a derivative thereof, (ii) transferrin, (iii) sodium selenite, (iv) ethanolamine, and (v) albumin or a derivative thereof; performing a γδ T cell transduction step; and, optionally, harvesting the population of cells comprising γδ T cells. In some embodiments, the IL-15 is human IL-15, an example of which is recombinant human IL-15. In some embodiments, the recombinant human IL-15 is added to the culture medium in an amount of about 10 ng / mL. In some embodiments, the insulin is human insulin. In some embodiments, the human insulin is added to the culture medium in an amount of about 20 mg / L. In some embodiments, the transferrin is human transferrin. In some embodiments, the human transferrin is added to the culture medium in an amount of about 11 mg / L. In some embodiments, sodium selenite is added to the culture medium in an amount of about 0.0134 mg / L. In some embodiments, ethanolamine is added to the culture medium in an amount of about 4 mg / L. In some embodiments, the albumin is human albumin. In some embodiments, human albumin is added to the culture medium in an amount of about 400 mg / L. In some embodiments, the culture medium comprises ITSEA. In some embodiments, the culture medium further comprises serum. In some embodiments, the serum is human AB serum. In some embodiments, human serum is added to the culture medium in an amount of about 1% (v / v). In some embodiments, the basal culture medium is NK MACS medium. In some embodiments, the culture is performed for about 14 days. In some embodiments, the culture step comprises a first culture step from about day 0 to about day 4, and transduction is performed around day 4. In some embodiments, the method includes a second culture step about 0 to about 10 days after transduction. In some embodiments, transduction includes introducing a CAR into the cells.In some embodiments, the population of manufactured cells comprises γδ T cells having a γδ T cell purity of at least about 85% (such as at least about 95%). In some embodiments, the population of manufactured cells comprises γδ T cells, including vδ2 γδ T cells, and optionally vδ1 γδ T cells. In some embodiments, the population of cells comprising γδ T cells has a vδ2 γδ T cell purity relative to the total T cells (such as γδ T cells) in the population of at least about 70%. In some embodiments, the population of manufactured cells comprising γδ T cells has a vδ2 γδ T cell purity relative to the total T cells (such as γδ T cells) in the population of at least about 95%. In some embodiments, the population of manufactured cells comprising γδ T cells comprises less than about 5% NK cells (such as less than about 2.5% NK cells). In some embodiments, the population of manufactured cells comprising γδ T cells comprises γδ T cells that comprise a CAR. In some embodiments, the CAR specifically recognizes globohexaosylceramide, AKR1C3, SSEA-4, or TROP-2. In some embodiments, the method further comprises cryopreserving the manufactured population of cells comprising γδ T cells after the harvesting step.

[0108] In some embodiments, a method for producing a population of cells comprising γδ T cells is provided. The method includes performing a step of depleting αβ T cells on an isolated pool of mixed immune cells (e.g., PBMCs), culturing the isolated pool of αβ T cells-depleted mixed immune cells, or a derivative thereof, in a culture medium comprising: (a) a basal culture medium; (b) IL-15; and (c) the following: (i) insulin or a derivative thereof, (ii) transferrin, (iii) sodium selenite, (iv) ethanolamine, and (v) albumin or a derivative thereof; and, optionally, harvesting the population of cells comprising γδ T cells. In some embodiments, the IL-15 is human IL-15, an example of which is recombinant human IL-15. In some embodiments, the recombinant human IL-15 is added to the culture medium in an amount of about 10 ng / mL. In some embodiments, the insulin is human insulin. In some embodiments, the human insulin is added to the culture medium in an amount of about 20 mg / L. In some embodiments, the transferrin is human transferrin. In some embodiments, human transferrin is added to the culture medium in an amount of about 11 mg / L. In some embodiments, sodium selenite is added to the culture medium in an amount of about 0.0134 mg / L. In some embodiments, ethanolamine is added to the culture medium in an amount of about 4 mg / L. In some embodiments, the albumin is human albumin. In some embodiments, human albumin is added to the culture medium in an amount of about 400 mg / L. In some embodiments, the culture medium comprises ITSEA. In some embodiments, the culture medium further comprises serum. In some embodiments, the serum is human AB serum. In some embodiments, human serum is added to the culture medium in an amount of about 1% (v / v). In some embodiments, the basal culture medium is NK MACS medium. In some embodiments, the culture is for about 14 days. In some embodiments, the step of depleting αβ T cells comprises antibody-based depletion.In some embodiments, the step of depleting αβ T cells comprises (a) exposing a pool of mixed immune cells to an anti-TCR α / β antibody; and (b) separating the antibody-conjugated αβ TCR+ T cells from other cells in the pool of mixed immune cells to obtain a population of αβ T cell-depleted cells. In some embodiments, the produced population of cells comprises γδ T cells having a γδ T cell purity of at least about 85% (e.g., at least about 95%). In some embodiments, the produced population of cells comprises γδ T cells, including vδ2 γδ T cells, and optionally vδ1 γδ T cells. In some embodiments, the population of cells comprising γδ T cells has a vδ2 γδ T cell purity relative to total T cells (e.g., γδ T cells) in the population of at least about 70%. In some embodiments, the produced population of cells comprising γδ T cells has a vδ2 γδ T cell purity relative to total T cells (e.g., γδ T cells) in the population of at least about 95%. In some embodiments, the population of manufactured cells comprising γδ T cells comprises less than about 5% NK cells (such as less than about 2.5% NK cells). In some embodiments, the population of manufactured cells comprising γδ T cells comprises γδ T cells comprising a CAR. In some embodiments, the CAR specifically recognizes globohexaosylceramide, AKR1C3, SSEA-4, or TROP-2. In some embodiments, the method further comprises cryopreserving the population of manufactured cells comprising γδ T cells after the harvesting step.

[0109] In some embodiments, a method for producing a population of cells comprising γδ T cells is provided. The method includes performing a step of depleting αβ T cells on an isolated pool of mixed immune cells (e.g., PBMCs), culturing the isolated pool of αβ T cells-depleted mixed immune cells, or a derivative thereof, in a culture medium comprising: (a) a basal culture medium; (b) IL-15; and (c) the following: (i) insulin or a derivative thereof, (ii) transferrin, (iii) sodium selenite, (iv) ethanolamine, and (v) albumin or a derivative thereof; performing a step of γδ T cell transduction; and optionally harvesting the population of cells comprising γδ T cells. In some embodiments, the IL-15 is human IL-15, an example of which is recombinant human IL-15. In some embodiments, the recombinant human IL-15 is added to the culture medium in an amount of about 10 ng / mL. In some embodiments, the insulin is human insulin. In some embodiments, the human insulin is added to the culture medium in an amount of about 20 mg / L. In some embodiments, the transferrin is human transferrin. In some embodiments, the human transferrin is added to the culture medium in an amount of about 11 mg / L. In some embodiments, sodium selenite is added to the culture medium in an amount of about 0.0134 mg / L. In some embodiments, ethanolamine is added to the culture medium in an amount of about 4 mg / L. In some embodiments, the albumin is human albumin. In some embodiments, human albumin is added to the culture medium in an amount of about 400 mg / L. In some embodiments, the culture medium comprises ITSEA. In some embodiments, the culture medium further comprises serum. In some embodiments, the serum is human AB serum. In some embodiments, human serum is added to the culture medium in an amount of about 1% (v / v). In some embodiments, the culture medium comprises basal culture medium. In some embodiments, the basal culture medium is NK MACS medium. In some embodiments, the culturing is performed for about 14 days. In some embodiments, the step of depleting αβ T cells comprises antibody-based depletion.In some embodiments, the step of depleting αβ T cells comprises: (a) exposing a pool of mixed immune cells to an anti-TCR α / β antibody; and (b) separating the antibody-conjugated αβ TCR+ T cells from other cells in the pool of mixed immune cells to obtain a population of αβ T cell-depleted cells. In some embodiments, the culturing step comprises a first culturing step from about day 0 to about day 4, and transduction is performed around day 4. In some embodiments, the method comprises a second culturing step from about day 0 to about 10 after transduction. In some embodiments, transduction comprises introducing a CAR into the cells. In some embodiments, the produced population of cells comprises γδ T cells having a γδ T cell purity of at least about 85% (e.g., at least about 95%). In some embodiments, the produced population of cells comprises γδ T cells, including vδ2 γδ T cells, and optionally vδ1 γδ T cells. In some embodiments, the population of cells comprising γδ T cells has a vδ2 γδ T cell purity relative to the total T cells (e.g., γδ T cells) in the population of at least about 70%. In some embodiments, the manufactured population of cells comprising γδ T cells has a vδ2 γδ T cell purity relative to the total T cells (e.g., γδ T cells) in the population of at least about 95%. In some embodiments, the manufactured population of cells comprising γδ T cells comprises less than about 5% NK cells (e.g., less than about 2.5% NK cells). In some embodiments, the manufactured population of cells comprising γδ T cells comprises γδ T cells comprising a CAR. In some embodiments, the CAR specifically recognizes globohexaosylceramide, AKR1C3, SSEA-4, or TROP-2. In some embodiments, the method further comprises cryopreserving the manufactured population of cells comprising γδ T cells after the harvesting step.

[0110] III. Populations of cells containing γδ T cells In certain aspects, provided herein are populations of cells comprising γδ T cells produced using the methods described herein. In some embodiments, the populations of cells comprising γδ T cells exhibit functional properties known to be important for supporting effective adoptive cell therapy activity. For example, T cells of memory or naive T cell lineages exhibit improved engraftment, proliferation, and anti-tumor activity over terminal effector T cells (see, e.g., Gattinoni et al. (2005) J Clin Invest 115(6):1616-1626; Janelle and Delisle (2021), Cancers (Basel) 13(4): 598; and Lopez-Cantillo et al (2022), Front Immunol 13:878209). Similarly, functional characteristics of T cell exhaustion and senescence are important indicators of downstream therapeutic efficacy, and cells that exhibit high levels of senescence markers (e.g., high levels of KLRG1 and CD57 and low levels of CD27 or CD28) or high levels of exhaustion markers (e.g., high levels of PD-1, CTLA-4, LAG-3, TIM-3, TIGIT, etc., and low levels of perforin, TNF-α, IL-2, IFN-γ, granzyme B, etc.) exhibit significantly reduced T cell proliferation or anti-tumor activity upon adoptive cell transfer. In some embodiments, the γδ T cells are not of the end effector T cell lineage. In some embodiments, the γδ T cells are not senescent. In some embodiments, the γδ T cells are not functionally exhausted.

[0111] In some embodiments, the population of cells is about 1 x 10 3 cells ~ approx. 1 x 10 10 In some embodiments, the population of cells comprises at least about 1 x 10 cells. 3 cells, approximately 2 x 10 3 cells, approximately 3 x 10 3 cells, approximately 4 x 10 3 cells, approximately 5 x 10 3 cells, approximately 6 x 10 3 cells, approximately 7 x 10 3 cells, approximately 8 x 10 3 cells, approximately 9 x 103 Cells, approximately 1 × 10 4 Cells, approximately 2 × 10 4 Cells, approximately 3 × 10 4 Cells, approximately 4 × 10 4 Cells, approximately 5 × 10 4 Cells, approximately 6 × 10 4 Cells, approximately 7 × 10 4 Cells, approximately 8 × 10 4 Cells, approximately 9 × 10 4 Cells, approximately 1 × 10 5 Cells, approximately 2 × 10 5 Cells, approximately 3 × 10 5 Cells, approximately 4 × 10 5 Cells, approximately 5 × 10 5 Cells, approximately 6 × 10 5 Cells, approximately 7 × 10 5 Cells, approximately 8 × 10 5 Cells, approximately 9 × 10 5 Cells, approximately 1 × 10 6 Cells, approximately 2 × 10 6 Cells, approximately 3 × 10 6 Cells, approximately 4 × 10 6 Cells, approximately 5 × 10 6 Cells, approximately 6 × 10 6 Cells, approximately 7 × 10 6 Cells, approximately 8 × 10 6 Cells, approximately 9 × 10 6 Cells, approximately 1 × 10 7 Cells, approximately 2 × 10 7 Cells, approximately 3 × 10 7 Cells, approximately 4 × 10 7 Cells, approximately 5 × 10 7 Cells, approximately 6 × 10 7 Cells, approximately 7 × 10 7 Cells, approximately 8 × 10 7 Cells, approximately 9 × 10 7 Cells, approximately 1 × 10 8 Cells, approximately 2 × 10 8 Cells, approximately 3 × 10 8 Cells, approximately 4 × 10 8 Cells, approximately 5 × 10 8 Cells, approximately 6 × 10 8 Cells, approximately 7 × 10 8 Cells, approximately 8 × 10 8 Cells, approximately 9 × 10 8 Cells, approximately 1 × 10 9 Cells, approximately 2 × 10 9cells, approximately 3 x 10 9 cells, approximately 4 x 10 9 cells, approximately 5 x 10 9 cells, approximately 6 x 10 9 cells, approximately 7 x 10 9 cells, approximately 8 x 10 9 cells, approximately 9 x 10 9 cells, or approximately 1 x 10 10 In some embodiments, the population of cells comprises about 1 x 10 3 γδ T cells ~ approx. 1×10 10 In some embodiments, the population of cells comprises at least 1 x 10 γδ T cells. 3 γδ T cells, approximately 2 × 10 3 γδ T cells, approximately 3 × 10 3 γδ T cells, approximately 4 × 10 3 γδ T cells, approximately 5 × 10 3 γδ T cells, approximately 6 × 10 3 γδ T cells, approximately 7 × 10 3 γδ T cells, approximately 8 × 10 3 γδ T cells, approximately 9 × 10 3 γδ T cells, approximately 1 × 10 4 γδ T cells, approximately 2 × 10 4 γδ T cells, approximately 3 × 10 4 γδ T cells, approximately 4 × 10 4 γδ T cells, approximately 5 × 10 4 γδ T cells, approximately 6 × 10 4 γδ T cells, approximately 7 × 10 4 γδ T cells, approximately 8 × 10 4 γδ T cells, approximately 9 × 10 4 γδ T cells, approximately 1 × 10 5 γδ T cells, approximately 2 × 10 5 γδ T cells, approximately 3 × 10 5 γδ T cells, approximately 4 × 10 5 γδ T cells, approximately 5 × 10 5 γδ T cells, approximately 6 × 10 5 γδ T cells, approximately 7 × 10 5 γδ T cells, approximately 8 × 10 5 γδ T cells, approximately 9 × 10 5 γδ T cells, approximately 1 × 10 6 γδ T cells, approximately 2 × 10 6γδ T cells, approximately 3 × 10 6 γδ T cells, approximately 4 × 10 6 γδ T cells, approximately 5 × 10 6 γδ T cells, approximately 6 × 10 6 γδ T cells, approximately 7 × 10 6 γδ T cells, approximately 8 × 10 6 γδ T cells, approximately 9 × 10 6 γδ T cells, approximately 1 × 10 7 γδ T cells, approximately 2 × 10 7 γδ T cells, approximately 3 × 10 7 γδ T cells, approximately 4 × 10 7 γδ T cells, approximately 5 × 10 7 γδ T cells, approximately 6 × 10 7 γδ T cells, approximately 7 × 10 7 γδ T cells, approximately 8 × 10 7 γδ T cells, approximately 9 × 10 7 γδ T cells, approximately 1 × 10 8 γδ T cells, approximately 2 × 10 8 γδ T cells, approximately 3 × 10 8 γδ T cells, approximately 4 × 10 8 γδ T cells, approximately 5 × 10 8 γδ T cells, approximately 6 × 10 8 γδ T cells, approximately 7 × 10 8 γδ T cells, approximately 8 × 10 8 γδ T cells, approximately 9 × 10 8 γδ T cells, approximately 1 × 10 9 γδ T cells, approximately 2 × 10 9 γδ T cells, approximately 3 × 10 9 γδ T cells, approximately 4 × 10 9 γδ T cells, approximately 5 × 10 9 γδ T cells, approximately 6 × 10 9 γδ T cells, approximately 7 × 10 9 γδ T cells, approximately 8 × 10 9 γδ T cells, approximately 9 × 10 9 γδ T cells, または approximately 1×10 10 γδ T cells are contained in the cells.

[0112] In some embodiments, a population of cells comprising γδ T cells produced using the methods provided herein has a γδ T cell purity of at least about 85%, examples of which include any of at least about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%, or about 100%. In some embodiments, a population of cells comprising γδ T cells produced using the methods provided herein comprises vδ2 γδ T cells, and optionally vδ1 γδ T cells. In some embodiments, a population of cells comprising γδ T cells has a vδ2 γδ T cell purity relative to total T cells (e.g., γδ T cells) in the population of at least about 70%, examples of which include at least about 75%, 80%, 85%, 90%, or 95%. In some embodiments, a population of cells comprising γδ T cells produced using the methods provided herein has a vδ2 γδ T cell purity relative to total T cells (e.g., γδ T cells) in the population of at least about 95%, examples of which include at least about 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100%. In any of the embodiments described herein, vδ2 γδ T cell purity is relative to total T cells in the population. In any of the embodiments described herein, vδ2 γδ T cell purity is relative to the total γδ T cells in the population.In some embodiments, a population of cells comprising γδ T cells produced using the methods provided herein comprises less than about 15% of NK cells, examples of which include any of: less than about 14% of NK cells, less than about 13% of NK cells, less than about 12% of NK cells, less than about 11% of NK cells, less than about 10% of NK cells, less than about 9% of NK cells, less than about 8% of NK cells, less than about 7% of NK cells, less than about 6% of NK cells, less than about 5% of NK cells, less than about 4.5% of NK cells, less than about 4% of NK cells, less than about 3.5% of NK cells, less than about 3% of NK cells, less than about 2.5% of NK cells, less than about 2% of NK cells, less than about 1.5% of NK cells, less than about 1% of NK cells, or less than about 0.5% of NK cells.

[0113] In some embodiments, the population of cells comprising γδ T cells comprises less than about 10% vδ1 γδ T cells, examples of which include any of less than about 9% vδ1 γδ T cells, less than about 8% vδ1 γδ T cells, less than about 7% vδ1 γδ T cells, less than about 6% vδ1 γδ T cells, less than about 5% vδ1 γδ T cells, less than about 4% vδ1 γδ T cells, less than about 3% vδ1 γδ T cells, less than about 2% vδ1 γδ T cells or less than about 1% vδ1 γδ T cells.

[0114] In some embodiments, the population of cells comprising γδ T cells comprises less than about 1% αβ T cells, examples of which include less than about 0.9% αβ T cells, less than about 0.8% αβ T cells, less than about 0.7% αβ T cells, less than about 0.6% αβ T cells, less than about 0.5% αβ T cells, less than about 0.4% αβ T cells, less than about 0.3% αβ T cells, less than about 0.2% αβ T cells, or less than about 0.1% αβ T cells.

[0115] IV. Pharmaceutical Compositions and Kits In certain aspects, provided herein is a pharmaceutical composition comprising a population of cells comprising γδ T cells produced using the methods described herein, and optionally a pharmaceutically acceptable carrier. Additionally, provided by the present application are kits and compositions useful for the methods provided herein, as well as kits and compositions obtained from the methods provided herein (e.g., kits and compositions of a population of cells comprising γδ T cells produced using the methods described herein).

[0116] A. Formulation of Pharmaceutical Compositions In some embodiments, pharmaceutical compositions may contain one or more pharmaceutically acceptable excipients or accessory ingredients, examples of which include, but are not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonicity agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, and other species. Also included may be excipients (such as waxes, butters, colorants, coating agents, flavors, and fragrances). Pharmaceutically acceptable excipients are well known in the art (see, e.g., Remington's The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006).

[0117] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and / or combinations thereof.

[0118] Preservatives include, but are not limited to, antioxidants, chelating agents, antibacterial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, acorbyl palmitate, butylhydroxyanisole, butylhydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, edetate disodium, edetate dipotassium, edetate, fumaric acid, malic acid, phosphoric acid, edetate sodium, tartaric acid, and / or edetate trisodium. Antibacterial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, benzyl alcohol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoic acid esters, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid.Other preservatives include, but are not limited to, tocopherol, tocopheryl acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN® II, NEOLONE™, KATHON™, and / or EUXYL®.

[0119] Examples of buffering agents include, but are not limited to, citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixture, tromethamine, aminosulfonic acid buffer (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof. Lubricants may be selected from the non-limiting group consisting of magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0120] In some embodiments, the pharmaceutical composition is formulated to have a pH in the range of about 4.5 to about 9.0, including, for example, any one of the following pH ranges: about 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0. In some embodiments, the pharmaceutical composition is formulated to have a pH in the range of about 4.5 to about 6.5. In some embodiments, the pharmaceutical composition is formulated to have a pH in the range of about 7.3 to about 7.5. In some embodiments, the pharmaceutical composition can be made isotonic with blood by adding a suitable tonicity adjuster.

[0121] The pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparation methods include associating a population of cells comprising γδ T cells produced using the methods described herein with an excipient and / or one or more other accessory ingredients. In some embodiments, the pharmaceutical compositions may be prepared, packaged, and / or sold in bulk as a single unit dose and / or as a plurality of single unit doses. As used herein, a "unit dose" refers to a discrete amount of a pharmaceutical composition comprising a predetermined amount of a population of cells comprising γδ T cells. The amount of γδ T cells may generally be equal to the dosage of γδ T cells administered to an individual, and / or a convenient fraction of such a dosage (including, but not limited to, one-half or one-third of such a dosage).

[0122] B. Administration Pharmaceutical compositions used for in vivo administration are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. In some embodiments, the compositions are pathogen-free. For parenteral administration, the pharmaceutical compositions can be in the form of liquid solutions, examples of which include physiologically compatible buffers such as Hank's solution or Ringer's solution.

[0123] In some embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for parenteral administration, examples of which include intravenous, subcutaneous, peritoneal, intravitreal, or intratumoral administration. In some embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration.

[0124] In some embodiments, pharmaceutical compositions are formulated according to conventional procedures as pharmaceutical compositions suitable for intravenous, intraperitoneal, subcutaneous, intramuscular, intratumoral, intradermal or intravitreal injection. Typically, injectable compositions are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also contain a solubilizer and a local anesthetic (such as lidocaine) to ease the pain at the injection site. Generally, the components are either supplied separately or mixed together in a unit dosage form.

[0125] In some embodiments, the pharmaceutical composition is suitable for administration to humans. In some embodiments, the pharmaceutical composition is suitable for administration to rodents (e.g., mice, rats) or non-human primates (e.g., cynomolgus monkeys). In some embodiments, the pharmaceutical composition is contained in a single-use vial, such as a single-use sealed vial. In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in a container in bulk. In some embodiments, the pharmaceutical composition is stored frozen.

[0126] Also provided are unit dosage forms of any of the populations of cells comprising γδ T cells produced using the methods described herein, or compositions thereof (such as pharmaceutical compositions). These unit dosage forms can be stored in suitable packaging in single or multiple unit dosage amounts, and may be sterilized and sealed. In some embodiments, the pharmaceutical composition is administered as a single dose. In some embodiments, the pharmaceutical composition is administered as multiple doses. In some embodiments, the pharmaceutical composition is administered once every four weeks, examples of which include about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, about once every ten weeks, about once every eleven weeks, about once every twelve weeks, about once every month, about once every two months, about once every three months, about once every four months, about once every five months, about once every six months, about once every seven months, about once every eight months, about once every nine months, about once every ten months, about once every eleven months, about once every 12 months, about once every thirteen months, about once every fourteen months, about once every fifteen months, about once every sixteen months, about once every seventeen months, about once every eighteen months, about once every year, about once every two years, about once every three years, or more. In some embodiments, the pharmaceutical composition is administered no more than once a year.

[0127] In some embodiments, the pharmaceutical composition is administered in combination with one or more therapies or within an overall treatment regimen using one or more therapies. In some embodiments, the pharmaceutical composition is administered simultaneously with one or more therapies. In some embodiments, the pharmaceutical composition is administered sequentially with one or more therapies. In some embodiments, the pharmaceutical composition is administered as a monotherapy. In some embodiments, the pharmaceutical composition is administered after one or more therapies for a disease or disorder have failed to treat or cure the individual.

[0128] In some embodiments, the pharmaceutical composition is administered to treat an individual diagnosed with or suspected of having cancer. In some embodiments, the pharmaceutical composition is administered in combination with or as part of an overall treatment regimen using one or more anti-cancer therapies. In some embodiments, the pharmaceutical composition and the one or more anti-cancer therapies are administered simultaneously. In some embodiments, the pharmaceutical composition and the one or more anti-cancer therapies are administered sequentially. In some embodiments, the one or more anti-cancer therapies are selected from chemotherapeutic agents, cytokines, immunotherapy, radiation therapy, anti-cancer vaccines, and therapeutic antibodies. For example, in some embodiments, the one or more anti-cancer therapies may include anti-PD-1, anti-PDL-1, and / or anti-PDL-2 monoclonal antibodies or biologically active fragments thereof. In some embodiments, the pharmaceutical composition is administered as adjuvant therapy after a surgical procedure to an individual diagnosed with cancer. In some embodiments, the cancer is a solid tumor malignancy. In some embodiments, the cancer is a liquid cancer, such as a blood cancer. In some embodiments, the cancer can range from early-stage to late-stage cancer.

[0129] In some embodiments, the pharmaceutical composition is administered to treat an individual diagnosed with or suspected of having an autoimmune disorder. In some embodiments, the pharmaceutical composition is administered in combination with or as part of an overall treatment regimen using one or more autoimmune treatments. In some embodiments, the pharmaceutical composition and the one or more autoimmune treatments are administered simultaneously. In some embodiments, the pharmaceutical composition and the one or more autoimmune treatments are administered sequentially. In some embodiments, the one or more autoimmune treatments are selected from corticosteroids, immunosuppressants including high doses, anti-inflammatory agents, and gene therapy agents. In some embodiments, the pharmaceutical composition and the corticosteroid or immunosuppressant are not administered simultaneously. In some embodiments, the pharmaceutical composition is administered as adjunctive therapy after an individual diagnosed with an autoimmune disorder has undergone a surgical procedure. In some embodiments, the individual may also use pain management agents or physical therapy.

[0130] In some embodiments, the pharmaceutical composition is administered for the prevention or treatment of an individual having or at risk of developing an infectious disease (such as a bacterial, fungal, viral, or parasitic disease). In some embodiments, the pharmaceutical composition is administered in combination with or within an overall treatment regimen using one or more infectious disease treatments, including prophylactic treatments. In some embodiments, the pharmaceutical composition and one or more infectious disease treatments are administered simultaneously. In some embodiments, the pharmaceutical composition and one or more infectious disease treatments are administered sequentially. In some embodiments, the one or more infectious disease treatments are administered up to 100 days before and / or after administration of the pharmaceutical composition described herein. In some embodiments, the infectious disease treatment, including prophylactic treatment, is selected from an antibiotic, an antiviral, an antifungal, or a vaccine.

[0131] In some embodiments, the pharmaceutical composition prevents, alleviates, ameliorates, treats, or cures any one of the diseases or disorders described herein. In some embodiments, the pharmaceutical composition does not induce GvHD. In some embodiments, the pharmaceutical composition does not induce cytokine release syndrome. In some embodiments, the pharmaceutical composition does not induce neurotoxicity.

[0132] C. Unit Dosage In some embodiments, the pharmaceutical compositions described herein contain about 1.0×10 5 ~1.0×10 10 For example, in some embodiments, the pharmaceutical compositions described herein are administered at a dose of about 4.0 x 10 cells / kg. 5 ~5.0×10 5 cells / kg, 4.5×10 5 ~5.5×10 5 cells / kg, 5.0×10 5 ~6.0×10 5 cells / kg, 5.5×10 5 ~6.5×10 5 cells / kg, 6.0×10 5 ~7.0×10 5cells / kg, 6.5×10 5 ~7.5×10 5 cells / kg, 7.0×10 5 ~8.0×10 5 cells / kg, 7.5×10 5 ~8.5×10 5 cells / kg, 8.0×10 5 ~9.0×10 5 cells / kg, 8.5×10 5 ~9.5×10 5 cells / kg, 9.0×10 5 ~1.0×10 6 In certain embodiments, the pharmaceutical composition is administered at a dose of about 7.0 x 10 cells / kg. 7 cells / kg, approximately 8.0×10 7 cells / kg, approximately 9.0×10 7 cells / kg, approximately 1.0×10 8 cells / kg, approximately 2.0×10 8 cells / kg, approximately 3.0×10 8 cells / kg, approximately 4.0×10 cells / kg, approximately 5.0×10 8 cells / kg, approximately 6.0×10 8 cells / kg, approximately 7.0×10 8 cells / kg, approximately 8.0×10 8 cells / kg, approximately 9.0×10 8 cells / kg, approximately 1.0×10 9 cells / kg, approximately 2.0×10 9 cells / kg, approximately 3.0×10 9 cells / kg, approximately 4.0×10 9 cells / kg, approximately 5.0×10 9 cells / kg, approximately 6.0×10 9 cells / kg, approximately 7.0×10 9 cells / kg, approximately 8.0×10 9 cells / kg, approximately 9.0×10 9 cells / kg or approximately 1.0 x 10 10 In one embodiment, the pharmaceutical composition is administered at a dose of about 7.5 x 10 cells / kg. 7 In certain embodiments, the pharmaceutical composition is administered at a dose of about 3.0-4.0 x 10 cells / kg. 8 In certain embodiments, the pharmaceutical composition is administered at a dose of about 3.5 to 4.5 x 10 cells.8 In certain embodiments, the pharmaceutical composition is administered at a dose of about 4.0 to 5.0 x 10 cells. 8 In certain embodiments, the pharmaceutical composition is administered at a dose of about 4.5 to 5.5 x 10 cells. 8 In certain embodiments, the pharmaceutical composition is administered at a dose of about 5.0 to 6.0 x 10 cells. 8 In certain embodiments, the pharmaceutical composition is administered at a dose of about 5.5 to 6.5 x 10 cells. 8 In certain embodiments, the pharmaceutical composition is administered at a dose of about 6.0 to 7.0 x 10 cells. 8 In certain embodiments, the pharmaceutical composition is administered at a dose of about 6.5 to 7.5 x 10 cells. 8 In certain embodiments, the pharmaceutical composition is administered at a dose of about 7.0 to 8.0 x 10 cells. 8 In certain embodiments, the pharmaceutical composition is administered at a dose of about 7.5 to 8.5 x 10 cells. 8 In certain embodiments, the pharmaceutical composition is administered at a dose of about 8.0 to 9.0 x 10 cells. 8 In certain embodiments, the pharmaceutical composition is administered at a dose of about 8.5 to 9.5 x 10 cells. 8 In certain embodiments, the pharmaceutical composition is administered at a dose of about 9.0 to 10.0 x 10 cells. 8 In certain embodiments, the pharmaceutical composition is administered at a dose of about 1.0 to 2.0 x 10 cells. 9 In certain embodiments, the pharmaceutical composition is administered at a dose of about 1.5 to 2.5 x 10 cells. 9 In certain embodiments, the pharmaceutical composition is administered at a dose of about 2.0 to 3.0 x 10 cells. 9 In certain embodiments, the pharmaceutical composition is administered at a dose of about 2.5 to 3.5 x 10 cells. 9 In certain embodiments, the pharmaceutical composition is administered at a dose of about 3.0 to 4.0 x 10 cells. 9 In certain embodiments, the pharmaceutical composition is administered at a dose of about 3.5 to 4.5 x 10 cells. 9 In certain embodiments, the pharmaceutical composition is administered at a dose of about 4.0 to 5.0 x 10 cells. 9 In certain embodiments, the pharmaceutical composition is administered at a dose of about 4.5 to 5.5 x 10 cells. 9In certain embodiments, the pharmaceutical composition is administered at a dose of about 5.0 to 6.0 x 10 cells. 9 In certain embodiments, the pharmaceutical composition is administered at a dose of about 5.5 to 6.5 x 10 cells. 9 In certain embodiments, the pharmaceutical composition is administered at a dose of about 6.0 to 7.0 x 10 cells. 9 In certain embodiments, the pharmaceutical composition is administered at a dose of about 6.5 to 7.5 x 10 cells. 9 In certain embodiments, the pharmaceutical composition is administered at a dose of about 7.0 to 8.0 x 10 cells. 9 In certain embodiments, the pharmaceutical composition is administered at a dose of about 7.5 to 8.5 x 10 cells. 9 In certain embodiments, the pharmaceutical composition is administered at a dose of about 8.0 to 9.0 x 10 cells. 9 In certain embodiments, the pharmaceutical composition is administered at a dose of about 8.5 to 9.5 x 10 cells. 9 In certain embodiments, the pharmaceutical composition is administered at a dose of about 9.0 to 10.0 x 10 cells. 9 The cells are administered in doses.

[0133] In some embodiments, the pharmaceutical compositions described herein are administered to a subject (such as a human patient) in an amount of about 1.0 x 10 5 ~1.0×10 10 For example, in some embodiments, the pharmaceutical compositions described herein are administered at a dose that results in a cell count of about 4.0 x 10 cells / kg in a subject (e.g., a human patient). 5 ~5.0×10 5 cells / kg, 4.5×10 5 ~5.5×10 5 cells / kg, 5.0×10 5 ~6.0×10 5 cells / kg, 5.5×10 5 ~6.5×10 5 cells / kg, 6.0×10 5 ~7.0×10 5 cells / kg, 6.5×10 5 ~7.5×10 5 cells / kg, 7.0×10 5 ~8.0×10 5 cells / kg, 7.5×10 5~8.5×10 5 cells / kg, 8.0×10 5 ~9.0×10 5 cells / kg, 8.5×10 5 ~9.5×10 5 cells / kg, 9.0×10 5 ~1.0×10 6 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 7.0 x 10 cells / kg in a subject (such as a human patient). 7 cells / kg, approximately 8.0×10 7 cells / kg, approximately 9.0×10 7 cells / kg, approximately 1.0×10 8 cells / kg, approximately 2.0×10 8 cells / kg, approximately 3.0×10 8 cells / kg, approximately 4.0×10 cells / kg, approximately 5.0×10 8 cells / kg, approximately 6.0×10 8 cells / kg, approximately 7.0×10 8 cells / kg, approximately 8.0×10 8 cells / kg, approximately 9.0×10 8 cells / kg, approximately 1.0×10 9 cells / kg, approximately 2.0×10 9 cells / kg, approximately 3.0×10 9 cells / kg, approximately 4.0×10 9 cells / kg, approximately 5.0×10 9 cells / kg, approximately 6.0×10 9 cells / kg, approximately 7.0×10 9 cells / kg, approximately 8.0×10 9 cells / kg, approximately 9.0×10 9 cells / kg or approximately 1.0 x 10 10 In one embodiment, the pharmaceutical composition is administered at a dose that results in a cell count of about 7.5 x 10 cells / kg in a subject (such as a human patient). 7 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell density of about 3.0 to 4.0 x 10 cells / kg in a subject (e.g., a human patient). 8 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell population of about 3.5 to 4.5 x 10 cells in a subject (e.g., a human patient). 8In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 4.0 to 5.0 x 10 cells in a subject (e.g., a human patient). 8 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell population of about 4.5 to 5.5 x 10 cells in a subject (e.g., a human patient). 8 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 5.0 to 6.0 x 10 cells in a subject (e.g., a human patient). 8 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 5.5 to 6.5 x 10 cells in a subject (e.g., a human patient). 8 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 6.0 to 7.0 x 10 cells in a subject (e.g., a human patient). 8 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell population of about 6.5 to 7.5 x 10 cells in a subject (e.g., a human patient). 8 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 7.0 to 8.0 x 10 cells in a subject (e.g., a human patient). 8 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 7.5 to 8.5 x 10 cells in a subject (e.g., a human patient). 8 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 8.0 to 9.0 x 10 cells in a subject (e.g., a human patient). 8 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 8.5 to 9.5 x 10 cells in a subject (e.g., a human patient). 8 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 9.0 to 10.0 x 10 in a subject (e.g., a human patient). 8 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 1.0 to 2.0 x 10 cells in a subject (e.g., a human patient). 9 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell population of about 1.5 to 2.5 x 10 cells in a subject (e.g., a human patient). 9 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 2.0 to 3.0 x 10 cells in a subject (e.g., a human patient). 9In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell population of about 2.5 to 3.5 x 10 cells in a subject (e.g., a human patient). 9 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 3.0 to 4.0 x 10 cells in a subject (e.g., a human patient). 9 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell population of about 3.5 to 4.5 x 10 cells in a subject (e.g., a human patient). 9 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 4.0 to 5.0 x 10 cells in a subject (e.g., a human patient). 9 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell population of about 4.5 to 5.5 x 10 cells in a subject (e.g., a human patient). 9 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 5.0 to 6.0 x 10 cells in a subject (e.g., a human patient). 9 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 5.5 to 6.5 x 10 cells in a subject (e.g., a human patient). 9 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 6.0 to 7.0 x 10 cells in a subject (e.g., a human patient). 9 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell population of about 6.5 to 7.5 x 10 cells in a subject (e.g., a human patient). 9 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 7.0 to 8.0 x 10 cells in a subject (e.g., a human patient). 9 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 7.5 to 8.5 x 10 cells in a subject (e.g., a human patient). 9 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 8.0 to 9.0 x 10 cells in a subject (e.g., a human patient). 9 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 8.5 to 9.5 x 10 cells in a subject (e.g., a human patient). 9 In certain embodiments, the pharmaceutical composition is administered at a dose that results in a cell count of about 9.0 to 10.0 x 10 in a subject (e.g., a human patient). 9The cells are administered in a dose that results.

[0134] In some cases, the subject methods include administering an effective amount of a pharmaceutical composition described herein to a subject (e.g., a human patient) in need thereof. In some embodiments, an "effective amount" of a pharmaceutical composition is an amount that, when administered to a subject (e.g., a human patient) in one or more doses in monotherapy or combination therapy, is effective to reduce symptoms of a disease (e.g., cancer) in a subject (e.g., a human patient) by at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold, compared to a subject (e.g., a human patient) not treated with the pharmaceutical composition.

[0135] In some embodiments, the kits and / or compositions comprise a population of cells comprising γδ T cells as described herein. In some embodiments, the kits may further comprise instruction(s) on how to use the composition, such as the methods of use described herein. The kits described herein may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, intravenous or dropper equipment, and package inserts containing instructions for carrying out any of the methods described herein.

[0136] In some embodiments, the kits and / or compositions comprise culture medium for use in the methods provided herein, whether provided as individual components, in any combination, or as culture medium mixed with cells. In some embodiments, the kits include reagents suitable for growing cells, examples of which include media, cytokines, ITSEA, human albumin serum. In some embodiments, the kits and / or compositions comprise culture medium for use in the methods provided herein mixed with PBMCs or derivatives thereof (e.g., intermediate compositions of the methods provided herein).

[0137] In some embodiments, the kit comprises one or more instruments for effecting transduction of a population of cells (such as PBMCs (or derivatives thereof) or γδ T cells) to express a transgene, such as a chimeric antigen receptor (CAR) construct (such instruments may be syringes, pipettes, forceps, and / or any such medically approved device). In some embodiments, the kit comprises reagents or devices for transduction of PBMCs (or derivatives thereof) or γδ T cells.

[0138] The components of the kit may be packaged either in aqueous medium or in lyophilized form. The container means of the kit will include at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit will generally also contain second, third, or other additional containers into which the additional components may be placed separately, although vials may contain various combinations of components. The kits of the present invention will also typically include a means for containing the population of cells (such as PBMCs (or derivatives thereof) or γδ T cells) and any other reagent containers in close confinement for commercial sale. Such containers may include, for example, injection- or blow-molded plastic containers into which the desired vials are retained.

[0139] In some embodiments, the kit may further include instructions or instructions regarding how to make or use the population of cells or composition thereof, examples of which include the methods of making or using described herein.

[0140] V. How to use In one aspect, a method of treating a disease in an individual is provided. The method includes obtaining an isolated pool of mixed immune cells depleted of αβ T cells (e.g., isolated PCMCs depleted of αβ T cells) or a derivative thereof from a donor individual, producing a population of cells comprising γδ T cells using a method described herein, and administering the population of cells comprising γδ T cells to the individual. In some embodiments, the donor individual is not the individual (e.g., in an allogeneic cell therapy setting). In some embodiments, the donor individual is an individual (e.g., in an autologous cell therapy setting).

[0141] In some embodiments, the disease is selected from any one of cancer, an infectious disease, an autoimmune disease, or another disease involving immune function (e.g., endometriosis). In some embodiments, the disease is cancer.

[0142] The method is applicable to cancers at any stage, including early stage, non-metastatic, primary, advanced, locally advanced, metastatic, or in remission. In some embodiments, the cancer has been refractory to previous therapies. In some embodiments, the cancer may be a solid tumor malignancy. In some embodiments, the cancer may be a liquid tumor. In some embodiments, the malignancy is acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, Kaposi's sarcoma (soft tissue sarcoma), AIDS-related lymphoma (lymphoma), primary central nervous system lymphoma (lymphoma), anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma of the skin, bile duct cancer, bladder cancer, bone cancer (including Ewing's sarcoma, osteosarcoma, and malignant fibrous histiocytoma), brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoma Id tumors, carcinomas, cardiac tumors, atypical teratoid / rhabdoid tumors, medulloblastoma, germ cell tumors, primary central nervous system lymphoma, cervical cancer, bile duct carcinoma, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma in situ (DCIS), endometrial cancer, ependymoma, esophageal cancer, nasal neuroblastoma, Ewing's sarcoma, extracranial germ cell tumors, extragonadal germ cell tumors, intraocular melanoma, retinoblastoma, oocyte Ductal carcinoma, osteofibrous histiocytoma, osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST) (soft tissue sarcoma), germ cell tumor, central nervous system germ cell tumor, extracranial germ cell tumor, extragonadal germ cell tumor, ovarian germ cell tumor, testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, histiocytosis (Langerhans cells), Hodgkin's lymphoma, hypopharyngeal carcinoma, islet cell tumor, pancreatic neuroendocrine tumor, Kaposi's sarcoma (soft tissue sarcoma), renal cell carcinoma, Langerhans Cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (non-small cell carcinoma, small cell carcinoma, pleuropulmonary blastoma, tracheobronchial tumor), lymphoma, male breast cancer, malignant fibrous histiocytoma and osteosarcoma of bone, Merkel cell carcinoma, mesothelioma, metastatic cancer, metastatic squamous cell neck cancer of unknown primary, midline carcinoma with NUT gene alteration, oropharyngeal cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides (lymphoma), myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm,Chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic myeloproliferative neoplasms, acute B-lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, blastic plasmacytoid dendritic cell neoplasm (BPDCN), systemic mastocytosis, nasal and paranasal cavity cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal and paranasal cavity cancer The cancer may include, but is not limited to, any one of the following: nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, primary peritoneal cancer, prostate cancer, recurrent carcinoma, rhabdomyosarcoma, salivary gland cancer, vascular tumor, small intestine cancer, soft tissue sarcoma, T-cell lymphoma, thymoma and thymic carcinoma, transitional cell carcinoma of the renal pelvis and ureter, vaginal cancer, vulvar cancer, or Wilms' tumor.

[0143] In some embodiments, the population of cells comprising γδ T cells produced using the methods described herein further comprises a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antigen binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain comprising a costimulatory domain and / or a primary signaling domain. In some embodiments, the population of cells comprising γδ T cells produced using the methods described herein further comprises an engineered T cell receptor (TCR). In some embodiments, the engineered TCR comprises an engineered antigen binding domain. In some embodiments, the CAR or engineered TCR antigen binding domain is selected from the group consisting of mesothelin, EGFRvIII, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, GD2, GD3, BCMA, TROP-2, Tn Ag, prostate-specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, interleukin-11 receptor α (IL-11Ra), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor receptor β (PDGFR-β), SSEA-4, CD20, folate receptor α (FRa), ERBB2 (Her2 / neu), HER3, MUC1, epidermal growth factor receptor (EGFR), TGF-β, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF -I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl-GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, CLDN6, CLDN18, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut It binds to a tumor antigen selected from the group consisting of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD5, CD7, nectin-4, PD-L1, HLA-E, HLA-G, MICA / B, AKR1C3, or ULBP1-6.

[0144] In some embodiments, the transmembrane domain of the CAR can be selected from the group consisting of CD8a, CD4, CD28, CD45, NKG2D, NKp30, NKp44, NKp46, DNAM1, 2B4, PD1, and CD152. In some embodiments, the intracellular signaling domain of the CAR can be selected from the group consisting of CD3, CD27, CD28, DNAM1, 2B4, DAP10, DAP12, CD54 (ICAM), CD134 (OX40), CD137 (41BB), CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), and CD278 (ICOS). In some embodiments, the hinge domain of the CAR can be selected from the group consisting of CD8, CD4, CD28, IgG, and CD34.

[0145] In some embodiments, the disease is an infectious disease, examples of which include bacterial, viral, fungal, or parasitic infections, hi some embodiments, the infectious disease is chronic.

[0146] Examples of bacterial infections include Mycobacteria (e.g., Mycobacterium tuberculosis, Mycobacterium leprae), Brucella (e.g., Brucella abortus), Corynebacterium (e.g., Corynebacterium diphtheriae), Vibrio (e.g., Vibrio cholerae), Bordetella (e.g., Bordetella pertussis), Clostridium (e.g., Clostridium tetani, Clostridium botulinum, and Clostridium difficile), Yersinia (e.g., Yersinia pestis, Yersinia enterocolitica), Neisseria (e.g., Neisseria gonorrhoeae, Neisseria meningitidis, etc.), Treponema (e.g., Treponema pallidum), Chlamydophila (Chlamydia pneumoniae and Chlamydia trachomatis), Aeromonas (Aeromonas kyphimurium), Examples of bacteria that may be present include, but are not limited to, Salmonella (e.g., Salmonella enterica spp., Salmonella enterica spp.), Legionella (e.g., Legionella pneumophila), Bacillus (e.g., Bacillus anthracis and Bacillus cereus), Leptospira (e.g., Leptospira species), Klebsiella (e.g., Klebsiella pneumoniae), Streptococcus (e.g., Streptococcus pneumoniae), Listeria (e.g., Listeria monocytogenes), Pseudomonas (e.g., Pseudomonas aeruginosa), Staphylococcus (e.g., Staphylococcus aureus), or Escherichia (e.g., Escherichia coli).

[0147] Examples of viral infections in mammals include, but are not limited to, infections caused by DNA viruses (e.g., herpesviruses such as herpes simplex virus, Epstein-Barr virus, and cytomegalovirus; poxviruses such as smallpox virus); hepadnaviruses (e.g., hepatitis B virus, papillomavirus, and adenovirus); RNA viruses (e.g., HIV I and II; HTLV I and II; poliovirus; hepatitis A; orthomyxoviruses (e.g., influenza virus); paramyxoviruses (e.g., measles virus); rabies virus; and hepatitis C); coronavirus (the cause of severe acute respiratory syndrome (SARS)), rhinovirus, respiratory syncytial virus, norovirus, West Nile virus, yellow fever, Rift Valley virus, Lassa fever virus, Ebola virus, and lymphocytic choriomeningitis virus).

[0148] Examples of fungal infections include Candida (e.g., Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida lusitaniae, Candida pseudotropicalis, Candida rugosa, Candida stellatoidea, Candida parapsilosis, Candida tropicalis, Candida auris), Cryptococcus (e.g., Cryptococcus neoformans), Aspergillus (e.g., Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus nidulans), Coccidioides (e.g., Coccidioides immitis and Coccidioides posadasii, also known as valley fever), Histoplasma (e.g., Histoplasma capsulatum), Blastomyces (e.g., Blastomyces dermatitidis), Pneumocystis (e.g., Pneumocystis jirovecii), Penicillium (e.g., Penicillium marneffei), Mucorales (e.g., Lithium Rhizopus oryzae, Rhizopus arrhizus, Rhizopus rhizopodiformis, Rhizomucor pusillus, Absidia oryzae, Absidia corymbifera, Absidia ramosa and Mucor circinelloides), Sporothrix (e.g., Sporothrix schenckii), Fusarium (e.g., Fusarium solanii, Fusarium oxysporum, Fusarium moniliforme, Fusarium zimelum, Fusarium cladosporium and Fusarium anthophyllum), Trichosporum These include, but are not limited to, infections caused by Trichosporon beigelii and Trichosporon capitatum, Geotrichum (e.g., Geotrichum candidum), Rhodotorula (e.g., Rhodotorula rubra), or black mold or pheohyphomycete (e.g., Pseudoalescheria boyzii (Scedosporium apiospermum), Bipolaris spp., Alternaria, and Scedosporium prolificans).

[0149] Examples of parasitic infections include Acanthamoeba (such as Acanthamoeba keratitis, granulomatous amebic encephalitis, or disseminated infections), African sleeping sickness or sleeping sickness (e.g., Trypanosoma brucei), echinococcosis (e.g., Echinococcus granulosus or Echinococcus multilocularis), amebic dysentery (e.g., Entamoeba histolytica), Chagas' disease (e.g., Trypanosoma cruzi), hookworms (e.g., Ancylostoma brasiliensis, Ancylostoma canis, Ancylostoma ceylanicum, and Ancylostoma stenocephalus), angiostrongyliasis (e.g., Angiostrongylus cantonensis), anisakiasis (e.g., Pseudoterranova decipiens larvae), ascariasis (e.g., Ascariasis lumbricoides), or Ascaris suum), babesiosis (e.g., Babesia microti), balantidiosis (e.g., Balantidium coli), balamuthiasis (e.g., Balamuthia mandrillus), raccoon ascariasis (e.g., Roundworm), bed bugs, schistosomiasis (such as Schistosoma mansoni, Schistosoma haematobium, or Schistosoma japonicum), Blastocystis hominis infection, body louse infestation, capillary disease (e.g., Capillaria hepatica or Capillaria philippinarum), cercarial dermatitis (e.g., Austrobilharzia varigrandis),variglandis), non-pathogenic intestinal protozoa (such as Meniere's flagellate, Amoeba diminuta, Amoeba coli, Amoeba dispar, Amoeba Hartmannii, Amoeba porrecchiata, and Amoeba iodoide), Clonorchiasis or liver flukes (such as Clonorchis sativus, Clonorchis viverrini, or Clonorchis cati), pubic lice, cryptosporidiosis (such as Cryptosporidium), cyclosporiasis (such as Cyclospora caetanensis) cis), cysticercosis (e.g., Taenia solium), cystoisosporiasis or isospora infection (e.g., Cystoisospora warblers), dientameba infection, diphyllobothriasis (e.g., Dirobothria lata), Dirobothriasis gonorrhea or canine / feline tapeworm, dirofilariasis (e.g., Dirobothria canis), dracunculiasis or Guinea worm disease (Dracunculiasis guinea worm), elephantiasis or filariasis (e.g., Wuchereria bancrofti or Brugia malayi), pinworm infection or wormworm worms (human pinworms), fascioliasis or common liver flukes (e.g., Fasciola hepatica or Fasciola gigantica), trematodiasis (e.g., Fasciola gigantica), giardiasis (e.g., Giardia lamblia), gnathostomiasis (e.g., Gnathostomiasis), heterophyiasis (e.g., Heterophagia heterophyia), hymenocoeliasis (e.g., Dimorpha taeniasis), common foodborne parasites (protozoa such as Cryptosporidium spp., Giardia lamblia, Cyclospora caetanensis and Toxoplasma gondii), roundworms (e.g. Trichinella and Anisakis), tapeworms (e.g. Diphyllobothria and Taenia), leishmaniasis (e.g. Leishmania promastigote), loiasis or African eyeworm disease (e.g. Loa loa), malaria (e.g. Plasmodium), microsporidiosis (e.g. Enterocytozoon bienoussi, E. cuniculi, E. herem, E. intestinalis, Ancaria algerae, algerae, A. connori, A. vesicularum, Microsporidium ceylonensis, Mycobacterium africanum, Trachipleistophora hominis, T. anthropophthera, Nosema ocularum, Pleistophora longnaephiaronneafiei, Vittaforma cornae or Tubulinosema acridofagus acridophagus), mite infestation (e.g., Sarcoptic mange), myiasis or fly maggot infection (e.g., Dermatophytidae), Naegleria infection (e.g., Naegleria fowleri), toxocariasis (e.g., Toxocara canis or Toxocara catis), onchocerciasis or river blindness (e.g., Onchocerciasis volvulus), Paragonimiasis or pulmonary fluke (e.g., Paragonimus westermani), Pneumocystis jirovecii pneumonia, Sappinia amoeba (e.g., Sappinia diploidea or Sappinia pedata), sarcocystis (e.g., Sarcocystis), strongyloidiasis (e.g., Strongyloides stercoralis), trichinosis (e.g., Trichinella spiralis), trichomoniasis (e.g., Trichomonas vaginalis), trichuriasis or whipworm (e.g., Trichuris trichiura), and the like.

[0150] In some embodiments, the disease is an autoimmune disorder. Examples of autoimmune disorders include graft-versus-host disease, autoimmune polyglandular syndrome, type 1 diabetes mellitus (TIDM), autoimmune gastritis, autoimmune uveoretinitis, autoimmune vasculitis, colitis, thyroiditis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune hepatitis, inner ear autoimmune diseases, axonal and neuronal neuropathy, Behcet's disease, bullous pemphigoid, Castleman's disease, celiac disease, Chagas' disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing polymyelitis, Churg-Strauss, cicatricial pemphigoid / benign mucous membrane pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, Devic's disease, discoid rash, Dressler's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, polyangiogenic granulomatosis, Graves' disease, Guillain-Barré syndrome group, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes gestationis or pemphigoid of pregnancy, hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis, Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus and atrophic psoriasis, lignified conjunctivitis, linear IgA disease, lupus, Lyme disease, chronic Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, Mooren's ulcer, Much-Habermann disease, multiple sclerosis, myasthenia gravis Myositis, narcolepsy, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism, PANDAS (pediatric autoimmune streptococcal neuropsychiatric disorders), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis,These conditions include, but are not limited to, progestational dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia, pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome, subacute bacterial endocarditis, Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis (polyangiogenic granulomatosis).

[0151] VI. Working Examples The following examples are intended to be purely illustrative of the present invention and therefore should not be considered as limiting in any way. The following examples and detailed descriptions are provided for illustrative purposes, not limiting. For embodiments in which experimental methods are not described in detail, such methods are carried out according to conventional conditions, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0152] A. Example 1: Expansion of γδ T Cells in Vitro Herein, we describe a novel method used to generate pure γδ T cell populations in vitro. To obtain the starting cell population, human peripheral blood cells (hPBMCs) were collected by apheresis. These collected cells were then stained with an anti-TCR α / β antibody (Miltenyi Biotec), followed by depletion of antibody-conjugated αβ TCR+ T cells using the CliniMACS plus system (Miltenyi Biotec). The remaining cells were cryopreserved for future γδ T cell expansion.

[0153] To expand γδ T cells, αβ TCR-depleted cells were thawed and cultured for 14 days in medium containing the following components: ·Basal medium: NK MACS medium (Miltenyi Biotec) IL-15 (10 ng / mL) Human AB serum (1%) ·ITSEA (outlined in Table 1 below). [Table 1]

[0154] The medium was refreshed either once on day 7 or twice on days 7 and 11 (or 12) by replacing approximately 80% of the old medium with an equal volume of new medium. γδ T cells were cultured in vitro for approximately 14 days. During cell culture, recombinant human IL-15 was added to the cell culture medium once every 2–3 days. Fresh ITSEA was added to the cell culture medium on day 7 or when the medium was refreshed on days 7 and 11. Cells were harvested on day 14 and cryopreserved.

[0155] Results showed that culturing αβ TCR-depleted cells in basal medium supplemented with IL-15 and ITSEA for 14 days led to a >2000-fold expansion of γδ T cells (Figure 2A), of which approximately 90% remained viable (Figure 1A) and had a purity of approximately 95% across two separate test runs (Figure 3A). As shown in Figure 1B, the majority of CD3+ T cells were vδ2 γδ T cells (>90%), with approximately 5% being vδ1 γδ T cells. This protocol is not only effective and efficient, but also consistent, which is crucial for scaling to meet the demands of downstream applications. These findings are significant when considering the expansion achieved using comparable methods. That is, for example, Dokouhaki et al. (2010) Cancer Lett 297(1): 126-136;Van Acker et al. (2016) J Hematol Oncol 9(1): 101;Li et al. (2010) J Immunother 33(3): 287-296;Salot et al. (2007) J Immunol Methods 326(1-2): 63-75; and Kondo et al. (2008) Cytotherapy 10(8): 842-856.

[0156] To stimulate γδ T cell proliferation, we tested the addition of IL-15 and ITSEA, either alone or in combination, to basal culture medium in the presence of 1% human AB serum. As shown in Figures 2A-2C, each single component failed to significantly expand γδ T cells or generate pure γδ T cells. Combining IL-15 and ITSEA in the above cell culture medium dramatically increased γδ T cell proliferation and achieved high γδ T purity in the final cell product. As shown in Figure 2A, in γδ T cells co-treated with IL-15 and ITSEA, proliferation increased from approximately 300-fold with IL-15 alone to approximately 2500-fold with the combination treatment. Figure 2B shows that NK cell proliferation in samples treated with IL-15 and ITSEA (approximately 10-fold) significantly decreased compared to IL-15 alone (approximately 60-fold). Figure 2C shows the lack of proliferation of CD3-CD56- cells (non-γδ T cells and non-NK cells).

[0157] Figure 3A shows that approximately 95% of the cells produced by culture with IL-15 and ITSEA treatment were γδ T cells. Figure 3B shows that NK cells predominate in the cell mixture when cultured with IL-15 alone, whereas NK cells account for approximately 5% of the total cell population when cultured with IL-15 and ITSEA. Figure 3C shows that non-γδ T cells and non-NK cells account for a significant proportion of the cell population when cultured with ITSEA alone, but are clearly absent when cultured with IL-15 and ITSEA in combination. These results indicate a high purity of γδ T cells when treated with IL-15 and ITSEA.

[0158] These results demonstrate that this protocol is effective in rapidly generating pure γδ T cell populations from a starting αβ TCR-depleted cell population, and is consistently achieved across multiple studies.

[0159] B. Example 2: Cellular Phenotype of In Vitro Expanded γδ T Cells γδ T cells were also tested for in vitro antitumor activity in 2D (Figures 4A-5B) and 3D (Figures 5A-5B) conditions. Briefly, in 2D assays, luciferase-expressing SKOV3 and A549 cancer cell lines were pretreated with vehicle or zoledronic acid (Zol) to enhance antigen expression within tumor cells. These cancer cell lines were then co-cultured with γδ T cells at an effector to target ratio (E / T) of 4:1 in the presence of 10 ng / mL recombinant human IL-15 (rhIL-15). In 3D assays, fluorescent protein-tagged SKOV3 and A549 tumor cells were seeded in 3D culture to allow tumor formation. Two days later, γδ T cells were added with 10 ng / mL rhIL-15 at an E / T ratio of 4:1.

[0160] In the 2D assay, cytotoxicity (% specific lysis) was measured on days 1 and 3 based on the disappearance of the luciferase signal. γδ T cells induced SKOV3 (Figure 4A) and A549 (Figure 4B) tumor cell death in a time-dependent manner. Pretreatment of tumor cells with zoledronic acid dramatically enhanced γδ T cell-mediated tumor cell killing to nearly 100% on day 3.

[0161] In the 3D assay, the fluorescence intensity, which indicates tumor mass, was measured on days 3 and 6. The results suggest that under 3D conditions, γδ T cells exhibited potent antitumor activity and completely destroyed SKOV3 (Fig. 5A) and A549 (Fig. 5B) tumor masses, even in the absence of zoledronic acid pretreatment, as shown by the almost complete disappearance of tumor masses in Figures 5A-B.

[0162] C. Example 3: Use of γδ T Cells for CAR-T Cell Therapy in an In Vitro Cancer Cell Co-culture Model γδ T cells were generated as described in Example 1 above. As shown in the schematic diagram in Figure 6A, cells were cultured for 4 days before viral transduction with a CAR construct targeting TROP-2. The light and heavy chain coding sequences of a TROP2 monoclonal antibody (hRS7) were converted into single-chain variable fragments (scFv) and inserted into a retroviral vector upstream of the remaining CAR domains (CD8 hinge, CD8 transmembrane domain (TM), intracellular CD28 costimulatory domain (CD28 ICD), and intracellular CD3 activation domain (CD3ζ)). The CAR-expressing retroviral vector was transfected into a retroviral packaging cell line to produce viral media, which were then used to transduce αβ TCR-depleted cells on day 4 of in vitro cell culture.

[0163] Transduction method 1: Cells were harvested on day 4 and retroviral transduction was performed by spinoculation. The viral medium was filled into a retronectin-coated cell culture bag. The bag was centrifuged at 2000G for 2 hours at 32°C. The virus-containing medium was then removed. Cells were added to γδ T cell medium (described in Example 1) and added to the virus-coated bag. The bag was then centrifuged at 1000G for 10 minutes at 32°C and then incubated at 37°C for 2 days.

[0164] Transduction method 2: Retroviral transduction was also performed using static incubation. Viral medium was loaded into a retronectin-coated cell culture bag. Without centrifugation, cells were added to γδ T cell medium (described in Example 1) and added to the virus-loaded bag. The bag was incubated at 37°C, followed by incubation at 37°C for 2 days.

[0165] On day 6, cells were harvested and replated in γδ T cell medium (described in Example 1) for cell expansion. The medium was refreshed once on day 11 and contained fresh ITSEA. Cells were cultured for an additional 3 days, for a total of 14 days. 10 ng / mL rhIL-15 was added every 2–3 days. On day 14, cells were harvested and cryopreserved.

[0166] Tumor killing assay: To evaluate the targeted cytotoxicity of γδ CAR-T cells, γδ CAR-T cells and untransduced control γδ T cells were cocultured with SKOV3 or A549 cancer cells. SKOV3 cancer cells express the TROP-2 antigen, which the CAR was engineered to recognize. In contrast, A549 cancer cells do not express the TROP-2 antigen. Both SKOV3 and A549 cell lines express luciferase, which was used to calculate the specific lysis of target cancer cells by γδ CAR-T cells.

[0167] As shown in Figure 6B, retroviral transduction yields a transduction rate of over 50% with both transduction approaches. Regardless of the transduction method, transduced γδ T cells achieved over 1000-fold expansion by day 14 (Figure 6C). When γδ CAR-T cells or non-transduced (NT) γδ T cells were cocultured with SKOV3 or A549 cancer cells (described above in Example 2), γδ CAR-T cells, but not NT γδ T cells, exhibited increased cancer cell killing specifically against TROP2-positive (TROP2+) SKOV3 tumor cells (Figure 6D) but not against TROP2-negative (TROP2-) A549 cells (Figure 6E) in a time-dependent manner, demonstrating successful targeting and cytotoxic activity against cancers expressing a specific antigen (i.e., TROP-2).

[0168] The above results confirm that the present invention provides a novel feeder cell-free and antibody-free approach (e.g., a γδ TCR-stimulating antibody-free approach) for expanding γδ T cells, which can further include a process for engineering γδ T cells to generate, for example, γδ CAR-T cells. The present invention eliminates extra manufacturing steps for feeder cells and activating antibodies, eliminates QA / QC steps to verify feeder cell content in the final product, and eliminates safety concerns regarding the introduction of residual feeder cells into human patients. The final product is highly pure γδ T cells. As a result, enrichment or isolation steps for γδ T cells after successful cell expansion are eliminated. No αβ T cell contamination was observed in the final cell product, further minimizing the potential patient risk of developing GvHD when administered as an allogeneic cell therapy.

[0169] γδ T cells demonstrated potent antitumor activity in 2D and 3D cell culture models. Moreover, the γδ T cell manufacturing protocol was compatible with cell engineering methods, such as retroviral transduction. γδ CAR-T cells generated by the γδ T cell protocol exhibited significantly improved tumor cell killing capacity compared with unmodified γδ T cells.

[0170] Taken together, these results indicate that the present invention may be used to produce highly pure and active γδ T cells and γδ CAR-T cells that can be used for further downstream applications, such as allogeneic or autologous cell therapy for patients with various diseases.

[0171] All references mentioned in this specification are incorporated herein by reference as if each of those references were individually incorporated by reference. Although the description refers to specific embodiments, it will be apparent to those skilled in the art that the present invention can be practiced with variations of these specific details. Therefore, the present invention should not be construed as being limited to the embodiments shown herein.

Claims

1. 1. A method for producing a population of cells comprising γδ T cells, comprising: Culturing the isolated pool of αβ T cell-depleted mixed immune cells or a derivative thereof in a culture medium, said culture medium comprising: (a) a basal medium; (b) a cytokine; and (c)(i) metabolic polypeptides; (ii) iron source; (iii) antioxidant enzyme cofactor; (iv) a lipid precursor, and (v) carrier protein and one or more of: and harvesting said population of cells comprising γδ T cells.

2. 2. The method of claim 1, wherein the population of cells comprising γδ T cells has a γδ T cell purity of at least about 85%.

3. 3. The method of claim 1 or 2, wherein the population of cells comprising γδ T cells has a γδ T cell purity of at least about 95%.

4. The method of any one of claims 1 to 3, wherein the population of cells comprising γδ T cells comprises vδ2 γδ T cells and optionally vδ1 γδ T cells.

5. The method of any one of claims 1 to 4, wherein the population of cells comprising γδ T cells has a purity of vδ2 γδ T cells relative to total γδ T cells in the population of at least about 70%.

6. The method of any one of claims 1 to 5, wherein the population of cells comprising γδ T cells has a purity of at least about 95% vδ2 γδ T cells relative to total γδ T cells in the population.

7. The method of any one of claims 1 to 6, wherein the population of cells comprising γδ T cells comprises less than about 15% of NK cells.

8. The method of any one of claims 1 to 7, wherein the population of cells comprising γδ T cells comprises less than about 2.5% NK cells.

9. The method according to any one of claims 1 to 8, wherein the basal culture medium is NK MACS medium.

10. 10. The method of any one of claims 1 to 9, wherein the cytokine is one or more of IL-15, IL-2, IL-7, SCF, TPO, FLT-3L, BMP4, VEGF or bFGF.

11. The method of claim 10, wherein the cytokine is IL-15.

12. The method of any one of claims 1 to 11, wherein the cytokine is added to the culture medium in an amount of about 100 pg / mL to about 500 ng / mL.

13. The method of any one of claims 1 to 12, wherein the metabolic polypeptide is insulin or an analog thereof.

14. 14. The method of claim 13, wherein the insulin is human insulin.

15. The method of any one of claims 1 to 14, wherein the metabolic polypeptide is added to the culture medium in an amount of about 1 mg / L to about 50 mg / L.

16. The method of any one of claims 1 to 15, wherein the iron source is transferrin.

17. 17. The method of claim 16, wherein the transferrin is human transferrin.

18. 18. The method of any one of claims 1 to 17, wherein the iron source is added to the culture medium in an amount of about 1 mg / L to about 50 mg / L.

19. The method of any one of claims 1 to 18, wherein the antioxidant enzyme cofactor is a selenium compound.

20. 20. The method of claim 19, wherein the selenium compound is sodium selenite.

21. 21. The method of any one of claims 1 to 20, wherein the antioxidant enzyme cofactor is added to the culture medium in an amount of about 0.001 mg / L to about 0.1 mg / L.

22. 22. The method of any one of claims 1 to 21, wherein the lipid precursor is ethanolamine.

23. 23. The method of any one of claims 1 to 22, wherein the lipid precursor is added to the culture medium in an amount of about 1 mg / L to about 10 mg / L.

24. The method of any one of claims 1 to 23, wherein the carrier protein is albumin.

25. 25. The method of claim 24, wherein the albumin is human albumin.

26. 26. The method of claim 25, wherein the human albumin is human serum albumin.

27. 27. The method of any one of claims 1 to 26, wherein the carrier protein is added to the culture medium in an amount of about 100 mg / L to about 5,000 mg / L.

28. The culture medium comprises: (i) the metabolic polypeptide; (ii) the iron source; (iii) the antioxidant enzyme cofactor; (iv) the lipid precursor, and (v) the carrier protein 28. The method of any one of claims 1 to 27, comprising two or more of:

29. The culture medium comprises: (i) the metabolic polypeptide; (ii) the iron source; (iii) the antioxidant enzyme cofactor; (iv) the lipid precursor, and (v) the carrier protein 29. The method of any one of claims 1 to 28, comprising three or more of:

30. The culture medium comprises: (i) the metabolic polypeptide; (ii) the iron source; (iii) the antioxidant enzyme cofactor; (iv) the lipid precursor, and (v) the carrier protein 30. The method of any one of claims 1 to 29, comprising four or more of:

31. The culture medium comprises: (i) the metabolic polypeptide; (ii) the iron source; (iii) the antioxidant enzyme cofactor; (iv) the lipid precursor, and (v) the carrier protein The method of any one of claims 1 to 30, comprising:

32. 32. The method of claim 31 , wherein the culture medium comprises ITSEA.

33. The method of any one of claims 1 to 32, wherein the culture medium further comprises serum.

34. 34. The method of claim 33, wherein the serum is an AB serotype (AB) serum.

35. 35. The method of claim 33 or 34, wherein the AB serum is human AB serum.

36. 36. The method of any one of claims 33 to 35, wherein the serum is added to the culture medium in an amount of about 0.1% (v / v) to about 10% (v / v).

37. 37. The method of any one of claims 1 to 36, wherein said culturing is for about 0 to about 14 days.

38. 38. The method of any one of claims 1 to 37, further comprising cryopreserving the harvested population of cells comprising γδ T cells.

39. 39. The method of any one of claims 1 to 38, wherein said isolated pool of mixed immune cells depleted of αβ T cells is derived from peripheral blood mononuclear cells (PBMCs).

40. 39. The method of any one of claims 1 to 38, wherein said isolated pool of mixed immune cells depleted of αβ T cells is derived from a stem cell bank or cell line.

41. 41. The method of any one of claims 1 to 40, further comprising performing a step of depleting αβ T cells on the isolated pool of mixed immune cells.

42. 42. The method of claim 41, wherein the step of depleting αβ T cells comprises antibody-based depletion.

43. 43. The method of claim 41 or 42, wherein the step of depleting αβ T cells comprises: (a) exposing the isolated pool of mixed immune cells to an anti-TCR α / β antibody; and (b) separating the antibody-conjugated αβ TCR+ T cells from other cells in the isolated pool of mixed immune cells to obtain a population of αβ T cell-depleted cells.

44. 44. The method of claim 43, wherein said separation is performed by a cell sorting technique.

45. 45. The method of any one of claims 41-44, further comprising cryopreserving cells obtained from the step of depleting αβ T cells from the isolated pool of mixed immune cells prior to culturing the isolated pool of mixed immune cells or a derivative thereof in a culture medium.

46. 46. ​​The method of any one of claims 1 to 45, further comprising isolating said pool of mixed immune cells from a donor.

47. 47. The method of claim 46, wherein the donor is not the intended recipient.

48. 47. The method of claim 46, wherein the donor is the intended recipient.

49. 49. The method of any one of claims 46 to 48, wherein said isolating comprises leukapheresis.

50. 50. The method of any one of claims 46 to 49, wherein said isolating is performed on blood from said donor.

51. The method of any one of claims 46 to 50, wherein the donor is a human individual.

52. 52. The method of any one of claims 46 to 51, further comprising withdrawing blood from the donor.

53. 53. The method of any one of claims 1 to 52, further comprising carrying out a step of γδ T cell transduction.

54. 54. The method of claim 53, wherein the culturing step is separated by a step of transducing the cells in culture to obtain transduced γδ T cells.

55. 55. The method of claim 54, wherein the culturing step comprises a first culturing step from about day 0 to about day 4, and wherein allowing transduction to occur is performed on or about day 4.

56. 56. The method of claim 55, wherein the culturing step comprises a second culturing step for about 0 to about 10 days.

57. 57. The method of any one of claims 53 to 56, wherein causing transduction comprises introducing one or more transgenes.

58. 58. The method of claim 57, wherein the one or more transgenes comprises a CAR.

59. The method of claim 58, wherein the CAR specifically recognizes globohexaosylceramide (GloboH), AKR1C3, SSEA-4, or TROP-2.

60. 60. The method of any one of claims 1 to 59, further comprising cryopreserving the produced population of cells comprising γδ T cells after the harvesting step.

61. 61. The method of any one of claims 1 to 60, which is a feeder-cell-free method for producing a population of cells comprising γδ T cells.

62. 62. The method of any one of claims 1 to 61, wherein culturing the pool of mixed immune cells or the derivative thereof after αβ T cell depletion does not involve the use of antibodies.

63. 63. The method of any one of claims 1 to 62, which does not involve the use of an aminobisphosphonate in culturing said pool of mixed immune cells or said derivatives thereof.

64. 64. The method of claim 63, wherein the aminobisphosphonate is zoledronic acid or pamidronic acid, or a mixture thereof.

65. A population of cells comprising γδ T cells produced using the method of any one of claims 1 to 64.

66. The population is at least about 1 x 10 ８ 61. The population of claim 60, comprising γδ T cells.

67. 62. The population of claim 60 or 61, wherein the population comprises one or more transgenes.

68. 63. The population of claim 62, wherein the one or more transgenes comprises a CAR.

69. 1. A method of treating a disease in an individual, comprising: Obtaining an isolated pool of αβ T cell-depleted mixed immune cells or a derivative thereof from a donor individual; Producing a population of cells comprising γδ T cells using a method according to any one of claims 1 to 64; administering to said individual said population of cells comprising γδ T cells.

70. 70. The method of claim 69, wherein the disease is cancer.

71. 71. The method of claim 69 or 70, wherein the donor individual is not the individual.

72. 71. The method of claim 69 or 70, wherein the donor individual is the individual.

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