Methods for expanding gamma delta T cells

Abstract

The present disclosure provides methods for expanding γδ T cells (e.g., vδ1 T cells), where the cells are contacted with IL-15. In some embodiments, the cells are not contacted with IL-4. In some embodiments, the cells are engineered, e.g., to express a chimeric antigen receptor. Further provided are populations of expanded and / or engineered γδ T cells and methods of their use.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of priority to UK application No. 2204926.6, filed April 4, 2022, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to methods for expanding and optionally manipulating γδ T-cells. The expanded and engineered γδ T-cells produced according to the methods described herein are used in the treatment of cancer, in particular solid tumors. The present invention also relates to both individual cells and populations of cells produced by the methods described herein. [Background technology]

[0003] Gamma delta T cells (γδ T cells) represent a subset of T cells that express a distinct and characteristic γδ T cell receptor (TCR) on their surface. This TCR is composed of one gamma (γ) chain and one delta (δ) chain. Human γδ TCR chains are selected from three major δ chains, namely Vδ1, Vδ2, and Vδ3, and six γ chains. Human γδ T cells can be broadly classified based on their TCR chains, since certain γ and δ types are found widely, but not exclusively, in cells of one or more tissue types. For example, many blood-resident γδ T cells express Vδ2 TCRs, e.g., Vγ9Vδ2, which are less common in tissue-resident γδ T cells, which more frequently use Vδ1 in the skin and Vγ4 in the gut.

[0004] Vδ1 γδ T cells are a subset of natural T cells defined by the expression of a T cell receptor composed of a γ chain paired with a Vδ1 chain. In mice, Vδ1 γδ T cells are primarily tissue resident and highly protective against a wide range of carcinomas by mediating antitumor responses via pattern and natural cytotoxicity receptor recognition. Similarly, in humans, Vδ1 γδ T cells reside primarily within epithelial tissues, mediate target cell recognition that is not MHC-restricted, and are not allo-HLA reactive. Thus, γδ T cell adoptive cell therapy does not require HLA matching of patients. The unique Vδ1 γδ T cell biology that allows for antigen-independent tumor recognition, the absence of a requirement for HLA matching, and the unique migration to and location in human tissues make Vδ1 γδ T cells an attractive platform for cell therapy.

[0005] There is therefore a need for methods to efficiently expand γδ T cells and enable their application as therapy, e.g., as adoptive T cell therapy, and potentially provide allogeneic "off-the-shelf" chimeric antigen receptor-expressing γδ T cell therapy, e.g., for the treatment of solid tumors.

[0006] WO2015189356 describes a composition for expanding lymphocytes obtained from a sample obtained by apheresis, comprising at least two cytokines selected from IL-2, IL-15 and IL-21. WO2016198480 describes a method for expanding V52-TCR γδ+ T cells, particularly those obtained from a blood sample, using a two-step culture method comprising culturing the cells in a first medium containing a T cell mitogen and interleukin-4 (IL-4) and then in a second medium containing a T cell mitogen and interleukin-15 (IL-15). WO2016081518 describes a method for expanding engineered and non-engineered γδ T cell populations using an antibody that binds to the delta chain of the TCR.

[0007] While these disclosures go some way to addressing the problems discussed above, improved methods for expanding γδ T cells remain necessary, particularly for producing γδ T cells that are effective for targeting solid tumors. Summary of the Invention

[0008] According to a first aspect of the present invention, there is provided a method for expanding γδ T cells, the method comprising the steps of: (1) preparing a composition enriched in γδ T cells by depleting αβ T cells from a sample obtained from a subject that contains γδ T cells; (2) administering the composition enriched for γδ T cells to (i) an anti-CD3 antibody or a fragment thereof, and (ii) in the presence of interleukin-15 (IL-15) and in the absence of interleukin-4 (IL-4) (e.g., IL-4 is never added during the method); Culturing from the first day of said culture; and (3) isolating a cultured cell population from the composition.

[0009] According to a further aspect of the invention, there is provided a method for manipulating γδ T cells, the method comprising the steps of: (i) preparing a composition enriched in γδ T cells using a method defined herein; (ii) transducing the composition with an exogenous nucleic acid for expression in the γδ T-cell; and (iii) culturing the transduced composition to expand the engineered γδ T-cells.

[0010] According to a further aspect of the present invention there is provided an expanded population of γδ T cells obtainable, e.g. obtained, by the method defined herein.

[0011] According to a further aspect of the invention there is provided a pharmaceutical composition comprising an expanded population of γδ T cells as defined herein.

[0012] According to a further aspect of the invention there is provided an expanded population of γδ T cells or a pharmaceutical composition as defined herein for use as a medicament.

[0013] According to a further aspect of the invention there is provided an expanded population of γδ T cells or a pharmaceutical composition as defined herein for use in the treatment of cancer.

[0014] In some embodiments, the expanded γδ T cells can have in vivo cytotoxicity for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, or at least about 21 days. In some embodiments, the expanded γδ T cells can have in vivo cytotoxicity for at least about 14 days. [Brief description of the drawings]

[0015] [Figure 1] The growth protocols for Method 1 and Method 2 described in the Examples are shown in the figures. [Diagram 2] 1 is a graphical representation of cell yield upon harvest of cell product produced using either the process of Method 1 or Method 2. [Diagram 3] 13 is a graphical representation of the analysis of immune cell composition by flow cytometry. [Figure 4] 13A-C graphically depict analysis of CD56 expression by flow cytometry in γδ T cell products generated by methods 1 or 2. [Diagram 5] 13A-C graphically depict comparison of cell surface marker expression in V51 T cell products generated by method 1 or 2. [Figure 6] 13A-C graphically depict comparison of chemokine receptor expression in V51 T cell products generated by method 1 or 2. [Figure 7]Figure 13 graphically depicts a deep phenotypic analysis of cell products generated by methods 1 or 2. A, Volcano plot showing cell surface markers differentially expressed between V51 T cell products generated by methods 1 or 2. B, Pie chart of top signaling pathways associated with significantly differentially expressed cell surface markers between V51 T cell products generated by methods 1 or 2. [Figure 8] A is a graphical representation of a comparison of secreted soluble factors in γδ T cell products generated by method 1 or 2. A is a principal component analysis. B is a graphical representation of a comparison of secreted soluble factors in γδ T cell products generated by method 1 or 2. B is a comparison of major secreted cytokines. C is a graphical representation of a comparison of secreted soluble factors in γδ T cell products generated by method 1 or 2. C is a comparison of chemokines. [Figure 9] 13 graphically depicts total cell recovery and viability following cryopreservation of cell products produced using methods 1 or 2. [Figure 10] 13 graphically depicts the cytotoxicity after cryopreservation of cell products generated by methods 1 or 2 against lung cancer cell lines (NCI-H226 and A549), gastric cancer cell line (GSU), colorectal cell line (HT-29), ovarian adenocarcinoma cell line (OvCAR3), skin cancer cell line (A375), and pancreatic cancer cell line (CAPAN2). [Figure 11] A is a graphical representation of chimeric antigen receptor (CAR) expression in cell products generated by method 1 or 2. A is a comparison of CAR expression in cell products generated by method 1 or 2. B is a graphical representation of chimeric antigen receptor (CAR) expression in cell products generated by method 1 or 2. B shows a representative flow plot of CAR expression in one donor in cell products generated by method 1 or 2. [Figure 12] 13 graphically depicts the functionality of CAR-engineered cell products generated by methods 1 or 2 in cytotoxicity assays against an ovarian adenocarcinoma cell line (OvCAR3) and a mesothelin-engineered lung cancer cell line (A549-Meso). [Figure 13] A graphically depicts tumor control and proliferation of CAR engineered cell products generated by methods 1 or 2 after repeated tumor challenge. A shows tumor control after repeated dosing every 3 / 4 days with OVCAR3 (top) or A549-Meso (bottom). B graphically depicts tumor control and proliferation of CAR engineered cell products generated by methods 1 or 2 after repeated tumor challenge. B shows proliferation of CAR engineered cell products during repeated tumor challenge assays with OVCAR3 (top) or A549-Meso (bottom). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Previous methods have been described for expanding γδ T cells derived from hematopoietic samples such as peripheral blood. However, while such methods result in γδ T cells with proven efficacy against multiple hematological targets, they show limited activity against solid tumor targets. The present invention provides a simple method for expanding γδ T cells, particularly those derived from blood samples, which results in comparable or superior total cell expansion folds and surprising efficacy against solid malignancies.

[0017] Thus, according to a first aspect of the present invention there is provided a method for expanding γδ T cells, the method comprising the steps of: (1) preparing a composition enriched in γδ T cells by depleting αβ T cells from a sample obtained from a subject that contains γδ T cells; (2) administering the composition enriched for γδ T cells to (i) an anti-CD3 antibody or a fragment thereof, and (ii) culturing in the presence of interleukin-15 (IL-15) and in the absence of interleukin-4 (IL-4) from the first day of the culture (e.g., IL-4 is not added at all during the method); and (3) isolating a cultured cell population from the composition.

[0018] In certain embodiments, the sample (i.e., the starting sample) is human. The present invention is particularly used with γδ T cells derived from blood or fractions thereof. Surprisingly, it has been found that the method of the present invention is capable of expanding γδ T cells derived from hematological samples, which further have improved cytotoxicity against solid tumors.

[0019] Therefore, in one embodiment, said sample is hematopoietic sample or its fraction.In a further embodiment, said sample is selected from peripheral blood, umbilical cord blood, lymphoid tissue, thymus, bone marrow, spleen, nodal tissue or its fraction, particularly peripheral blood or its fraction.In a further embodiment, said sample is composed of peripheral blood mononuclear cell (PBMC) or low density mononuclear cell (LDMC).

[0020] In alternative embodiments, the sample is a non-hematopoietic tissue. References herein to "non-hematopoietic tissue" or "non-hematopoietic tissue sample" include skin (e.g., human skin) and intestine (e.g., human intestine). Non-hematopoietic tissue is a tissue other than blood, bone marrow, or thymus tissue. In one embodiment, the non-hematopoietic tissue sample is skin (e.g., human skin). In a further embodiment, the non-hematopoietic tissue sample is intestine or gastrointestinal tract (e.g., human intestine or human gastrointestinal tract). In some embodiments, the non-hematopoietic tissue sample is skin (e.g., human skin), which can be obtained by methods known in the art. Alternatively, the non-hematopoietic tissue sample is selected from gastrointestinal tract (e.g., colon or intestine), mammary gland, lung, prostate, liver, spleen, pancreas, uterus, vagina, and other skin, mucosa, or serosa.

[0021] The sample may be a cancer tissue sample, for example from a breast or prostate tumor, in particular a human cancer tissue sample. In other embodiments, the sample is not obtained from a cancer tissue (e.g., tissue that does not have a substantial number of tumor cells). For example, the sample may be derived from a separate skin area (e.g., healthy skin) from nearby or adjacent cancer tissue. Thus, in some embodiments, the γδ T cells are not obtained from a human cancer tissue.

[0022] In one embodiment, the sample is obtained from a human, hi an alternative embodiment, the sample is obtained from a non-human animal subject.

[0023] Methods for obtaining such tissues are known in the art. Examples of such methods include scalpel explants or punch biopsies, which may vary in size depending on the method. In some embodiments, the non-hematopoietic tissue sample is obtained by punch biopsy.

[0024] The methods described herein are carried out outside the human or animal body, i.e., they are in vitro and / or ex vivo. Thus, in one embodiment, the methods described herein are in vitro methods. In a further embodiment, the methods described herein are ex vivo methods.

[0025] As used herein, reference to "expanded", "expanded population" or "expanded γδ T-cells" includes a population of cells that is larger or contains a greater number of cells than a non-expanded population. Such populations may be more numerous, less numerous or may be mixed populations with an increase in the percentage or a particular cell type within the population. It will be understood that the term "expansion step" refers to a process that results in an expanded or expanded population. Thus, an expanded or expanded population may be more numerous or contain a greater number of cells compared to a population that has not undergone an expansion step or the population prior to any expansion step. Furthermore, it will be understood that any numbers provided herein to indicate expansion (e.g., fold increase or fold expansion) reflect an increase in the number or size of a population of cells or number of cells and are indicative of the amount of expansion.

[0026] In one preferred embodiment, the γδ T cells expanded by the method defined herein comprise a population of Vδ1 T cells.

[0027] In other embodiments, the composition enriched in γδ T cells comprises NK cells. As described herein, step (i) comprises depletion of αβ T cells, i.e. the composition enriched in γδ T cells is prepared by depletion of αβ T cells. In a further embodiment, preparing a composition enriched in γδ T cells according to step (i) comprises depleting αβ T cells from a mixed cell population obtained from a starting sample, such as a hematopoietic sample as described herein. The presence of NK cells in the composition may be advantageous as these cells are also effective cytotoxic cells.

[0028] NK cells (also known as large granular lymphocytes (LGLs)) are cytotoxic lymphocytes of the innate immune system. They respond rapidly, for example, to virus-infected and tumor cells, independent of MHC expression on the surface of the target cells. Thus, similar to γδ T cells, the recognition of target cells by NK cells is not MHC-restricted, and they are not allo-HLA reactive, meaning that NK cell-based therapy does not require HLA matching of patients.

[0029] In some embodiments, step (i) (i.e., preparing a composition enriched for γδ T cells) further or alternatively comprises positively selecting γδ T cells from a sample obtained from the subject that contains γδ T cells.

[0030] In one embodiment, the method comprises freezing the expanded γδ T-cells. Such frozen expanded γδ T-cells can then be thawed for downstream processing (e.g. further culture and expansion steps) and / or use (e.g. therapeutic use). Freezing allows easy transport and long-term storage of expanded γδ T-cells and is well known in the art. Thus, a method that provides cells that exhibit good viability and activity after freezing and thawing is advantageous, as not all expansion methods result in such cells.

[0031] In one embodiment, the composition of γδ T cells is derived from a single donor, in an alternative embodiment, the composition is derived from multiple donors, i.e., the composition is a "pooled" composition.

[0032] In one embodiment, the single or multiple donors may include a subject to be treated with a cell population or composition of the invention. Alternatively, the single or multiple donors do not include a subject to be treated with a cell population or composition of the invention.

[0033] Culture conditions The method comprises culturing the γδ T cells in the presence of a TCR agonist, in particular an anti-CD3 antibody or fragment thereof. The antibody can specifically bind to CD3. Preferred antibody clones include anti-CD3 antibodies, such as OKT-3 and UCHT-1 clones. In some embodiments, Retronectin is used in combination with OKT-3.

[0034] The term "antibody" includes any antibody protein construct that contains at least one antibody variable domain that contains at least one antigen binding site (ABS). Antibodies include, but are not limited to, immunoglobulins of the IgA, IgG, IgE, IgD, and IgM types (and their subtypes). The overall structure of the immunoglobulin G (IgG) antibody, assembled from two identical heavy (H) chains and two identical light (L) chain polypeptides, is well established and highly conserved in mammals (Padlan (1994) Mol. Immunol. 31:169-217).

[0035] As used herein, a fragment of an antibody (which may also be referred to as an "antibody fragment", "immunoglobulin fragment", "antigen-binding fragment" or "antigen-binding polypeptide") refers to a portion of an antibody (or a construct comprising said portion) that specifically binds to a target, i.e., the CD3 protein that is part of the T-cell receptor (TCR) complex (e.g., a molecule in which one or more immunoglobulin chains are not full length but which specifically binds to said target). Examples of binding fragments encompassed by the term antibody fragment include: (i) Fab fragment (a monovalent fragment consisting of the VL, VH, CL, and CH1 domains); (ii) F(ab')2 fragment (a bivalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region); (iii) Fd fragment (consisting of the VH and CH1 domains); (iv) Fv fragment (consisting of the VL and VH domains of a single arm of an antibody); (v) single chain variable fragments (scFv), which consist of VL and VH domains linked by a synthetic linker that allows the VL and VH regions to be produced using recombinant methods as a single protein chain that pairs to form a monovalent molecule; (vi) VH (a variable domain of an immunoglobulin chain consisting of a VH domain), (vii) VL (a variable domain of an immunoglobulin chain consisting of a VL domain), (viii) domain antibodies (consisting of either a dAb, VH or VL domain); (ix) a minibody (consisting of a pair of scFv fragments linked via a CH3 domain); and (x) diabodies, which consist of a non-covalent dimer of scFv fragments consisting of the VH domain from one antibody linked by a short peptide linker to the VL domain from another antibody.

[0036] "Specificity" refers to the number of different types of antigens or antigenic determinants that a particular antibody or fragment thereof can bind. The specificity of an antibody is the ability of the antibody to recognize a particular antigen as a unique molecular entity and distinguish it from other antigens. An antibody that "specifically binds" to an antigen or epitope is a term well understood in the art. A molecule is said to exhibit "specific binding" if it reacts more frequently, more rapidly, with a longer duration, and / or with a higher affinity to a particular target antigen or epitope than it reacts with other targets. An antibody "specifically binds" to a target antigen or epitope if it binds with higher affinity, avidity, more readily and / or for a longer duration than it binds to other substances. An antibody (or fragment thereof) may be considered to specifically bind to a target if the binding is statistically significant compared to unrelated binders.

[0037] In one embodiment, the anti-CD3 antibody or fragment thereof is OKT3. This antibody, also known as muromonab-CD3, is a mouse monoclonal antibody that targets an epitope located on the CD3 epsilon chain.

[0038] In one embodiment, the anti-CD3 antibody or fragment thereof is soluble or immobilized. For example, the antibody or fragment thereof may be added to the composition in soluble form. Alternatively, the antibody or fragment thereof may be added to the composition, and the antibody or fragment thereof is bound or covalently bound to a surface, for example, a bead or a plate (i.e., immobilized). In one embodiment, the antibody is immobilized to a surface, for example, an Fc-coated well. Alternatively, the antibody or fragment thereof is bound to the surface of a cell (for example, immobilized to the surface of an antigen-presenting cell (APC)). In another embodiment, the antibody is not immobilized to a surface when the composition contacts the antibody.

[0039] It will be appreciated that the culturing of the composition of γδ T cells is for a period of time effective to generate an expanded population of γδ T cells. In one embodiment, the period of time effective to generate an expanded population of γδ T cells is at least 7 days. Thus, in one embodiment, the composition of γδ T cells is cultured for at least 7 days. In a further embodiment, the composition is cultured for 7-21 days, e.g., 9-15 days. In a further embodiment, the composition is cultured for about 10, 11, 12, 13, or 14 days. In some aspects, the composition is cultured for at least about 7 days. In some aspects, the composition is cultured for at least about 8 days. In some aspects, the composition is cultured for at least about 9 days. In some aspects, the composition is cultured for at least about 10 days. In some aspects, the composition is cultured for at least about 11 days. In some aspects, the composition is cultured for at least about 12 days. In some aspects, the composition is cultured for at least about 13 days. In some aspects, the composition is cultured for at least about 14 days.

[0040] In further embodiments, the composition is cultured for at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or at least 21 days, such as about 14 days or about 21 days, to produce a population of expanded γδ T cells. In one embodiment, the composition is cultured for about 10, 11, 12, 13, or 14 days to produce a population of expanded γδ T cells. In some aspects, the composition is cultured for at least about 5 days to produce a population of expanded γδ T cells. In some aspects, the composition is cultured for at least about 6 days to produce a population of expanded γδ T cells. In some aspects, the composition is cultured for at least about 7 days to produce a population of expanded γδ T cells. In some aspects, the composition is cultured for at least about 8 days to produce a population of expanded γδ T cells. In some embodiments, the composition is cultured for at least about 9 days to produce an expanded population of γδ T cells. In some embodiments, the composition is cultured for at least about 10 days to produce an expanded population of γδ T cells. In some embodiments, the composition is cultured for at least about 11 days to produce an expanded population of γδ T cells. In some embodiments, the composition is cultured for at least about 12 days to produce an expanded population of γδ T cells. In some embodiments, the composition is cultured for at least about 13 days to produce an expanded population of γδ T cells. In some embodiments, the composition is cultured for at least about 14 days to produce an expanded population of γδ T cells. In some embodiments, the composition is cultured for at least about 15 days to produce an expanded population of γδ T cells. In some embodiments, the composition is cultured for at least about 16 days to produce an expanded population of γδ T cells. In some embodiments, the composition is cultured for at least about 17 days to produce an expanded population of γδ T cells. In some embodiments, the composition is cultured for at least about 18 days to produce an expanded population of γδ T cells.In some embodiments, the composition is cultured for at least about 19 days to produce an expanded population of γδ T cells. In some embodiments, the composition is cultured for at least about 20 days to produce an expanded population of γδ T cells. In some embodiments, the composition is cultured for at least about 21 days to produce an expanded population of γδ T cells.

[0041] In certain embodiments of the invention, the sample is cultured in a medium that is substantially free of serum (e.g., serum-free medium or medium containing a serum replacement (SR)). Thus, in one embodiment, the sample is cultured in a serum-free medium. Such serum-free medium may also include a serum replacement medium, the serum replacement being based on chemically defined components to avoid the use of serum of human or animal origin. In an alternative embodiment, the sample is cultured in a medium containing serum (e.g., human AB serum or fetal bovine serum (FBS)). In one embodiment, the sample is cultured in a medium containing a serum replacement. In one embodiment, the sample is cultured in a medium that does not contain animal-derived products. It will be appreciated that an embodiment according to the invention in which the sample is cultured in a serum-free medium has the advantage of avoiding problems related to filtration, precipitation, contamination, and serum supply. Furthermore, animal-derived products are not suitable for use in the manufacture of clinical grade human therapeutics.

[0042] In one embodiment, the methods defined herein are carried out in a vessel (e.g., a growth vessel) comprising a gas-permeable material. Such a material is permeable to gases such as oxygen, carbon dioxide, and / or nitrogen, allowing gas exchange between the contents of the vessel and the surrounding atmosphere. References herein to "vessels" include culture dishes, culture plates, single-well dishes, multi-well dishes, multi-well plates, flasks, multi-layer flasks, bottles (e.g., roller bottles), bioreactors, bags, tubes, and the like. Such vessels are known in the art for use in methods involving the growth of non-adherent cells and other lymphocytes. However, vessels comprising gas-permeable materials have surprisingly also found utility in the isolation and growth of γδ T cells, which are normally considered to be adherent. It has been found that the use of such vessels in culture significantly increases the yield of expanded γδ T cells. It has also been found that such vessels preferentially support γδ T cells and other lymphocytes over fibroblasts and other stromal cells (e.g., epithelial cells), including adherent cell types. See, e.g., International Publication Nos. WO2020095058 and WO2020095059, each of which is incorporated herein by reference in its entirety. Thus, in one embodiment, a container comprising a gas permeable material as defined herein preferentially supports γδ T cells and other lymphocytes (e.g., αβ T cells and / or NK cells). In a further embodiment, fibroblasts and / or other stromal cells (e.g., epithelial cells) are absent from cultures performed in containers comprising gas permeable material.

[0043] Such a container that includes a gas permeable material may further include a gas permeable material that is non-porous. Thus, in one embodiment, the gas permeable material is non-porous. In some embodiments, the gas permeable material is a membrane film such as silicone, fluoroethylene polypropylene, polyolefin, or ethylene vinyl acetate copolymer. Furthermore, such a container may only partially include a gas permeable material, a gas permeable membrane film, or a non-porous gas permeable material. Thus, according to a further embodiment, the container includes a top, a bottom, and at least one sidewall, and at least a portion of the bottom of the container includes a gas permeable material, which is in a substantially horizontal plane when the top is on the bottom. In one embodiment, the container includes a top, a bottom, and at least one sidewall, and at least a portion of the bottom includes a gas permeable material, which is in a horizontal plane when the top is on the bottom. In further embodiments, the container comprises a top, a bottom, and at least one sidewall, the at least one sidewall comprising a gas permeable material, which may be in a vertical plane if the top is above the bottom, or in a horizontal plane if the top is not above the bottom. In such embodiments, it will be understood that only the bottom or a portion of the sidewall may comprise a gas permeable material. Alternatively, the entire bottom or the entire sidewall may comprise a gas permeable material. In further embodiments, the top of the container comprising a gas permeable material may be sealed, for example, using an O-ring. It will be understood that such embodiments prevent the contents of the container from spilling or reduce their evaporation. Thus, in certain embodiments, the container comprises a liquid-tight container comprising a gas permeable material that allows gas exchange. In alternative embodiments, the top of the container comprising a gas permeable material is in a horizontal plane, above the bottom, and is not sealed. Thus, in certain embodiments, the top is configured to allow gas exchange from the top of the container. In a further embodiment, the bottom of the gas permeable container is configured to allow gas exchange from the bottom of the container, hi a further embodiment, the container comprising a gas permeable material may be a liquid tight container and may further comprise inlet and outlet ports or tubes.Thus, in certain embodiments, the container comprising a gas permeable material comprises a top, a bottom, and optionally at least one sidewall, wherein at least a portion of the top and bottom comprises a gas permeable material, and when present, at least a portion of the at least one sidewall comprises a gas permeable material. Exemplary containers are described in WO2005035728 and US9255243, which are incorporated herein by reference. These containers are also commercially available, for example, G-REX® cell culture devices provided by Wilson Wolf Manufacturing, such as G-REX 6-well plates, G-REX 24-well plates, and G-REX 10 containers.

[0044] Suitably expanding the population of γδ T cells provides at least a 5-fold, particularly at least a 10-fold, or at least a 15-fold, particularly at least a 20-fold increase in the number of γδ T cells, in some embodiments, the fold change is relative to the starting population of γδ T cells.

[0045] The composition is cultured in a medium containing IL-15. As used herein, "IL-15" refers to natural or recombinant IL-15 or variants thereof (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof) that act as agonists for one or more IL-15 receptor (IL-15R) subunits. IL-15, like IL-2, is a known T cell growth factor and can support the proliferation of CTLL-2, an IL-2-dependent cell line. IL-15 was first reported by Grabstein, et al. (Grabstein, et al. Science 1994.264.5161:965-969) as a 114 amino acid mature protein. As used herein, the term "IL-15" refers to native or recombinant IL-15 and its muteins, analogs, subunits, or complexes thereof (e.g., receptor complexes, e.g., sushi peptides, as described in WO2007 / 046006), each of which can stimulate proliferation of CTLL-2 cells. In a CTLL-2 proliferation assay, supernatants of cells transfected with recombinantly expressed precursor and in-frame fusions of the mature form of IL-15 can induce proliferation of CTLL-2 cells.

[0046] Human IL-15 can be obtained according to the procedure described by Grabstein, et al., or by conventional procedures, such as polymerase chain reaction (PCR). The cDNA for human IL-15 was deposited with the ATCC on February 19, 1993, and assigned accession number 69245.

[0047] The amino acid sequence of human IL-15 (gene ID 3600) is found in Genbank under accession locators NP000576.1 GI:10835153 (isoform 1) and NP_751915.1 GI:26787986 (isoform 2). The mouse (Mus musculus) IL-15 amino acid sequence (gene ID 16168) is found in Genbank under accession locator NP_001241676.1 GI:363000984.

[0048] IL-15 can also refer to IL-15 from various mammalian species, including, for example, human, monkey, cow, pig, horse, and mouse. As referred to herein, a "mutated protein" or "variant" of IL-15 is a polypeptide having an amino acid sequence that is substantially homologous to the sequence of a native mammalian IL-15, but differs from the native mammalian IL-15 polypeptide due to amino acid deletion, insertion, or substitution. A variant may include a conservatively substituted sequence, i.e., a given amino acid residue may be replaced by a residue having similar physiochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue for another, e.g., Ile, Val, Leu, or Ala for each other, or the substitution of one polar residue for another, e.g., between Lys and Arg, Glu and Asp, or Gln and Asn. Other such conservative substitutions are known, e.g., the substitution of whole regions with similar hydrophobicity. Naturally occurring IL-15 variants are also encompassed by the present invention. Examples of such variants are proteins resulting from alternative mRNA splicing events or from proteolytic cleavage of the IL-15 protein, where the binding properties of IL-15 are retained. Alternative splicing of the mRNA can result in truncated but biologically active IL-15 proteins. Mutations resulting from proteolysis include, for example, differences in the N-terminus or C-terminus upon expression in different types of host cells due to proteolytic removal of one or more terminal amino acids (typically 1-10 amino acids) of the IL-15 protein. In some embodiments, the termini of the protein can be modified with chemical groups, such as, for example, polyethylene glycol, to alter its physical properties (Yang, et al. Cancer 1995.76:687-694). In some embodiments, the termini or interiors of the protein can be modified with additional amino acids (Clark-Lewis, et al. PNAS 1993.90:3574-3577).

[0049] In some embodiments, the methods defined herein involve administering IL-15 to a subject, typically at a concentration of at least 0.1 ng / mL, such as at least 10 ng / mL (e.g., 0.1 ng / mL to 10,000 ng / mL, 1.0 ng / mL to 1,000 ng / mL, 5 ng / mL to 800 ng / mL, 10 ng / mL to 750 ng / mL, 20 ng / mL to 500 ng / mL, 50 ng / mL to 400 ng / mL, or 100 ng / mL to 250 ng / mL, such as ... In some embodiments, the method as defined herein comprises IL-15 at a concentration of less than 500 ng / mL, e.g., less than 250 ng / mL. In some embodiments, the concentration of IL-15 is about 100 ng / mL. In some embodiments as defined herein, the IL-15 is present at a concentration of 5 ng / mL to 300 ng / mL (e.g., 5 ng / mL to 150 ng / mL) (e.g., 10 ng / mL to 150 ng / mL) (e.g., 10 ng / mL to 100 ng / mL).In some embodiments, the IL-15 is administered at a concentration of about 5 ng / mL to 250 ng / mL, about 5 ng / mL to 200 ng / mL, about 5 ng / mL to 150 ng / mL, about 10 ng / mL to 250 ng / mL, about 10 ng / mL to 200 ng / mL, about 10 ng / mL to 150 ng / mL, about 20 ng / mL to 250 ng / mL, about 20 ng / mL to 200 ng / mL, about 20 ng / mL to 150 ng / mL, about 30 ng / mL to 250 ng / mL, about 30 The IL-15 is contained at a concentration of about 5 ng / mL to about 150 ng / mL. In some embodiments, the IL-15 is contained at a concentration of about 5 ng / mL to about 125 ng / mL. In some embodiments, the IL-15 is contained at a concentration of about 5 ng / mL to about 100 ng / mL. In some embodiments, the IL-15 is contained at a concentration of about 5 ng / mL to about 125 ng / mL. In some embodiments, the IL-15 is contained at a concentration of about 5 ng / mL to about 100 ng / mL. In some embodiments, the IL-15 is contained at a concentration of about 10 ng / mL to about 150 ng / mL. In some embodiments, the IL-15 is contained at a concentration of about 10 ng / mL to about 125 ng / mL. In some embodiments, the IL-15 is contained at a concentration of about 10 ng / mL to about 100 ng / mL. In some embodiments, the IL-15 is contained at a concentration of about 15 ng / mL to about 150 ng / mL. In some embodiments, the IL-15 is contained at a concentration of about 5 to 300 (e.g., the range is 10 to 150). In some embodiments, the IL-15 is contained at a concentration of about 15 ng / mL to about 125 ng / mL. In some embodiments, the IL-15 is contained at a concentration of about 15 ng / mL to about 100 ng / mL. In some embodiments, the IL-15 is present at a concentration of about 20 ng / mL to about 150 ng / mL. In some embodiments, the IL-15 is present at a concentration of about 20 ng / mL to about 125 ng / mL. In some embodiments, the IL-15 is present at a concentration of about 20 ng / mL to about 100 ng / mL.In some embodiments, the IL-15 is present at a concentration of about 25 ng / mL to about 150 ng / mL. In some embodiments, the IL-15 is present at a concentration of about 25 ng / mL to about 125 ng / mL. In some embodiments, the IL-15 is present at a concentration of about 25 ng / mL to about 100 ng / mL.

[0050] In some embodiments, the IL-15 is included at a concentration of about 5 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 10 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 15 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 20 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 21 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 25 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 30 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 35 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 40 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 45 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 50 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 60 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 70 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 80 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 90 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 100 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 110 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 120 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 130 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 140 ng / mL. In some embodiments, the IL-15 is included at a concentration of about 150 ng / mL.

[0051] In one embodiment, the composition is cultured in a medium consisting essentially of an anti-CD3 antibody or fragment thereof, and IL-15. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding any other element of essential importance to the composition for the intended purpose. Thus, a composition consisting essentially of elements as defined herein does not exclude standard media components, such as media (CTS OpTmizer, Thermo Fisher), plasma, serum, and essential amino acids, such as L-glutamine (e.g., Glutamax).

[0052] Alternatively, the method (particularly, for example, step (ii)) may comprise culturing the composition enriched for γδ T cells in the presence of one or more further cytokines selected from IL-2, IL-21, and IL-1β.

[0053] In some embodiments, the cells are seeded in a medium comprising an anti-CD3 antibody and IL-15, the medium not comprising additional exogenous cytokines. In some embodiments, the cells are seeded in a medium comprising an anti-CD3 antibody and IL-15, the medium not comprising IL-4. In some embodiments, the cells are seeded in a medium comprising an anti-CD3 antibody and IL-15, the medium not comprising IL-1β. In some embodiments, the cells are seeded in a medium comprising an anti-CD3 antibody and IL-15, the medium not comprising IL-21. In some embodiments, the cells are seeded in a medium comprising an anti-CD3 antibody and IL-15, the medium not comprising IFNγ. In some embodiments, the cells are seeded in a medium comprising an anti-CD3 antibody and IL-15, the medium not comprising IL-2. In some embodiments, the cells are seeded in a medium comprising an anti-CD3 antibody and IL-15, the medium not comprising IL-9.

[0054] In some embodiments, the hematopoietic-derived γδ cells are seeded in a medium comprising an anti-CD3 antibody and IL-15, the medium being free of additional exogenous cytokines. In some embodiments, the hematopoietic-derived γδ cells are seeded in a medium comprising an anti-CD3 antibody and IL-15, the medium being free of IL-4. In some embodiments, the hematopoietic-derived γδ cells are seeded in a medium comprising an anti-CD3 antibody and IL-15, the medium being free of IL-1β. In some embodiments, the hematopoietic-derived γδ cells are seeded in a medium comprising an anti-CD3 antibody and IL-15, the medium being free of IL-21. In some embodiments, the hematopoietic-derived γδ cells are seeded in a medium comprising an anti-CD3 antibody and IL-15, the medium being free of IFNγ. In some embodiments, the hematopoietic-derived γδ cells are seeded in a medium comprising an anti-CD3 antibody and IL-15, the medium being free of IL-2. In some aspects, the hematopoietic derived γδ cells are plated in a medium comprising an anti-CD3 antibody and IL-15, wherein the medium does not comprise IL-9.

[0055] In some embodiments, additional IL-15 is added to the cell culture medium after seeding. In some embodiments, the additional IL-15 is added at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days (e.g., the IL-15 is added in an amount of 5 to 150 ng / ml) (e.g., 15 to 115 ng / ml). In some embodiments, the additional IL-15 is added to the culture medium on the first day after seeding (e.g., the IL-15 is added in an amount of 5 to 150 ng / ml) (e.g., 15 to 115 ng / ml). In some embodiments, the additional IL-15 is added to the culture medium on the second day after seeding (e.g., the IL-15 is added in an amount of 5 to 150 ng / ml) (e.g., 15 to 115 ng / ml). In some embodiments, additional IL-15 is added to the medium on the third day after seeding (e.g., the IL-15 is added in an amount of 5-150 ng / ml) (e.g., 15-115 ng / ml). In some embodiments, additional IL-15 is added to the medium on the fourth day after seeding (e.g., the IL-15 is added in an amount of 5-150 ng / ml) (e.g., 15-115 ng / ml). In some embodiments, additional IL-15 is added to the medium on the fifth day after seeding (e.g., the IL-15 is added in an amount of 5-150 ng / ml) (e.g., 15-115 ng / ml). In some embodiments, additional IL-15 is added to the medium on the sixth day after seeding (e.g., the IL-15 is added in an amount of 5-150 ng / ml) (e.g., 15-115 ng / ml). In some embodiments, additional IL-15 is added to the medium on the 7th day after seeding (e.g., the IL-15 is added in an amount of 5-150 ng / ml) (e.g., 15-115 ng / ml). In some embodiments, additional IL-15 is added to the medium on the 8th day after seeding (e.g., the IL-15 is added in an amount of 5-150 ng / ml) (e.g., 15-115 ng / ml). In some embodiments, additional IL-15 is added to the medium on the 9th day after seeding (e.g., the IL-15 is added in an amount of 5-150 ng / ml) (e.g., 15-115 ng / ml).In some embodiments, additional IL-15 is added to the medium on the 10th day after seeding (e.g., the IL-15 is added in an amount of 5 to 150 ng / ml) (e.g., 15 to 115 ng / ml). In some embodiments, additional IL-15 is added to the medium on the 11th day after seeding (e.g., the IL-15 is added in an amount of 5 to 150 ng / ml) (e.g., 15 to 115 ng / ml). In some embodiments, additional IL-15 is added to the medium on the 12th day after seeding (e.g., the IL-15 is added in an amount of 5 to 150 ng / ml) (e.g., 15 to 115 ng / ml). In some embodiments, additional IL-15 is added to the medium on the 13th day after seeding (e.g., the IL-15 is added in an amount of 5 to 150 ng / ml) (e.g., 15 to 115 ng / ml). In some embodiments, additional IL-15 is added on day 14 post-seeding (eg, the IL-15 is added in an amount of 5-150 ng / ml) (eg, 15-115 ng / ml).

[0056] In some embodiments, additional IL-15 is added on days 4, 5, 6, and 7 after seeding (e.g., the IL-15 is added in an amount of 5-150 ng / ml) (e.g., 15-115 ng / ml). In some embodiments, additional IL-15 is added on days 10, 11, and 12 after seeding (e.g., the IL-15 is added in an amount of 5-150 ng / ml) (e.g., 15-115 ng / ml). In some embodiments, additional IL-15 is added on days 16, 17, and 18 after seeding (e.g., the IL-15 is added in an amount of 5-150 ng / ml) (e.g., 15-115 ng / ml).

[0057] In some embodiments, no other cytokines are added along with the additional IL-15.Thus, in some embodiments, the cells are cultured in the absence of exogenously added IL-4, IL-1β, IL-21, IFNγ, or any combination thereof, for the duration of the culture.

[0058] In some embodiments, the cells are cultured according to the methods disclosed herein for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, or at least about 21 days. In some embodiments, the cells are cultured according to the methods disclosed herein for at least about 7 days. In some embodiments, the cells are cultured according to the methods disclosed herein for at least about 8 days. In some embodiments, the cells are cultured according to the methods disclosed herein for at least about 9 days. In some embodiments, the cells are cultured according to the methods disclosed herein for at least about 10 days. In some embodiments, the cells are cultured according to the methods disclosed herein for at least about 11 days. In some embodiments, the cells are cultured according to the methods disclosed herein for at least about 12 days. In some embodiments, the cells are cultured according to the methods disclosed herein for at least about 13 days. In some embodiments, the cells are cultured according to the methods disclosed herein for at least about 14 days. In some embodiments, the cells are cultured according to the methods disclosed herein for at least about 15 days. In some embodiments, the cells are cultured according to the methods disclosed herein for at least about 16 days. In some embodiments, the cells are cultured according to the methods disclosed herein for at least about 17 days. In some embodiments, the cells are cultured according to the methods disclosed herein for at least about 18 days. In some embodiments, the cells are cultured according to the methods disclosed herein for at least about 19 days. In some embodiments, the cells are cultured according to the methods disclosed herein for at least about 20 days. In some embodiments, the cells are cultured according to the methods disclosed herein for at least about 21 days.

[0059] In some embodiments, the cells are contacted with IL-21. As used herein, "IL-21" refers to native or recombinant IL-21 or variants thereof (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof) that act as agonists for one or more IL-21 receptor (IL-21R) subunits. Such agents include natural killer (NK) and cytotoxic (CD8 + IL-21 can support the proliferation of IL-21R T cells. Mature human IL-21 occurs as a sequence of 133 amino acids (minus a signal peptide consisting of an additional 22 N-terminal amino acids). IL-21 muteins are polypeptides in which certain substitutions have been made in the interleukin-21 protein while retaining the ability to bind to IL-21Rα, such as those described in US 9,388,241. The IL-21 muteins can be characterized by insertions, deletions, substitutions and modifications of amino acids at one or more sites of the native IL-21 polypeptide chain or at other residues. According to the present disclosure, any such insertions, deletions, substitutions and modifications result in IL-21 muteins that retain IL-21R binding activity. Exemplary muteins can include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0060] Nucleic acids encoding human IL-21 can be obtained by conventional procedures, for example, polymerase chain reaction (PCR). The amino acid sequence of human IL-21 (gene ID 59067) is found in Genbank under the accession locator NC_000004.12. The mouse (Mus musculus) IL-21 amino acid sequence (gene ID 60505) is found in Genbank under the accession locator NC_000069.6.

[0061] IL-21 can also refer to IL-21 from various mammalian species, including, for example, human, monkey, cow, pig, horse, and mouse. Variants may include conservatively substituted sequences, i.e., a given amino acid residue may be replaced by a residue with similar physiochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue for another, such as Ile, Val, Leu, or Ala for each other, or the substitution of one polar residue for another, such as Lys for Arg, Glu for Asp, or Gln for Asn. Other such conservative substitutions, such as the substitution of whole regions with similar hydrophobicity, are well known. Naturally occurring IL-21 variants are also encompassed by the present invention. Examples of such variants are proteins resulting from alternative mRNA splicing events or from proteolytic cleavage of IL-21 protein, where the binding properties of IL-21 are retained. Alternative splicing of mRNA can result in truncated but biologically active IL-21 proteins. Mutations due to proteolysis include, for example, differences in the N-terminus or C-terminus upon expression in different types of host cells due to proteolytic removal of one or more terminal amino acids (typically 1-10 amino acids) of the IL-21 protein. In some embodiments, the termini of the protein can be modified with chemical groups, such as, for example, polyethylene glycol, to alter its physical properties (Yang, et al. Cancer 1995. 76:687-694). In some embodiments, the termini or interiors of the protein can be modified with additional amino acids (Clark-Lewis, et al. PNAS 1993. 90:3574-3577).

[0062] In a further embodiment, the methods defined herein typically include IL-21 at a concentration of at least 0.1 ng / mL, such as at least 1.0 ng / mL (e.g., 0.1 ng / mL to 1,000 ng / mL, 1.0 ng / mL to 100 ng / mL, 1.0 ng / mL to 50 ng / mL, 2 ng / mL to 50 ng / mL, 3 ng / mL to 10 ng / mL, 4 ng / mL to 8 ng / mL, 5 ng / mL to 10 ng / mL, 6 ng / mL to 8 ng / mL, such as, 0.1 ng / mL to 10 ng / mL, 1.0 ng / mL to 5 ng / mL, 1.0 ng / mL to 10 ng / mL, 1.0 ng / mL to 20 ng / mL). In a further embodiment, the methods defined herein typically include IL-21 at a concentration of less than 100 ng / mL, such as less than 50 ng / mL. In some embodiments, the concentration of IL-21 is between 3 ng / mL and 40 ng / mL, between 4 ng / mL and 20 ng / mL, between 5 ng / mL and 15 ng / mL, or between 6 ng / mL and 10 ng / mL. In some embodiments, the cells are not contacted with IL-21.

[0063] In some embodiments, the cells are contacted with IL-2. As used herein, "IL-2" refers to natural or recombinant IL-2 or variants thereof (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof) that act as agonists for one or more IL-2 receptor (IL-2R) subunits. Such agents can support the growth of CTLL-2 (33, American Type Culture Collection (ATCC®) TIB214), an IL-2-dependent cell line. Mature human IL-2 occurs as a sequence of 133 amino acids (minus a signal peptide of 20 additional N-terminal amino acids) as described in Fujita, et al. Cell 1986.46.3:401-407. IL-2 muteins are polypeptides in which certain substitutions have been made in the interleukin-2 protein while retaining the ability to bind to IL-2Rβ, such as those described in US 2014 / 0046026. The IL-2 muteins may be characterized by amino acid insertions, deletions, substitutions and modifications at one or more sites in the native IL-2 polypeptide chain or at other residues. According to the present disclosure, any such insertions, deletions, substitutions and modifications result in an IL-2 mutein that retains IL-2Rβ binding activity. Exemplary muteins may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions.

[0064] Nucleic acids encoding human IL-2 can be obtained by conventional procedures, for example, polymerase chain reaction (PCR). The amino acid sequence of human IL-2 (gene ID 3558) is found in Genbank under accession locator NP_000577.2 GI:28178861. The mouse (Mus musculus) IL-2 amino acid sequence (gene ID 16183) is found in Genbank under accession locator NP_032392.1 GI:7110653.

[0065] IL-2 can also refer to IL-2 from various mammalian species, including, for example, human, monkey, cow, pig, horse, and mouse. Variants may include conservatively substituted sequences, i.e., a given amino acid residue may be replaced by a residue with similar physiochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue for another, e.g., Ile, Val, Leu, or Ala for each other, or the substitution of one polar residue for another, e.g., Lys for Arg, Glu for Asp, or Gln for Asn. Other such conservative substitutions, e.g., substitution of entire regions with similar hydrophobicity, are well known. Naturally occurring IL-2 variants are also encompassed by the present invention. Examples of such variants are proteins resulting from alternative mRNA splicing events or from proteolytic cleavage of the IL-2 protein, where the binding properties of IL-2 are retained. Alternative splicing of mRNA can result in truncated but biologically active IL-2 proteins. Mutations due to proteolysis include, for example, differences in the N-terminus or C-terminus upon expression in different types of host cells due to proteolytic removal of one or more terminal amino acids (typically 1-10 amino acids) of the IL-2 protein. In some embodiments, the termini or interior of the protein may be modified with chemical groups, such as, for example, polyethylene glycol, to alter its physical properties (Yang, et al. Cancer 1995.76:687-694). In some embodiments, the termini or interior of the protein may be modified with additional amino acids (Clark-Lewis, et al. PNAS 1993.90:3574-3577).

[0066] In certain embodiments, the methods defined herein involve administering IL-2 to a subject, typically at a concentration of at least 10 IU / mL, such as at least 100 IU / mL (e.g., 10 IU / mL to 1,000 IU / mL, 20 IU / mL to 800 IU / mL, 25 IU / mL to 750 IU / mL, 30 IU / mL to 700 IU / mL, 40 IU / mL to 600 IU / mL, 50 IU / mL to 500 IU / mL, 75 IU / mL to 250 IU / mL, or 10 0IU / mL to 200IU / mL, e.g., 10IU / mL to 20IU / mL, 20IU / mL to 30IU / mL, 30IU / mL to 40IU / mL, 40IU / mL to 50IU / mL, 50IU / mL to 75IU / mL, 75IU / mL to 100IU / mL, 100IU / mL to 150IU / mL, 150IU / mL to 200IU / mL, 200IU / mL to 500IU / mL, or 500IU / mL to 1,000IU / mL). In certain embodiments, the methods defined herein include IL-2 at a concentration typically less than 1,000IU / mL, e.g., less than 500IU / mL. In some embodiments, the concentration of IL-2 is about 100IU / mL. In some embodiments, the cells are not contacted with IL-2.

[0067] In some embodiments, the cells are contacted with IL-1β. As used herein, "IL-1β" refers to natural or recombinant IL-1β or variants thereof (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof) that act as agonists for one or more IL-1 receptor (IL-1R) subunits. IL-1 is a proinflammatory cytokine that plays a major role in a wide range of diseases, including inflammatory diseases. It consists of two molecular species, IL-1α and IL-1β, which share only limited sequence identity but exert similar biological activities through binding to the IL-1 receptor (type I and type II). Mature human IL-1β arises from the N-terminus of the precursor polypeptide as a 153 amino acid sequence after cleavage of 116 amino acids by CASP1, as described in Andrei et al. (2004) PNAS 101(26):9745-9750. An IL-1β mutein is a polypeptide in which certain substitutions have been made in the IL-1β protein while retaining the ability to bind to IL-1R. The IL-1β mutein may be characterized by insertions, deletions, substitutions and modifications of amino acids at one or more sites of the native IL-1β polypeptide chain or at other residues. According to the present disclosure, any such insertions, deletions, substitutions and modifications result in an IL-1β mutein that retains IL-1R binding activity. Exemplary muteins may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions.

[0068] Nucleic acids encoding human IL-1β can be obtained by conventional procedures, for example, polymerase chain reaction (PCR). The amino acid sequence of human IL-1β (gene ID 3553) is found in Genbank under accession locator NP_000567 or in UniProt under accession number P01584. The mouse (Mus musculus) IL-1β amino acid sequence (gene ID 16176) is found in Genbank under accession locator NP_032387 or in UniProt under accession number P10749.

[0069] IL-1β can also refer to IL-1β derived from various mammalian species, including, for example, human, monkey, cow, pig, horse, and mouse. Variants may include conservatively substituted sequences, i.e., a given amino acid residue may be replaced by a residue with similar physiochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue for another, e.g., Ile, Val, Leu, or Ala for each other, or the substitution of one polar residue for another, e.g., Lys for Arg, Glu for Asp, or Gln for Asn. Other such conservative substitutions, e.g., substitution of entire regions with similar hydrophobicity, are well known. Naturally occurring IL-1β variants are also encompassed by the present invention. Examples of such variants are proteins resulting from alternative mRNA splicing events or from proteolytic cleavage of IL-1β protein, where the binding properties of IL-1β are retained.

[0070] In certain embodiments, the methods defined herein include IL-1β, typically at a concentration of at least 100 IU / mL, e.g., at least 1,000 IU / mL (e.g., 100 IU / mL to 8,000 IU / mL, 250 IU / mL to 7,000 IU / mL, 500 IU / mL to 6,000 IU / mL, or 1,000 IU / mL to 5,000 IU / mL). In certain embodiments, the methods defined herein include IL-1β, typically at a concentration of less than 8,000 IU / mL, e.g., less than 5,000 IU / mL. In some embodiments, the concentration of IL-1β is about 4,500 IU / mL. In some aspects, the cells are not contacted with IL-1β.

[0071] In contrast to previous methods for expanding γδ T cells described in the art, the present method cultures a composition enriched for γδ T cells in the absence of IL-4, and thus the method of the invention is performed in medium that does not contain (and is not supplemented with) IL-4.

[0072] In some embodiments, the cells are not contacted with IL-4. As used herein, "IL-4" refers to native or recombinant IL-4 or variants thereof (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof) that act as agonists for one or more IL-4 receptor (IL-4R) subunits. Such agents can support the differentiation of naive helper T cells (Th0 cells) into Th2 cells. Mature human IL-4 occurs as a sequence of 129 amino acids (minus a signal peptide consisting of an additional 24 N-terminal amino acids). IL-4 muteins are polypeptides in which certain substitutions have been made in the interleukin-4 protein while retaining the ability to bind to IL-4Rα, such as those described in U.S. Pat. No. 6,313,272. The IL-4 muteins can be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites of the native IL-4 polypeptide chain or at other residues. According to the present disclosure, any such insertions, deletions, substitutions and modifications result in an IL-4 mutein that retains IL-2Rα binding activity. Exemplary muteins may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions.

[0073] Nucleic acids encoding human IL-4 can be obtained by conventional procedures, for example, polymerase chain reaction (PCR). The amino acid sequence of human IL-4 (gene ID 3565) is found in Genbank under the accession locator NG_023252. The mouse (Mus musculus) IL-4 amino acid sequence (gene ID 16189) is found in Genbank under the accession locator NC_000077.6.

[0074] IL-4 can also refer to IL-4 from various mammalian species, including, for example, human, monkey, cow, pig, horse, and mouse. Variants may include conservatively substituted sequences, i.e., a given amino acid residue may be replaced by a residue with similar physiochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue for another, e.g., Ile, Val, Leu, or Ala for each other, or the substitution of one polar residue for another, e.g., Lys for Arg, Glu for Asp, or Gln for Asn. Other such conservative substitutions, e.g., substitution of entire regions with similar hydrophobicity, are well known. Naturally occurring IL-4 variants are also encompassed by the present invention. Examples of such variants are proteins resulting from alternative mRNA splicing events or from proteolytic cleavage of the IL-4 protein, where the binding properties of IL-4 are retained. Alternative splicing of mRNA can result in truncated but biologically active IL-4 proteins. Variations due to proteolysis include, for example, differences in the N- or C-terminus upon expression in different types of host cells due to proteolytic removal of one or more terminal amino acids (generally 1-10 amino acids) of the IL-4 protein.

[0075] Compositions and Markers In some embodiments, at least 80%, e.g., 85%, 90%, 95%, 97%, 98%, 99%, 99.5% of the cells present in the cell population comprise NK cells and γδ T cells (e.g., Vδ1 T cells). In some embodiments, at least 80%, e.g., 85%, 90%, 95%, 97%, 98%, 99%, 99.5% of the cells present in the cell population obtained by the methods described herein comprise NK cells and γδ T cells. In one embodiment, at least 85% of the cells present in the cell population comprise NK cells and γδ T cells. In a further embodiment, at least 90% of the cells present in the cell population comprise NK cells and γδ T cells. In a further embodiment, at least 95% of the cells present in the cell population comprise NK cells and γδ T cells.

[0076] In some embodiments, at least 50% of the cells present in the composition are γδ T cells. In some embodiments, the composition comprises at least about 50% γδ T cells, such as at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% γδ T cells. In further embodiments, the composition comprises at least about 60% γδ T cells, such as more than about 70% γδ T cells. In other embodiments, at least 80%, such as at least 90%, of the cells present in the composition are γδ T cells. In one embodiment, the composition comprises at least about 82% γδ T cells.

[0077] In some embodiments, the composition comprises at least about 30% V51 T cells, e.g., at least about 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50% V51 T cells. In further embodiments, the composition comprises at least about 40% V51 T cells, e.g., at least about 50% V51 T cells. In some embodiments, the composition comprises about 50%-70% V51 T cells. In one embodiment, the composition comprises about 60% V51 T cells.

[0078] In some embodiments, the composition comprises less than about 1% αβ T cells. In some embodiments, the composition comprises less than about 1% αβ T cells, 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, less than about 0.1% αβ T cells, less than about 0.09% αβ T cells, less than about 0.08% αβ T cells, less than about 0.07% αβ T cells, less than about 0.09% αβ T cells, less than about 0.08 ... The composition may comprise less than about 0.06% αβ T cells, less than about 0.05% αβ T cells, less than about 0.04% αβ T cells, less than about 0.05% αβ T cells, less than about 0.04% αβ T cells, less than about 0.03% αβ T cells, less than about 0.02% αβ T cells, less than about 0.01% αβ T cells, less than about 0.009% αβ T cells, less than about 0.008% αβ T cells, less than about 0.007% αβ T cells, less than about 0.006% αβ T cells, less than about 0.005% αβ T cells. In some embodiments, the composition comprises less than about 0.9% αβ T cells. In some embodiments, the composition comprises less than about 0.8% αβ T cells. In some embodiments, the composition comprises less than about 0.7% αβ T cells. In some embodiments, the composition comprises less than about 0.6% αβ T cells. In some embodiments, the composition comprises less than about 0.5% αβ T cells. In some embodiments, the composition comprises less than about 0.4% αβ T cells. In some embodiments, the composition comprises less than about 0.3% αβ T cells. In some embodiments, the composition comprises less than about 0.2% αβ T cells. In some embodiments, the composition comprises less than about 0.1% αβ T cells. In some embodiments, the composition comprises less than about 0.09% αβ T cells. In some embodiments, the composition comprises less than about 0.08% αβ T cells. In some embodiments, the composition comprises less than about 0.07% αβ T cells. In some embodiments, the composition comprises less than about 0.06% αβ T cells. In some embodiments, the composition comprises less than about 0.05% αβ T cells. In some embodiments, the composition comprises less than about 0.04% αβ T cells. In some embodiments, the composition comprises less than about 0.03% αβ T cells.In some embodiments, the composition comprises less than about 0.02% αβ T cells. In some embodiments, the composition comprises less than about 0.01% αβ T cells. In some embodiments, the composition comprises a concentration of αβ T cells below the limit of detection.

[0079] In some embodiments, at least about 50% of the cells isolated prior to the expansion methods disclosed herein are αβT cells, and in the composition after expansion by the methods disclosed herein, less than about 1% of the cells are αβT cells. In some embodiments, at least about 45% of the cells isolated prior to the expansion methods disclosed herein are αβT cells, and in the composition after expansion by the methods disclosed herein, less than about 1% of the cells are αβT cells. In some embodiments, at least about 40% of the cells isolated prior to the expansion methods disclosed herein are αβT cells, and in the composition after expansion by the methods disclosed herein, less than about 1% of the cells are αβT cells. In some embodiments, at least about 35% of the cells isolated prior to the expansion methods disclosed herein are αβT cells, and in the composition after expansion by the methods disclosed herein, less than about 1% of the cells are αβT cells. In some embodiments, at least about 30% of the cells isolated prior to the expansion methods disclosed herein are αβT cells, and in the composition after expansion by the methods disclosed herein, less than about 1% of the cells are αβT cells. In some embodiments, at least about 25% of the cells isolated prior to the expansion methods disclosed herein are αβT cells, and in the composition after expansion by the methods disclosed herein, less than about 1% of the cells are αβT cells. In some embodiments, at least about 20% of the cells isolated prior to the expansion methods disclosed herein are αβT cells, and in the composition after expansion by the methods disclosed herein, less than about 1% of the cells are αβT cells. In some embodiments, at least about 15% of the cells isolated prior to the expansion methods disclosed herein are αβT cells, and in the composition after expansion by the methods disclosed herein, less than about 1% of the cells are αβT cells. In some embodiments, at least about 10% of the cells isolated prior to the expansion methods disclosed herein are αβ T cells, and in the composition after expansion by the methods disclosed herein, less than about 1% of the cells are αβ T cells. In some embodiments, at least about 5% of the cells isolated prior to the expansion methods disclosed herein are αβ T cells, and in the composition after expansion by the methods disclosed herein, less than about 1% of the cells are αβ T cells.

[0080] In one embodiment, at least 50% of the expanded γδ T cells present in the cell population express CD56. In one embodiment, at least about 60%, such as at least 65%, 70%, 75%, or 80%, of the expanded γδ T cells present in the cell population express CD56. In a further embodiment, at least 80% of the expanded γδ T cells present in the cell population express CD56, such as after a culture period of about 14 days (e.g., 14 days).

[0081] CD56, also known as neural cell adhesion molecule (NCAM), is an adhesion molecule of the immunoglobulin (Ig) superfamily that correlates with high cytotoxicity in NK cells, αβT cells, and γδT cells. It has been shown to be involved in cis and trans binding to itself, which contributes to lymphocyte activation. It has also been shown to be fundamental for the formation of immunological synapses between lymphocytes, between lymphocytes and antigen-presenting cells (APCs), and between lymphocytes and target cells (Nussbaumer and Thurnher (2020) Cells 9(3):772). Cell phenotypes can also be defined by the cell surface density of CD56. Thus, CD56 bright Cells and CD56 dim There are recognized subtypes of γδ T cells known in the art, CD56. Increased expression and / or intensity of CD56 on γδ T cells has been shown to correlate with improved killing. Surface expression of CD56 can be identified using methods known in the art, for example, by analysis of staining intensity via flow cytometry. Cells can be classified as CD56 bright and CD56 dim Separating a sample population into a set of CD56-specific expression levels is understood in the art and described, for example, in Van Acker et al. (2017) Front. Immunol. 8:892. In such methods, the sample population is compared to a reference population of known CD56 expression levels, e.g., a cell population that includes NK cells. Because NK cells have different CD56 expression levels that can be distinguished as bright and dark, this reference population is used to establish a gating strategy to separate the sample population into a set of CD56-specific expression levels. bright and CD56dim They can also be divided into fractions.

[0082] In one embodiment, the cell population comprises γδ T cells expressing CD56, NKp30, CD57, GITR, TIGIT, CCR6, CCR2, CCR5 and / or CXCR6, e.g., CD56, NKp30, CD57, GITR and / or TIGIT. In further embodiments, the cell population comprises γδ T cells expressing CD56, NKp30, CD57, GITR and TIGIT. In some aspects, the cell population comprises γδ T cells expressing NKp30. In some aspects, the cell population comprises γδ T cells expressing NKG2D. In some aspects, the cell population comprises γδ T cells expressing DNAM-1. In some aspects, the cell population comprises γδ T cells expressing CD57. In some aspects, the cell population comprises γδ T cells expressing GITR. In some aspects, the cell population comprises γδ T cells expressing TIGIT. In some embodiments, the cell population comprises γδ T cells expressing CXCR3. In some embodiments, the cell population comprises γδ T cells expressing CXCR4. In some embodiments, the cell population comprises γδ T cells expressing CCR4. In some embodiments, the cell population comprises γδ T cells expressing CCR6. In some embodiments, the cell population comprises γδ T cells expressing CCR2. In some embodiments, the cell population comprises γδ T cells expressing CCR5. In some embodiments, the cell population comprises γδ T cells expressing CXCR6.

[0083] In some embodiments, the cell population comprises γδ T cells that secrete IFNδ. In some embodiments, the cell population comprises γδ T cells that secrete IFNδ at a higher level than cells grown using standard techniques. In some embodiments, the cell population comprises γδ T cells that secrete GM-CSF. In some embodiments, the cell population comprises γδ T cells that secrete GM-CSF at a higher level than cells grown using standard techniques. In some embodiments, the cell population comprises γδ T cells that secrete CXCL9. In some embodiments, the cell population comprises γδ T cells that secrete CXCL9 at a higher level than cells grown using standard techniques. In some embodiments, the cell population comprises γδ T cells that secrete CXCL10. In some embodiments, the cell population comprises γδ T cells that secrete CXCL10 at a higher level than cells grown using standard techniques. In some embodiments, the cell population comprises γδ T cells that secrete CCL3. In some embodiments, the cell population comprises γδ T cells that secrete CCL3 at a higher level than cells grown using standard techniques. In some embodiments, the cell population comprises γδ T cells that secrete CCL4. In some embodiments, the cell population comprises γδ T cells that secrete CCL4 at a higher level than cells grown using standard techniques. In some embodiments, the cell population comprises γδ T cells that secrete CCL7. In some embodiments, the cell population comprises γδ T cells that secrete CCL7 at a higher level than cells grown using standard techniques. In some embodiments, the cell population comprises γδ T cells that secrete CCL20. In some embodiments, the cell population comprises γδ T cells that secrete CCL20 at a higher level than cells grown using standard techniques.

[0084] The expansion methods provided herein, in some embodiments, result in an expanded cell population of γδ T cells (particularly Vδ1 T cells) having high expression of a particular marker compared to a reference population (e.g., a population of isolated γδ T cells prior to the expansion step). Such markers may include, for example, CD56, NKp30, CD57, GITR and / or TIGIT. In some embodiments, the expanded cell population of γδ T cells (particularly Vδ1 T cells) may have a frequency of CD56+ cells of more than about 20%, e.g., more than about 40%, more than about 50%, or more than about 60%. In some embodiments, more than about 60% of the cell population comprises γδ T cells (particularly Vδ1 T cells) expressing CD56. In some embodiments, the expanded cell population of γδ T cells (particularly Vδ1 T cells) may have a frequency of NKp30+ cells of more than about 20%, e.g., more than about 25%, more than about 30%, or more than about 35%. In some embodiments, more than about 35% of the cell population comprises γδ T cells expressing NKp30. In some embodiments, the expanded cell population of γδ T cells (particularly Vδ1 T cells) may have a frequency of CD57+ cells of more than about 20%, such as more than about 30%, more than about 40%, or more than about 50%. In some embodiments, more than about 50% of the cell population comprises γδ T cells expressing CD57. In some embodiments, the expanded cell population of γδ T cells (particularly Vδ1 T cells) may have a frequency of GITR+ cells of more than about 20%, such as more than about 30%, more than about 40%, or more than about 50%. In some embodiments, more than about 50% of the cell population comprises γδ T cells expressing GITR. In some embodiments, the expanded cell population of γδ T cells (particularly Vδ1 T cells) may have a frequency of TIGIT+ cells greater than about 25%, such as greater than about 30%, greater than about 35%, greater than about 40%, or greater than about 45%. In some embodiments, greater than about 45% of the cell population comprises γδ T cells that express GITR.

[0085] In one embodiment, the cell population comprises γδ T cells with low (or undetectable) expression of LAG-3, PD-1, and CTLA-4. In some embodiments, less than about 15% of the cell population comprises γδ T cells that express LAG-3, PD-1, or CTLA-4 (i.e., >15% of γδ T cells are LAG-3+, PD-1+, or CTLA-4+).

[0086] The expansion methods provided herein, in some embodiments, result in an expanded cell population of γδ T cells (particularly Vδ1 T cells) that has low expression of a particular marker compared to a reference population (e.g., a population of isolated γδ T cells prior to the expansion step). Such markers may include, for example, CD27. In some embodiments, the expanded cell population of γδ T cells (particularly Vδ1 T cells) may have a frequency of CD27+ cells of less than about 90%, e.g., less than about 85%, less than about 80%, or less than about 75%. In certain embodiments, the expanded cell population of γδ T cells (particularly Vδ1 T cells) has a frequency of CD27+ cells of less than 75%. In some embodiments, less than about 75% of the cell population comprises γδ T cells expressing CD27.

[0087] In one embodiment, the cell population comprises γδ T cells that have low (or undetectable) expression of LAG-3, PD-1, and CTLA-4. In some embodiments, less than about 15% of the cell population comprises γδ T cells that express LAG-3, PD-1, or CTLA-4 (i.e., has a frequency of LAG-3+, PD-1+, or CTLA-4+ cells less than 15%).

[0088] In one embodiment, the cell population comprises γδ T cells that have low (or undetectable) expression of CD62L and CCR7. In some embodiments, less than about 15% of the cell population comprises γδ T cells that express CD62L or CCR7 (i.e., has a frequency of CD62L+ or CCR7+ cells less than 15%).

[0089] In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) has increased persistence of in vivo cytotoxicity compared to similarly engineered cells obtained by standard methods. In some embodiments, a γδ T cell population obtained by the methods described herein can have in vivo cytotoxicity for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, or at least about 21 days. In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least about 7 days. In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least about 8 days. In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least about 9 days. In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least about 10 days. In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least about 11 days. In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least about 12 days. In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least about 13 days. In some embodiments, a γδ T cell population (e.g., a Vδ1 γδ T cell population) obtained by the methods described herein can have in vivo cytotoxicity for at least about 14 days.In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least about 15 days. In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least about 16 days. In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least about 17 days. In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least about 18 days. In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least about 19 days. In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least about 20 days. In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least about 21 days.

[0090] In some embodiments, a γδ T cell population (e.g., a Vδ1 γδ T cell population) obtained by the methods described herein is capable of in vivo cytotoxicity despite little proliferation. The efficacy of αβ T cell therapy depends on T cell proliferation, but the methods disclosed herein generate γδ T cells capable of highly effective cytotoxicity despite very little proliferation. In some embodiments, the γδ T cell population (e.g., a Vδ1 γδ T cell population) has an expansion rate of less than about 15-fold, less than about 14-fold, less than about 13-fold, less than about 12-fold, less than about 11-fold, less than about 10-fold, less than about 9-fold, less than about 8-fold, less than about 7-fold, less than about 6-fold, less than about 5-fold, less than about 4-fold, less than about 3-fold, or less than about 2-fold. In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least 7 days, wherein said γδ T cell population (e.g., a Vδ1 γδ T cell population) has a proliferation rate of less than 15 fold increase over said at least 7 days. In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least 10 days, wherein said γδ T cell population (e.g., a Vδ1 γδ T cell population) has a proliferation rate of less than 15 fold increase over said at least 10 days. In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least 14 days, wherein said γδ T cell population (e.g., a Vδ1 γδ T cell population) has a proliferation rate of less than 15 fold increase over said at least 14 days. In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least 18 days, wherein said γδ T cell population (e.g., a Vδ1 γδ T cell population) has a proliferation rate of less than 15 fold increase over said at least 18 days. In some embodiments, a γδ T cell population obtained by the methods described herein (e.g., a Vδ1 γδ T cell population) can have in vivo cytotoxicity for at least 21 days, wherein said γδ T cell population (e.g., a Vδ1 γδ T cell population) has a proliferation rate of less than 15 fold increase over said at least 21 days.

[0091] In some embodiments, the in vivo cytotoxicity is against cancer cells, hi some embodiments, the cancer cells are solid tumor cells. In some embodiments, the cancer is bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)), uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia (AML) (e.g., relapsed or refractory AML), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia (ALL), chronic myelogenous leukemia, leukemia), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer (including asbestos induced), or any combination thereof. In some embodiments, the cancer is locally advanced. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is refractory. In some embodiments, the cancer is recurrent. In some embodiments, the cancer is refractory or recurrent after one or more previous anti-cancer treatments. In some embodiments, the one or more previous anti-cancer treatments include standard of care.

[0092] Engineered cells The γδ T-cells described herein can also be genetically engineered to enhance therapeutic properties, for example, the cells can be engineered to express exogenous nucleic acids encoding cell surface receptors and / or secreted proteins.

[0093] One example of cell engineering is CAR-T therapy. This can include the generation of engineered receptors (e.g., chimeric antigen receptors or engineered T cell receptors) to reprogram T cells with new specificities, such as the specificity of monoclonal antibodies. The engineered receptors can make T cells specific to malignant cells, and thus useful for cancer immunotherapy. For example, the T cells may recognize cancer cells that express tumor antigens, such as tumor-specific antigens that are not expressed by normal somatic cells of the target tissue, tumor-associated antigens that are preferentially overexpressed in cancer cells compared to healthy somatic cells, or antigens that are expressed in the context of stress events, such as oxidative stress, DNA damage, UV irradiation, EGF receptor stimulation, or other means to distinguish between cancerous and non-cancerous cells. Thus, the CAR-engineered T cells can be used, for example, in adoptive T cell therapy for cancer patients.

[0094] According to a further aspect of the invention, there is provided a method for manipulating γδ T cells, the method comprising the steps of: (i) preparing a composition enriched for γδ T cells using a method described herein; (ii) transducing the composition with an exogenous nucleic acid for expression in the γδ T-cell; and (iii) culturing the transduced composition to expand the engineered γδ T-cells.

[0095] It will be appreciated that transducing the composition with an exogenous nucleic acid for expression in a γδ T-cell according to step (ii) produces an engineered γδ T-cell expressing the exogenous nucleic acid. In some embodiments, the exogenous nucleic acid encodes a surface receptor. In further embodiments, the surface receptor is a chimeric antigen receptor (CAR) that recognizes a tumor antigen.

[0096] In one embodiment, the method described herein comprises transducing the composition of γδ T cells to express a CAR that recognizes a surface receptor of interest, e.g., a tumor antigen. Any such CAR can be used in the present invention, including CARs that target CD19, mesothelin (MSLN), or other known tumor-associated antigens.

[0097] According to a further aspect of the invention, there is provided a method for manipulating γδ T cells, the method comprising the steps of: (i) preparing a composition enriched for γδ T cells according to the methods described herein; (ii) transducing the composition to express a chimeric antigen receptor (CAR); and (iii) culturing the transduced composition to expand the engineered γδ T-cells.

[0098] Thus, in some embodiments, step (iii) comprises culturing the transduced composition in the presence of feeder cells.

[0099] In certain embodiments, the composition is transduced using a viral vector. Such viral vectors are known in the art, and the skilled artisan will be able to recognize the appropriate viral vector to be used according to the cells to be transduced. In one embodiment, the viral vector is a lentiviral vector or a retroviral vector, for example, a gamma retroviral vector. In a further embodiment, the viral vector is a gamma retroviral vector, for example, a murine stem cell virus (MSCV) or a Moloney murine leukemia virus (MLV). In a further embodiment, the viral vector is pseudotyped with an envelope other than vesicular stomatitis virus G (VSV-G), for example, a beta retroviral envelope such as baboon endogenous virus (BaEV) or RD114.

[0100] In some embodiments, step (ii) comprises 1x10 6 ~1x108 TU / ml, e.g., about 1x10 6 , about 5x10 6 , Approx. 1x10 7 , about 5x10 7 , or about 1x10 8 In a particular embodiment, step (ii) is carried out using 1x10 TU / ml of viral vector. 7 TU / ml of viral vector. In other embodiments, step (ii) is performed using an MOI of 0.5-50 of the viral vector, e.g., an MOI of about 0.5, about 1, about 1.5, about 2.5, about 5, about 10, about 25, about 40, or about 50. In one embodiment, step (ii) is performed using an MOI of 2.5 of the viral vector. In another embodiment, step (ii) is performed using an MOI of 5 of the viral vector. In a further embodiment, step (ii) is performed using an MOI of 10 of the viral vector.

[0101] In one embodiment, the tumor-associated antigen is an antigen associated with a solid tumor. Thus, in some embodiments, the tumor and / or cancer is a solid tumor. Constitutive expression of CD70, a member of the tumor necrosis family, has been described in both hematological and solid tumors, and increases the survival of tumor cells and regulatory T cells in these tumor microenvironments by signaling through its receptor, CD27. Thus, in a further embodiment, the solid tumor expresses CD70. + It will be appreciated that CD70 can be used to target the engineered γδ T-cells to the tumor. Thus, in a further embodiment, the tumor-associated antigen is CD70.

[0102] In an alternative embodiment, the tumor-associated antigen is mesothelin (also referred to herein as "MSLN"). Mesothelin is a 40 kDa protein expressed in mesothelial cells and is overexpressed in several tumors, including mesothelioma, ovarian cancer, pancreatic adenocarcinoma, lung adenocarcinoma, and cholangiocarcinoma. It has therefore been proposed as a tumor marker or tumor-associated antigen that can be targeted in immunotherapy (Hassan et al. Clin. Cancer Res., 2004, 10(12):3937-3942). The expression of mesothelin in these tumors may contribute to tumor implantation and intraperitoneal spread through cell adhesion (Rump et al., Biological Chemistry, 2004, 279(10):9190-9198).

[0103] Expanded cell population According to one aspect of the invention there is provided an expanded population of γδ T cells obtained by the methods described herein. According to a further aspect there is provided an engineered population of γδ T cells obtained by the methods described herein.

[0104] In some embodiments, the expanded / engineered γδ T cell population comprises more than 50% γδ T cells, such as more than 75% γδ T cells, in particular more than 85% γδ T cells. In one embodiment, the expanded / engineered population comprises V51 cells, and less than 50% of the V51 cells express TIGIT. In one embodiment, the expanded / engineered population comprises V51 cells, and more than 50%, such as more than 60%, of the V51 cells express CD27.

[0105] In some embodiments, the expanded / engineered γδ T cell population comprises less than about 1% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises less than about 1% αβ T cells, 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, less than about 0.1% αβ T cells, less than about 0.09% αβ T cells, less than about 0.08% αβ T cells, less than about 0.07% αβ T cells. β T cells, less than about 0.06% αβ T cells, less than about 0.05% αβ T cells, less than about 0.04% αβ T cells, less than about 0.05% αβ T cells, less than about 0.04% αβ T cells, less than about 0.03% αβ T cells, less than about 0.02% αβ T cells, less than about 0.01% αβ T cells, less than about 0.009% αβ T cells, less than about 0.008% αβ T cells, less than about 0.007% αβ T cells, less than about 0.006% αβ T cells, less than about 0.005% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises less than about 0.9% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises less than about 0.8% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises less than about 0.7% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises less than about 0.6% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises less than about 0.5% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises less than about 0.4% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises less than about 0.3% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises less than about 0.2% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises less than about 0.1% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises less than about 0.09% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises less than about 0.08% αβ T cells.In some embodiments, the expanded / engineered γδ T cell population comprises less than about 0.07% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises less than about 0.06% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises less than about 0.05% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises less than about 0.04% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises less than about 0.03% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises less than about 0.02% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises less than about 0.01% αβ T cells. In some embodiments, the expanded / engineered γδ T cell population comprises a concentration of αβ T cells below the limit of detection.

[0106] The expanded / engineered γδ T cell population obtained by the methods described herein may be used as a medicine, for example for adoptive T cell therapy. This involves transplantation of the expanded / engineered population obtained by the methods into a patient. The therapy may be autologous, i.e., the γδ T cells may be transplanted into the same patient from which they were obtained, or the therapy may be allogeneic, i.e., γδ T cells from one person may be transplanted into a different patient. In cases involving allogeneic transplantation, the expanded / engineered population may be substantially free of αβ T cells. For example, αβ T cells may be depleted from the expanded / engineered population, for example after manipulation, using any suitable means known in the art (e.g., negative selection, e.g., using magnetic beads). The method of treatment may include obtaining a sample from a donor individual, expanding and / or manipulating the γδ T cells as described herein to produce an expanded / engineered population, and administering the expanded / engineered population of γδ T cells to a recipient individual.

[0107] In some embodiments, the expanded / engineered γδ T cell populations obtained by the methods described herein have increased persistence of in vivo cytotoxicity compared to similarly engineered cells obtained by standard methods. In some embodiments, the expanded / engineered γδ T cell populations obtained by the methods described herein can have in vivo cytotoxicity for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, or at least about 21 days. In some embodiments, the expanded / engineered γδ T cell populations obtained by the methods described herein can have in vivo cytotoxicity for at least about 7 days. In some embodiments, the expanded / engineered γδ T cell populations obtained by the methods described herein can have in vivo cytotoxicity for at least about 8 days. In some embodiments, the expanded / engineered γδ T cell population obtained by the methods described herein can have in vivo cytotoxicity for at least about 9 days. In some embodiments, the expanded / engineered γδ T cell population obtained by the methods described herein can have in vivo cytotoxicity for at least about 10 days. In some embodiments, the expanded / engineered γδ T cell population obtained by the methods described herein can have in vivo cytotoxicity for at least about 11 days. In some embodiments, the expanded / engineered γδ T cell population obtained by the methods described herein can have in vivo cytotoxicity for at least about 12 days. In some embodiments, the expanded / engineered γδ T cell population obtained by the methods described herein can have in vivo cytotoxicity for at least about 13 days. In some embodiments, the expanded / engineered γδ T cell population obtained by the methods described herein can have in vivo cytotoxicity for at least about 14 days.In some embodiments, the expanded / engineered γδ T cell population obtained by the methods described herein can have in vivo cytotoxicity for at least about 15 days. In some embodiments, the expanded / engineered γδ T cell population obtained by the methods described herein can have in vivo cytotoxicity for at least about 16 days. In some embodiments, the expanded / engineered γδ T cell population obtained by the methods described herein can have in vivo cytotoxicity for at least about 17 days. In some embodiments, the expanded / engineered γδ T cell population obtained by the methods described herein can have in vivo cytotoxicity for at least about 18 days. In some embodiments, the expanded / engineered γδ T cell population obtained by the methods described herein can have in vivo cytotoxicity for at least about 19 days. In some embodiments, the expanded / engineered γδ T cell population obtained by the methods described herein can have in vivo cytotoxicity for at least about 20 days. In some embodiments, the expanded / engineered γδ T cell population obtained by the methods described herein can have in vivo cytotoxicity for at least about 21 days.

[0108] In one embodiment, the expanded / engineered γδ T-cell population obtained by the methods described herein is for use in the treatment of cancer. As used herein, "cancer" refers to the abnormal growth or division of cells. Generally, the proliferation and / or lifespan of cancer cells exceeds and is uncoordinated with the proliferation and / or lifespan of the surrounding normal cells and tissues. Cancer can be benign, pre-cancerous, or malignant. Cancers occur in a variety of cells and tissues, including the oral cavity (e.g., mouth, tongue, pharynx, etc.), digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, bile duct, gallbladder, pancreas, etc.), respiratory system (e.g., larynx, lungs, bronchi, etc.), bones, joints, skin (e.g., basal cell, squamous cell, meningioma, etc.), breast, reproductive system, (e.g., uterus, ovaries, prostate, testes, etc.), urinary system (e.g., bladder, kidneys, ureters, etc.), eyes, nervous system (e.g., brain, etc.), endocrine system (e.g., thyroid, etc.), and hematopoietic system (e.g., lymphoma, myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, etc.).

[0109] In further embodiments, the cancer is a malignant solid tumor (also referred to herein as a solid tumor). A solid tumor may occur in several locations, such as tissue, bone, muscle, and / or organs. It is understood that a solid tumor does not include blood cancer (i.e., blood cancer). In alternative embodiments, the cancer is a blood cancer.

[0110] In some embodiments, the cancer is bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)), uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia (ALL), chronic myelogenous leukemia, leukemia), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer (including asbestos induced), or any combination thereof. In some embodiments, the cancer is locally advanced. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is refractory. In some embodiments, the cancer is recurrent. In some embodiments, the cancer is refractory or recurrent after one or more previous anti-cancer treatments. In some embodiments, the one or more previous anti-cancer treatments include standard of care.

[0111] The patient or subject to be treated is preferably a human cancer patient (e.g., a human cancer patient undergoing treatment for a solid tumor) or a virally infected patient (e.g., a CMV- or HIV-infected patient). In some cases, the patient has a solid tumor and / or is undergoing treatment for a solid tumor. Because tissue-resident V51 T cells normally reside in non-hematopoietic tissues, they are more likely to home to and reside within tumor masses than their systemic blood-resident counterparts, and adoptive transfer of these cells may be more effective in targeting solid tumors and potentially other non-hematopoietic tissue-associated immunopathologies.

[0112] γδ T cells are not MHC restricted and therefore do not recognise the host into which they are transplanted as foreign, meaning that they are unlikely to cause graft-versus-host disease, which means that they can be used "off the shelf" and transplanted into any recipient, for example for allogeneic adoptive T cell therapy.

[0113] The γδ T cells obtained by the methods described herein express NKG2D and respond to NKG2D ligands (e.g., MICA) that are strongly associated with malignant tumors. They also express a cytotoxic profile without activation, and therefore may be effective in killing tumor cells. For example, the expanded / engineered γδ T cells obtained as described herein may express one or more, preferably all, of IFN-γ, TNF-α, GM-CSF, CCL4, IL-13, granulysin, granzymes A and B, and perforin without activation. IL-17A may not be expressed.

[0114] The expanded / engineered γδ T cells obtained by the methods described herein may be suitable as "off-the-shelf" immunotherapeutics: these cells have innate-like killing, are not MHC-restricted, and show improved tumor homing and / or retention over other T cells.

[0115] In some embodiments, a method of treating an individual with a solid tumor in a non-hematopoietic tissue may comprise expanding and / or manipulating γδ T-cells from a sample from the individual to produce an expanded / engineered population as described herein, and administering the expanded / engineered population of γδ T-cells to the individual. In an alternative embodiment, the method of treatment comprises expanding and / or manipulating γδ T-cells from a sample from a different individual to produce an expanded / engineered population as described herein, and administering the expanded / engineered population of γδ T-cells to the individual with a solid tumor. In one embodiment, the amount of expanded / engineered γδ T-cells administered to the individual is a therapeutically effective amount.

[0116] In further embodiments, the therapeutic methods and / or therapeutically effective amounts include those disclosed in WO2020095058 or WO2020095059, the contents of which are incorporated in their entirety.

[0117] A pharmaceutical composition may comprise the expanded and / or engineered γδ T-cells described herein in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers, e.g., neutral buffered saline, phosphate buffered saline, etc., carbohydrates, e.g., glucose, mannose, sucrose or dextran, mannitol, proteins, polypeptides or amino acids, e.g., glycine, antioxidants, chelating agents, e.g., EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. Cryopreservation solutions that may be used in the pharmaceutical compositions of the invention include, e.g., DMSO. The compositions may be formulated, e.g., for intravenous administration.

[0118] Thus, according to another aspect of the invention there is provided a pharmaceutical composition comprising an expanded or engineered γδ T cell population as described herein.

[0119] In one embodiment, the pharmaceutical composition is substantially free (eg, completely free) of detectable levels of contaminants, such as endotoxin or mycoplasma.

[0120] According to a further aspect of the invention, there is provided an expanded γδ T cell population, an engineered γδ T cell population, or a pharmaceutical composition as described herein for use as a medicament. In another aspect, there is provided an expanded γδ T cell population, an engineered γδ T cell population, or a pharmaceutical composition as described herein for use in the treatment of cancer. In a further embodiment, the cancer is a solid tumor.

[0121] It will be understood that all embodiments described herein are applicable to all aspects of the invention.

[0122] As used herein, the term "about" includes up to, including 10% greater than, and up to, including 10% less than, the specified value, suitably including up to, including 5% greater than, and up to, including 5% less than, the specified value, and particularly including the specified value. The term "between" includes the boundary values ​​specified.

[0123] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology that are within the skill of those in the art and are fully described in the literature. For example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press), Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY), DNGlover ed., (1985) DNA Cloning, Volumes I and II, Gait, ed. (1984) Oligonucleotide. Synthesis, Mullis et al. U.S. Patent No. 4,683,195, Hames and Higgins, eds. (1984) Nucleic Acid Hybridization, Hames and Higgins, eds. (1984) Transcription And Translation, Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.), Immobilized Cells And Enzymes (IRL Press)(1986), Perbal(1984)A Practical Guide To Molecular Cloning, the treatise, Methods In Enzymology (Academic Press, Inc., NY), Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory), Wu et al., eds., Methods In Enzymology, Vols.154 and 155, Mayer and Walker, eds.(1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London), Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV, Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986)), Crooke, Antisense drug Technology: Principles, Strategies and See Applications, 2nd Ed. CRC Press (2007) and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.). .

[0124] All references cited above, and all references cited herein, are incorporated herein by reference in their entirety.

[0125] Certain aspects and embodiments of the present invention will now be described by way of the following examples and with reference to the above-mentioned figures. EXAMPLES

[0126] Example 1. Materials and Methods cell culture V51+ γδ T cell enriched cultures were generated according to the methods described below and summarized in Figure 1.

[0127] Method 1: Vδ1+γδ T cells were generated from αβ-TCR+ depleted peripheral blood mononuclear cells using serum-free medium (CTS OpTmizer, Thermo Fisher) supplemented with 2.5% pooled allogeneic plasma (Octaplas, Octapharma) and Glutamax (ThermoFisher). Isolated cells were seeded in the presence of recombinant IL-4 (Miltenyi), IL-1β (Miltenyi), IL-21 (CellGenix), IFNγ (Bio-Techne), and soluble anti-CD3 (clone OKT3) antibody (Biolegend). After setup, cultures were incubated at 37 °C and 5% CO in a humidified incubator. 2 The medium was supplemented with fresh IL-21, 39 ng / ml (CellGenix) and IL-15, 21 ng / ml (Bio-Techne) on day 7, and IL-15, 100 ng / ml (Bio-Techne) on day 11. After 14 days of culture, cells were harvested and cryopreserved as a mixed population in a Cryostor5 (STEMCELL Technologies).

[0128] Method 2: Vδ1+γδ T cells were generated from αβ-TCR+ depleted peripheral blood mononuclear cells using serum-free medium (CTS OpTmizer, Thermo Fisher) supplemented with 2.5% pooled allogeneic plasma (Octaplas, Octapharma) and Glutamax (ThermoFisher). Isolated cells were seeded in the presence of recombinant IL-15, 100 ng / ml (Bio-Techne), and soluble anti-CD3 (clone OKT3) antibody (Biolegend) without or with the exclusion of IL-21, e.g., IL-21. After setup, cultures were incubated at 37 °C and 5% CO in a humidified incubator. 2 The cells were incubated at 4°C for 14 days. The expanding cells were fed with fresh IL-15, 100 ng / ml (Bio-Techne). After 14 days of culture, the cells were harvested and cryopreserved as a mixed population in a Cryostor5 (STEMCELL Technologies).

[0129] Flow cytometric assessment of cell purity and surface phenotype Standard characterization: Immunophenotyping was performed using MACSQuant16 or BD FACSLyric™ flow cytometry instruments. The same protocol was applied to characterize both cell products at harvest and after cryopreservation. Dead cells were excluded using the LIVE / DEAD™ Fixable Aqua Dead Cell Stain kit (Invitrogen). Cells were analyzed for expression of surface markers using BV421-CD27, BV421-DNAM-1, BV605-PD-1, FITC-Mesothelin, PE-NKG2D, PE-NKp30, PE-Cy7-CD56, PE-Cy7-TIGIT, APC-TCRγδ, APC-Cy7-Vδ1 antibodies available from Miltenyi, BioLegend and ACRO Biosystems.

[0130] Deep characterization: Deep immunophenotypic characterization was performed using Biolegend's LEGENDScreen™ Human PE Kit (#700007), which included investigating the cell surface expression of 361 markers in cell products generated according to methods 1 or 2.

[0131] Quantification of secreted factors: Secreted proteins were quantified after cryopreservation. Briefly, V51+ γδ T cell products were thawed and co-cultured with OVCAR3 adherent tumor targets overnight. Supernatants were then collected and labeled using a 65-Plex human ProcartaPlex™ panel (catalog number: EPX650-10065-901) according to the manufacturer's instructions. Labeled samples were analyzed using the Luminex® FLEXMAP 3D platform. This assay allowed for the simultaneous detection and quantification of 65 secreted proteins, including cytokines and chemokines, during co-culture with target cells. Effector alone was used as a control.

[0132] Transduction with gammaretroviral vectors encoding anti-MSLN chimeric antigen receptors Cells were grown according to method 1 or 2 and transduced with a gamma-retroviral vector encoding a mesothelin (MSLN)-targeted chimeric antigen receptor in the presence of retronectin (20 μg / mL). The viral vector was mixed with immune cells diluted in a CTS OpTmizer overnight at 37°C. Transduction efficiency was determined by flow cytometry 4-9 days after transduction.

[0133] Thawing of cryopreserved cell products Frozen cryovials were thawed in a 37°C water bath and added to pre-warmed OpTmizer + 2.5% allogeneic plasma. Cells were spun down at 300g for 7 minutes, counted to assess viability, and then aliquoted at 2x10 for phenotyping and downstream assays. 6 Resuspended at 100 cells / mL.

[0134] Cytotoxicity assay The expanded cells were co-cultured with adherent tumor targets (e.g., ovarian adenocarcinoma cell line, OvCAR3), some of which express firefly luciferase, at various effector-to-target ratios. Target cells in the absence of effector served as a control (Control). Maximal lysis was determined using target cells treated with staurosporine. After 20 hours, quantification of target survival was assessed using the ONE-Glo™ Luciferase Assay System Kit (Promega) or the CellTiter-Glo® Luminescent Cell Viability Assay (Promega). Luminescence was measured on a Biotek Synergy H4 plate reader.

[0135] Repeated antigen stimulation assay Cryopreserved V51+ γδ T cell products were thawed and co-cultured with GFP-expressing adherent tumor targets (e.g., lung cancer cell line, A549-Meso+) in the presence of cytokines at an effector to target ratio of 2.5 per target. Cells were incubated at 37°C and 5% CO 2Cultures were incubated at 4 °C for 3 h in an Incucyte® Live Cell Analysis System (Sartorius). Vδ1+γδ T cell product was administered by adding new tumor targets to the cultures every 3-4 days. % GFP was used as a measure of remaining tumor targets at each time point of the assay. To calculate Vδ1+γδ fold expansion, cells were counted at each time point and flow cytometry was performed to identify enrichment of Vδ1+γδ.

[0136] Example 2. Comparison of cell products αβ-TCR+ depleted peripheral blood mononuclear cells were expanded using either method 1 or 2 as described in Example 1 and summarized in Figure 1. The expanded cell products were then analyzed and compared.

[0137] Cell yields at harvest are shown in Figure 2. Expanding cells according to method 2 gave comparable (or slightly less) cell yields than method 1. Method 2 reduced the variability in yields between donors.

[0138] Immune cell composition was analyzed by flow cytometry (Figure 3). Both expansion methods resulted in a pure γδ T cell product that was highly enriched (>50%) in Vδ1 T cells.

[0139] Vδ1 T cells generated using method 2 upregulated CD56, a key marker of cell product generated by this method. Representative flow plots are shown in Figure 4. Vδ1 T cells expanded by method 2 displayed distinct cell surface marker expression, with significantly lower expression of CD27 and higher expression of CD56, NKp30, CD57, GITR and TIGIT, consistent with a more effector-like phenotype. Markers associated with exhaustion, such as LAG-3, PD-1 and CTLA-4, remained low or were undetectable (Figure 5). Furthermore, expanding cells according to method 2 altered the expression of various chemokine receptors (Figure 6). CD62L and CCR7, two markers associated with lymph node retention, were significantly downregulated, while CCR6, a marker associated with peripheral retention, was significantly upregulated.

[0140] The cell products generated by methods 1 and 2 were deeply phenotyped using LegendScreen technology, which allows for the simultaneous analysis of 364 cell surface markers. Flow cytometry results were analyzed using ANOVA and represented as volcano plots. The analysis revealed distinct differentially expressed surface markers between the two growth methods (Figure 7A). Each individual dot in the volcano plot represents an individual cell surface marker analyzed. The higher the position of that dot in the y-axis range, the greater the significance of the differential expression of that marker between cells generated by method 2 when compared to method 1. The more dispersed the position of that dot in the x-axis range (higher or lower than 0), the greater the fold exchange expression of that individual marker between cells generated by method 2 when compared to method 1. Cell surface markers significantly altered by growing cells according to method 2 when compared to method 1 are mainly involved in signaling pathways of co-activation, adhesion / migration / retention, and immune organization (Figure 7B). The most relevant and significantly altered markers are listed in tabular form in Table 1 below. In summary, altered marker expression contributes to the acquisition of distinct functionality of cells generated by Method 2 when compared to Method 1.

[0141] [Table 1]

[0142] Cell products generated by methods 1 and 2 were analyzed for factor secretion using Luminex technology, which allows for the simultaneous detection and quantification of 65 proteins per sample. Principal component analysis (PCA) was performed to measure the variance in soluble factor secretion between samples prepared by methods 1 and 2 (Figure 8A). PCA analysis showed that samples generated by method 2 (closed circles) clustered separately from samples generated by method 1 (open circles), revealing a distinct soluble factor secretion profile of γδ T cell products generated by method 2 when compared to method 1. Vδ1 T cells expanded by method 2 showed a significant increase in secretion of effector cytokines, e.g., IFNγ and GM-CSF, compared to method 1 (Figure 8B). Furthermore, method 2 resulted in a significant increase in secretion of chemokines, e.g., CXCL9 and CXCL10, as well as a near-significant increase in CXCL11, CCL4, CCL7, CCL20 (Figure 8C).

[0143] Example 3. Functionality of cell products after storage The functionality of the cells was also examined. The frozen cell products were thawed according to Example 1. The total cell recovery and viability after cryopreservation were comparable between cell products generated using method 1 or 2. However, cells generated according to method 2 had slightly higher viability after cryopreservation (Figure 9). Importantly, the immune cell composition and cell surface phenotype of the cell products were retained after cryopreservation (data not shown).

[0144] The cytotoxicity of the cell products after cryopreservation was also examined. Cell products generated according to method 2 were significantly more cytotoxic against a broad range of solid tumor cancer cell lines across a range of effector-to-target ratios compared to cells generated using method 1 (Figure 10).

[0145] Example 4. Functionality of cell products after transduction Expanding cells were transduced with a gamma-retroviral vector as described in Example 1, and gene transfer efficiency was determined by flow cytometry. Expression of chimeric antigen receptor was identical between cells expanded by method 1 or 2, suggesting that the change in expansion method does not alter the permissiveness of V51 T cells to gammaretroviral transduction (Figure 11A). Representative flow characterization of CAR expression in cells transduced and expanded by method 1 or 2 can be seen in Figure 11B.

[0146] Furthermore, the functionality of the engineered cell products was also investigated in overnight cytotoxicity assays (Figure 12) and repeated tumor administration (Figure 13) in two different models using OVCAR3 and A549-Meso tumor cell lines. In the overnight cytotoxicity assays, CAR-transduced cells showed comparable cytotoxicity regardless of the method by which they were grown. Transduction with CAR significantly enhanced the overnight cytotoxicity of cell products grown according to method 1 (Figure 12). However, in a 21-day repeated antigen stimulation (RAS) assay in which our cell products were administered to tumors every 3-4 days, only cells grown according to method 2 transduced with CAR were able to control tumor growth up to day 21 in all models used (Figure 13A). Moreover, products grown according to method 2 were able to show superior tumor control during this assay with a significantly lower fold growth when compared to cell products grown according to method 1 (Figure 13B). This indicates that the cell product expanded by method 2 is even more capable of effective multiple rounds of serial killing and tumor control over time. Collectively, these observations highlight that endowing V51 T cells with natural cytotoxic function against tumor targets synergizes with engineering strategies. Thus, the culture process of method 2 enhances the functionality of the γδ cell product prior to and apart from engineering.

[0147] Example 5. Analysis of IL-15 concentrations V51+ γδ T cells are generated from αβ-TCR+ depleted peripheral blood mononuclear cells as described in Example 1. Isolated cells are seeded in the presence of various concentrations of recombinant IL-15 and soluble anti-CD3 (clone OKT3) antibody (Biolegend) without the presence of IL-21, e.g., omitting IL-21. After setup, cultures are maintained at 37°C and 5% CO in a humidified incubator. 2 The cells are incubated at 4°C for 12 h. The proliferating cells are fed with fresh IL-15 at various concentrations. After 14 days of culture, the cells are harvested and cryopreserved for further analysis. The concentrations of IL-15 examined include, but are not limited to, about 5 ng / mL to about 150 ng / mL (e.g., 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, and 150 ng / mL).

Claims

1. 1. A method for expanding γδ T cells, comprising: (1) preparing a composition enriched in γδ T cells by depleting αβ T cells from a sample obtained from a subject that contains γδ T cells; (2) administering the composition enriched for γδ T cells to (i) an anti-CD3 antibody or a fragment thereof, and (ii) culturing in the presence of interleukin-15 (IL-15) and in the absence of interleukin-4 (IL-4) from the first day of the culture; and (3) isolating the cultured cell population from the composition. The method comprising:

2. The method of claim 1 , wherein the sample is a hematopoietic sample or a fraction thereof.

3. The method according to claim 2, wherein the sample is selected from peripheral blood, umbilical cord blood, lymphoid tissue, thymus, bone marrow, spleen, lymph node tissue or a fraction thereof, in particular peripheral blood or a fraction thereof.

4. The method of claim 3, wherein the sample consists of peripheral blood mononuclear cells (PBMCs) or low density mononuclear cells (LDMCs).

5. The method of claim 1 , wherein the sample is a non-hematopoietic tissue.

6. The method according to any one of claims 1 to 5, wherein the subject is a human.

7. The method of any one of claims 1 to 6, comprising culturing the composition for 7 to 21 days.

8. 8. The method of claim 7, comprising culturing the composition for about 10, 11, 12, 13, or 14 days.

9. The method of any one of claims 1 to 8, wherein the anti-CD3 antibody is OKT3.

10. The method according to any one of claims 1 to 9, wherein expanding the population of γδ T cells provides at least a 5-fold, particularly at least a 10-fold, particularly at least a 20-fold increase in the number of γδ T cells.

11. The method of any one of claims 1 to 10, wherein at least 50% of the expanded γδ T cells present in the cell population express CD56.

12. The method of any one of claims 1 to 11, wherein the cell population comprises γδ T cells expressing NKp30, CD57, GITR, TIGIT, CCR6, CCR2, CCR5 and / or CXCR6.

13. The method of any one of claims 1 to 12, wherein the γδ T cells are derived from a single donor.

14. The method of any one of claims 1 to 12, wherein the γδ T cells are derived from multiple donors.

15. The method of any one of claims 1 to 14, comprising freezing the expanded γδ T cells.

16. 1. A method for manipulating γδ T cells, comprising: (i) preparing a composition enriched in γδ T cells using the method according to any one of claims 1 to 15, (ii) transducing the composition with an exogenous nucleic acid for expression in the γδ T-cell; and (iii) culturing the transduced composition to expand the engineered γδ T-cells. The method comprising:

17. 17. The method of claim 16, wherein the exogenous nucleic acid encodes a chimeric antigen receptor (CAR) that recognizes a tumor antigen.

18. 18. The method of claim 17, wherein the tumor antigen is a tumor-specific antigen that is not expressed by normal somatic cells of the target tissue.

19. 19. The method of claim 17 or claim 18, wherein the tumor antigen is a tumor-associated antigen that is preferentially overexpressed in cancer cells compared to healthy somatic cells.

20. The method according to any one of claims 17 to 19, wherein the tumor antigen is an antigen that is expressed in association with a stress event, such as oxidative stress, DNA damage, UV irradiation, EGF receptor stimulation.

21. The method according to any one of claims 17 to 20, wherein the tumor antigen is an antigen associated with a solid tumor.

22. The method of any one of claims 16 to 21, wherein the composition is transduced using a viral vector, such as a retroviral vector, such as a gamma retroviral vector or a lentiviral vector.

23. 23. The method of claim 22, wherein the viral vector is a gammaretroviral vector, such as murine stem cell virus (MSCV) or Moloney murine leukemia virus (MLV).

24. 24. The method of claim 22 or claim 23, wherein the viral vector is pseudotyped with an envelope other than vesicular stomatitis virus G (VSV-G), for example a betaretrovirus envelope such as baboon endogenous virus (BaEV) or RD114.

25. The method of any one of claims 16 to 24, wherein step (iii) comprises culturing the transduced composition in the absence of feeder cells.

26. The method of any one of claims 16 to 24, wherein step (iii) comprises culturing the transduced composition in the presence of feeder cells.

27. 27. An expanded γδ T cell population obtainable, eg obtained, by a method according to any one of claims 1 to 26.

28. 28. A pharmaceutical composition comprising the expanded γδ T cell population described in claim 27.

29. 29. The expanded γδ T cell population of claim 27 or the pharmaceutical composition of claim 28 for use as a medicament.

30. 29. The expanded γδ T cell population of claim 27 or the pharmaceutical composition of claim 28 for use in the treatment of cancer.

31. 31. The expanded γδ T-cell population or pharmaceutical composition for use according to claim 30, wherein the cancer is a solid tumor.

32. 29. The expanded γδ T-cell population of claim 27 or the pharmaceutical composition of claim 28, wherein the expanded γδ T-cells are capable of in vivo cytotoxicity for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, or at least about 21 days.

33. 29. The expanded γδ T cell population of claim 27 or the pharmaceutical composition of claim 28, wherein the expanded γδ T cells are capable of in vivo cytotoxicity for at least about 14 days.

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