Methods of expanding and production of gammadelta t cells

Abstract

The present disclosure relates to expanding and production of gamma delta (γδ) T cells, e.g., Vδ1+ γδ T cells. In particular, the present disclosure provides improved methods and processes for activating and expanding γδ T cells (e.g., Vδ1+ γδ T cells) using combined CD3 and CD30 agonists and cytokines.

Description

Attorney Docket No. MIL-051WO1METHODS OF EXPANDING AND PRODUCTION OF GAMMADELTA T CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U. S. Provisional Application No. 63 / 736,185 filed on December 19, 2024, the entire contents of which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] Immune cells (e.g., T cells) are a critical component of the body’s defense against foreign pathogens (e.g., bacteria and viruses) and malignant cancer cells. T cells can be divided into two subclasses: “conventional” alpha beta (aP) T cells and “unconventional” gamma delta (y5) T cells. y5 T cells comprise approximately 1-5% of the total population of PBMCs in the blood and can be found in higher proportions in secondary lymphoid organs (e.g., gut mucosa and skin). These immune cells have also been found in various cancerous tissue types, including lung and melanoma to a similar or greater extent that CD8+ tumor infiltrating lymphocytes have been found. Particularly, y5 T cells have a demonstrated capacity for cytotoxicity by secreting large amounts of pro-inflammatory cytokines (e.g., TNFs and ILs). Recent studies also demonstrate that the majority of y5 T cells infiltrating cancer tissues is V51 T cells. These observations suggest great potential of y5 T cells, e.g., V51 T cells as effectors for next-generation immunotherapies.

[0003] y5 T cells can be derived from ex vivo from pluripotent stem cells (i.e., iPSCs) or isolated from primary blood cells. To obtain y5 T cells for use in therapeutic applications, processes and methods to expand y5 T cells are useful for expanding y5 T cells mediated cell therapy. The inventors herein designed processes for preparing viable and highly active y5 T cells from non-stem cells, such as whole blood and skin.SUMMARY OF INVENTION

[0004] The present invention provides, among other things, methods and processes of expanding gamma delta (y5) T cells comprising activating gamma delta (y5) T cells in a cell population in the presence of a CD3 agonist and a CD30 agonist prior to expanding said gamma delta (y5) T cells. The present invention also provides y5 T cells (e.g., V51+ y5 T cells) prepared using methods and processes disclosed herein, and compositions comprising y5 T cells (e.g., V51+ y5 T cells) and methods of use thereof. The cell population is not aAttorney Docket No. MIL-051WO1pluripotent cell population (e.g., induced pluripotent stem cell (iPSC), or a cell population derived from the pluripotent cell population) (e.g., the cell population comprises primary cells derived from blood and / or non-hematopoietic tissue).

[0005] In some embodiments, the cell population is from a blood sample (e.g., a peripheral blood sample, a cord blood sample), or a non- hematopoietic tissue (e.g., skin). In one embodiment, the cell population is from a human peripheral blood sample. In one embodiment, the cell population is from a human cord blood sample. In some embodiments, the cell population is from a human skin explant.

[0006] In some embodiments, the gamma delta (y5) T cells are V51+ y5 T cells.

[0007] In some embodiments, the CD3 agonist is an anti-CD3 antibody, or a functional fragment thereof.

[0008] In some embodiments, the CD30 agonist is an anti-CD30 antibody, or a functional fragment thereof.

[0009] In some embodiments, the y5 T cells are activated in the presence of the CD3 agonist and the CD30 agonist for a period of 1 to 5 days. In some embodiments, the y5 T cells are activated in the presence of the CD3 agonist and the CD30 agonist for 1 day, 2 days, 3 days, 4 days or 5 days.

[0010] In some embodiments, the cell population is cultured in a cell culture medium comprising one or more cytokines from IL-7, IL- 15, IL- 18 IL-21 and combinations thereof, for activating the y5 T cells.

[0011] In some embodiments, the method comprises expanding the activated y5 T cells in the presence of one or more cytokines from IL-2, IL-7, IL-15, IL-18 IL-21 and combinations thereof, and / or a CD30 agonist.

[0012] In some embodiments, the cell population is from a blood sample (e.g., a peripheral blood sample, a cord blood sample). The y5 T cells from blood is activated in the culture medium comprising IL-18 and IL-21.

[0013] In some embodiments, the activated y5 T cells are expanded in the culture medium comprising the CD30 agonist, IL-2, IL-15 and IL-21, or in the culture medium comprising IL-2, IL-7, IL-15, IL-21 and optionally, with a CD30 agonist.

[0014] In some embodiments, the mixed cell population is from a non- hematopoietic tissue (e.g., skin). The y5 T cells are activated in the culture medium comprising IL-7, IL-15, IL-18 and IL-21.Attorney Docket No. MIL-051WO1

[0015] In some embodiments, the activated y5 T cells are expanded in the culture medium comprising IL-7 and IL-15.

[0016] In some embodiments, the expanded y5 T cells are harvested at Day 14.

[0017] In some embodiments, the y5 T cells are expanded at least 100-fold as compared to y5 T cells before the activation and expansion. In some embodiments, the gamma-delta T cells from blood are expanded 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold or more.

[0018] In some embodiments, the y5 T cells have increased post-thaw survival rate.

[0019] In some embodiments, the y5 T cells have increased cytotoxicity.

[0020] In another aspect, the present invention provides methods of expanding y5 T cells from a mixed cell population derived from a non-hematopoietic tissue comprising activating y5 T cells in the mixed cell population in the presence of a CD3 agonist and a CD30 agonist, in a culture medium comprising one or more cytokines selected from IL-7, IL-15, IL-18 and IL-21; and expanding the activated y5 T cells in a culture medium comprising IL-7 and IL-15.

[0021] In some embodiments, the non-hematopoietic tissue is a skin tissue.

[0022] In some embodiments, the culture medium for activating the y5 T cells comprises cytokines IL-7, IL- 15, IL- 18 and IL-21.

[0023] In some embodiments, the y5 T cells are activated for 1 to 3 days. In some embodiments, the y5 T cells are activated for 1 day. In some embodiments, the y5 T cells are activated for 2 days. In some embodiments, the y5 T cells are activated for 3 days.

[0024] In some embodiments, the activated y5 T cells are expanded in the cell culture medium comprising IL-7 and IL-15 for 1 to 14 days.

[0025] In some embodiments, the method comprises repeating the activation and expansion steps to further expand the y5 T cells.

[0026] In some embodiments, the y5 T cells from skin are expanded by at least 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold or more.

[0027] In another aspect, the present invention provides a method of expanding y5 T cells from a mixed cell population derived from a blood sample comprising activating y5 T cells in the mixed cell population in the presence of a CD3 agonist and a CD30 agonist, in a culture medium comprising one or more cytokines selected from IL-7, IL-15, IL-18 and IL-Attorney Docket No. MIL-051WO121; and expanding the activated y5 T cells in a cell culture medium comprising the CD30 agonist and one or more cytokines selected from IL-2, IL-7, IL- 15 and IL-21.

[0028] In some embodiments, the blood sample is a peripheral blood sample, or a cord blood sample.

[0029] In some embodiments, the cell culture medium for activating the y5 T cells comprises cytokines IL-7, IL-15, IL-18 and IL-21.

[0030] In some embodiments, the cell culture medium for activating the y5 T cells comprises cytokines IL- 15, IL- 18 and IL-21.

[0031] In some embodiments, the activated y5 T cells are expanded in the cell culture medium comprising cytokines IL-7 and IL-15.

[0032] In some embodiments, the activated y5 T cells are expanded in the cell culture medium comprising cytokines IL-2, IL-7 and IL-15.

[0033] In some embodiments, the activated y5 T cells are expanded in the cell culture medium comprising cytokines IL-2, IL-7, IL- 15 and IL-21.

[0034] In some embodiments, the activated y5 T cells are expanded in the cell culture medium comprising cytokines IL-2, IL- 15 and IL-21.

[0035] In some embodiments, the total fold expansion of gamma delta T cells is between 200 to 1600-fold.

[0036] In some embodiments, the CD3 agonist is an anti-CD3 antibody, or a functional fragment thereof; and wherein the CD30 agonist is an anti-CD30 antibody, or a functional fragment thereof.

[0037] In some embodiments, the y5 T cells are V51+ y5 T cells.

[0038] In some embodiments, the method further comprises depleting other non- y5 T cells from the mixed cell population prior to activating and expanding the y5 T cells.

[0039] In some embodiments, the cell culture medium comprises 5% human platelet lysate (HPL) and 5% OctaPlas.

[0040] In some embodiments, the V51+ y5 T cells have increased cytotoxicity.

[0041] In another aspect, the present invention provides a composition comprising the y5 T cells manufactured by the method of any one of the preceding claims.Attorney Docket No. MIL-051WO1

[0042] In some embodiments, the y5 T cells are for allogeneic cell therapy.

[0043] In another aspect, the present invention provides a method for treatment of a disease using y5 T cells produced by a method described herein.

[0044] In some embodiments, the y5 T cells are used for allogeneic cell therapy

[0045] In another aspect, the present invention provides a method of treating a disease; the method comprises administering to a subject in need thereof, a composition comprising the y5 T cells manufactured by a method described herein.

[0046] In some embodiments, the disease is a cancer, an infectious disease, an autoimmune disease, or an inflammatory disease.BRIEF DESCRIPTION OF DRAWINGS

[0047] The drawings are for illustrative purposes and should not be limiting.

[0048] FIGS. 1A - 1B show flow diagrams of exemplary conditions in which immune cells of interest were expanded from blood. FIG. 1A depicts the expansion conditions for conditions 1-3, wherein FIG. 1B shows conditions 4-6.

[0049] FIGS. 2A - 2B show that the addition of the exemplary agonist antibody, anti- CD30, boosts blood-derived y5 T cell expansion between Days 5 and 14. Compared to anti-CD30 isotype control or without anti-CD30 antibody controls, fold expansions of y5 T cells were augmented when cultured in the media with the anti-CD30 antibody. FIG. 2A shows expansion data from healthy Donor- 1, whereas FIG. 2B shows expansion data from healthy Donor-2. The term ‘w / o’ in the figures is defined herein as without; the term ‘w’ in the figures is defined herein as with; and the term ‘Ab’ in the figures is defined herein as antibody.

[0050] FIGS. 3A -3B demonstrate that the addition of the exemplary agonist anti-CD30 antibody promotes post-thaw survival of blood-derived y5 T cells. Frozen y5 T cell products were thawed and cultured in the basal media with 70 ng / mL IL- 15 for 7 days. y5 T cells manufactured with anti-CD30 antibody appeared better post-thaw proliferation rates compared to anti-CD30 isotype or without anti-CD30 antibody controls. FIG. 3A shows expansion data from healthy Donor- 1, whereas FIG. 3B shows expansion data from healthy Donor-2. The term ‘w / o’ in the figures is defined herein as without; the term ‘w’ in theAttorney Docket No. MIL-051WO1figures is defined herein as with; and the term ‘Ab’ in the figures is defined herein as antibody.

[0051] FIGS. 4A - 4B show that the addition of the exemplary agonist anti-CD30 antibody enhances y5 T cell cytotoxicity against tumor cells. Blood-derived y5 T cells were co-cultured with GSU cells (target cells) in the basal media containing 10 ng / mL IL-15 for 14 days. GSU cells were added every 3-4 days to rechallenge y5 T cells. GSU cell numbers were measured using Incucyte software every 4 hours. y5 T cells manufactured with anti-CD30 antibody appeared better cytotoxicity against tumor cells compared to anti-CD30 isotype or without anti-CD30 antibody controls. FIG. 4A shows cytotoxicity data from healthy Donor-1, whereas FIG. 4B shows cytotoxicity data from healthy Donor-2. The term ‘w / o’ in the figures is defined herein as without; the term ‘w’ in the figures is defined herein as with; and the term ‘Ab’ in the figures is defined herein as antibody.

[0052] FIGS. 5A - 5B demonstrate that the addition of the exemplary agonist anti-CD30 antibody improves antigen stimulated proliferation of V51 subset of y5 T cells. y5 T cells were co-cultured with GSU cells in the basal media containing 10 ng / mL IL-15 for 14 days. GSU cells were added every 3-4 days to rechallenge y5 T cells. V51 cells in total viable y5 T cells were analyzed using flow cytometry at Day 0, Day 7 and Day 14. Compared to anti-CD30 isotype or without anti-CD30 antibody controls, V51 cells manufactured with anti-CD30 antibody showed higher antigen stimulated proliferation. FIG. 5A shows expansion data from healthy Donor- 1, whereas FIG. 5B shows expansion data from healthy Donor-2. The term ‘w / o’ in the figures is defined herein as without; the term ‘w’ in the figures is defined herein as with; and the term ‘Ab’ in the figures is defined herein as antibody.

[0053] FIGS. 6A - 6B visualize exemplary flow charts for the expansion of cells utilizing a cocktail of cytokines and the anti-CD3 (RN / OKT3) and anti-CD30 agonist antibodies for activation on after seeding from Day 0 to Day 3, with expansion upon incubation with the cytokines IL-7 and IL-15 from Day 4 to Day 14 in fetal bovine serum (FBS). FIG. 6A exemplifies one cycle of such an expansion protocol, whereas FIG. 6B shows the option of repeated activation on Day 14 to Day 17, and expansion from Day 17 to Day 28 in the same manner in FBS.

[0054] FIGS. 7A - 7B show that addition of exemplary agonist antibodies, anti-CD3 (RN / OKT3) and anti-CD30, to the expansion protocol as compared to just cytokine-basedAttorney Docket No. MIL-051WO1control expansion significantly improves overall total live-cell fold expansion (FIG. 7A) and has comparable viability at harvest (FIG. 7B) for all skin donors.

[0055] FIGS. 8A - 8D show that the exemplary protocol for expanding y5 T cells, including the V51 subset, with the anti-CD3 and anti-CD30 agonist antibodies cultured from skin explants have a significantly increased fold expansion (FIG. 8A - 8B) and comparable viability (FIG. 8C - 8D) relative to the cytokine-based control expansion protocols.

[0056] FIG. 9 demonstrates that by using the exemplary cell expansion with the anti-CD3 / anti-CD30 process enables repeat activation and extension of culture period up to 28 days to enhance total cell yield.

[0057] FIGS. 10A - 10B show that both of the exemplary agonist antibodies, anti-CD3 and anti-CD30, are required in for optimal cell expansion. FIG. 10A exemplifies that removal of either agonist antibody decreases the average fold-expansion. FIG. 10B shows that the anti-CD30 isotype control does not maintain high cell expansion.

[0058] FIGS. 11A - 11B show that y5 T cells expanded in the exemplary anti-CD3 / anti-CD30 expansion process have comparable or higher overnight cytotoxicity (FIG. 11A) measured as percent killing against OVCAR-3 cells (n=6, mean+ / - SEM) and perform well in repeat antigen stimulation assay in which target cells are repeatedly added (marked with arrows) in continuous culture and measured on the incucyte (n=l representative donor) (FIG.11B)

[0059] FIGS. 12A - 12B demonstrate that the fetal bovine serum (FBS) used in the anti-CD3 / anti-CD30 activation and expansion process can be replaced by 5% human platelet lysate (HPL) and 5% OctaPlas to enable a xeno-free process without impaction to expansion (FIG. 12A) or overnight cytotoxicity (FIG. 12B).DETAILED DESCRIPTION

[0060] The present disclosure provides, among other things, improved processes and methods for expanding y5 T cells which are derived from non-stem cells. In particular, the present disclosure provides methods for activating and expanding y5 T cells from primary cells like blood cells and non-hematopoietic tissues (e.g., skin). The y5 T cells are V51+ y5 T cells. The present disclosure also provides compositions comprising y5 T cells and methods of use thereof. The improved processes as described herein, relate to a strategy for activation of y5 T cells (e.g., V51+ y5 T cells) with dual agonists including a CD3 agonist and a CD30Attorney Docket No. MIL-051WO1agonist and subsequent expansion by specific cytokines. In some aspects, the improved methods increase recovery of y5 T cells after cryopreservation. In some aspects, the improved methods can shorten the time required for expanding y5 T cells, e.g., V51+ y5 T cells.DEFINITIONS

[0061] In order that the present disclosure can be more readily understood, certain terms are first defined Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art.

[0062] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. The terms “comprise”, “comprises” and “comprising” and variations thereof (e.g., “comprises / comprising”, “includes / including”) should be understood to imply the inclusion of a stated component, feature, element or step or group of components, features, elements or steps but not the exclusion of any other component, feature, element or step or group of components, features, elements or steps. Any one of the terms "comprising," consisting essentially of, "and" consisting of may be substituted with either of the other two terms, while retaining their ordinary meaning.

[0063] Activation: As used herein, the term “activation” refers to the process by which a cell is stimulated for an intended function. In preferred embodiments, the term “activation” may refer to a stimulus that induces cellular division.

[0064] Administer'. As used herein, the “administer” and its various grammatical forms refers to giving, applying or bringing a composition into contact with a subject.Administration can be accomplished by any of a number of routes, such as, for example, topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal, and intradermal.

[0065] Allogeneic.' The term “allogeneic” as used herein means that a donor and a recipient are of different genetic makeup, but of the same species. As used herein, an “allogeneic cell” refers to a cell that is not derived from the individual to which the cell is to be administered, that is, it has a genetic constitution different from the recipient individual. An allogeneic cell is generally obtained from the same species as the recipient individual to which the cell is to be administered. For example, the allogeneic cell can be a human cell, as disclosed herein, for administering to a human patient.Attorney Docket No. MIL-051WO1

[0066] Allogeneic cell therapy: As used herein, the term “allogeneic cell therapy” refers to a type of cellular therapy that uses cells from a donor (e.g., a healthy person) to treat a patient. Cells from a single donor can be used to treat one or more patients. Donor cells (e.g., V51 y5 T cells isolated from a donor’s blood) can undergo extensive expansion and screening before establishing a master cell bank, which is the basis for generating allogeneic cell therapies. Cells from the donor can be modified (e.g., expressing a chimeric antigen receptor (CAR)) or unmodified. The cells can be derived from multiple potential cell sources, including but not limited to donated tissues, umbilical cord blood, placenta, bone marrow, and induced pluripotent and embryonic stem cells. An advantage of allogeneic strategies, among other things, is “off-the-shelf’ supply. For example, in urgent medical situations, allogeneic therapies utilize donor’s cells, allowing storage and immediate availability to treat multiple patients. Exemplary allogeneic cell therapies include CAR-T cell therapy.Allogeneic CAR-T cell therapy involves engineering T cells from a donor to express chimeric receptors that target cancer cells which are then infused into patients. In the context of the present invention, the y5 T cells may be produced for the manufacture of a medicament for treating various cancers.

[0067] Antibody, as used herein, the term “antibody,” refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The term is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigenbinding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), diabodies, and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, heteroconjugate antibodies, multispecific (e.g., bispecific) antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.

[0068] Agonist'. As used herein, the term “agonist” refers to a molecule that initiates a physiological response when in complex with a receptor. In various embodiments, anti-CD3Attorney Docket No. MIL-051WO1and anti-CD30 agonist antibodies may be used for the activation and proliferation of primary immune cells, such as y5 T cells.

[0069] Cancer-. As used herein, the term “cancer” refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, leukemia, lymphoma, multiple myeloma, ovarian cancer, breast cancer, endometrial cancer, colon cancer (colorectal cancer), rectal cancer, bladder cancer, urothelial cancer, lung cancer (non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung), bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, gall bladder cancer, bile duct cancer, esophageal cancer, renal cell carcinoma, thyroid cancer, squamous cell carcinoma of the head and neck (head and neck cancer), testicular cancer, cancer of the endocrine gland, cancer of the adrenal gland, cancer of the pituitary gland, cancer of the skin, cancer of soft tissues, cancer of blood vessels, cancer of brain, cancer of nerves, cancer of eyes, cancer of meninges, cancer of oropharynx, cancer of hypopharynx, cancer of cervix, and cancer of uterus, glioblastoma, meduloblastoma, astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma, melanoma, myelodysplastic syndrome, and a sarcoma and the like.

[0070] Cell population'. As used herein, the term "cell population" refers to a number of cells obtained by isolation directly from a suitable source, usually from a mammal. The isolated cell population may be subsequently cultured in vitro. Those of ordinary skill in the art will appreciate that various methods for isolating and culturing cell populations for use with the present invention and various numbers of cells in a cell population that are suitable for use in the present invention. In the context of the present invention, a cell population may be, for example, a mixed heterogeneous cell population derived from a peripheral blood sample, a cord blood sample, a tissue, a lymph, or from epithelial sites of a subject directly contacting the external milieu.

[0071] Cryopreserve'. As used herein, the term “cry opreserve”, “cry opreservation”, or “cryopreserved” refers to a process including at least one step of lowering the temperature of a biological material (e.g., a cell therapy product) from a temperature that is above the freezing temperature of the biological material (or of a mixture of the biological material and a cry opreservation composition) to a temperature that is below that freezing temperature. Cryopreservation encompasses freezing, vitrification and lyophilization. TheAttorney Docket No. MIL-051WO1term “cryopreservation of cells” means to freeze and preserve cells for the purpose of maintaining the cells over a desired period of time without sub-culturing.

[0072] Culture'. The term “culture” or “cell culture” or “culturing” refers to the maintenance, growth and / or differentiation of cells in an in vitro environment. In various methods described herein, cells are cultured in a particular cell culture medium (or “media” in case of a plural) which facilitates or promotes the growth or differentiation of one type of cell into a different type of cell. A cell culture medium acts as a source of nutrients, hormones and / or other factors helpful to propagate and / or sustain the cells.

[0073] Cytokine-. As used herein, the term “cytokine” refers to a large class of secreted proteins, peptides, and / or glycoproteins that are essential in cell-to-cell communication. In the broadest sense of the term, cytokines are proteins that are secreted cells of the immune system to control growth and function of other immune cells, as well as the inflammatory response. The term also encompasses several classes of proteins, including but not limited to, tumor necrosis factors (TNFs), interferons (IFNs), and interleukins (ILs). In various embodiments, the cytokines used for activation and expansion of immune cells include, but are not limited to IL-7, IL- 15, IL- 18, and IL-21.

[0074] Derived: As used herein, the term “derived” refers to obtaining something from a specified source. In the present disclosure, the term “derived” refers to obtaining y5 cells from either blood or non-hematopoietic tissue (e.g., skin).

[0075] Donor-. As used herein, the term “donor” refers to a person that willingly gives blood, tissue, or organs for use in scientific studies.

[0076] Enhance'. As used herein, the term "enhance" refers to an increase or improvement in the function or activity of a cell after administration or contacting witha combination described herein compared to the cell prior to such administration or contact.

[0077] Expansion: As used herein, the term “expansion” or “expanding” refers to a method of growing a cell population in tissue culture that increases the number of cells in that population. Cells that have undergone ex vivo expansion are referred to as “expanded”. A synonym that may be used for “expansion” in this context is “proliferation”.

[0078] Expanded population of Gamma Delta T cells'. As used herein, “expanded” or “expanded population of gamma delta T cells” includes populations of cells which are larger or contain a larger number of cells than a non-expanded population. Such populations may be large in number, small in number or a mixed population with the expansion of a proportion or particular cell type within the population. It will be appreciated that the term “expansionAttorney Docket No. MIL-051WO1step” refers to processes which result in expansion or an expanded population. Thus, expansion or an expanded population may be larger in number or contain a larger number of cells compared to a population which has not had an expansion step performed or prior to any expansion step. It will be further appreciated that any numbers indicated herein to indicate expansion (e.g., fold-increase or fold-expansion) are illustrative of an increase in the number or size of a population of cells or the number of cells and are indicative of the amount of expansion.

[0079] Immune cells'. As used herein, the term “immune cell” or “immune cells” refers to cells of the immune system, including, but not limited to, aP T cells, y5 T cells, NK cells, T / NK cells, dendritic cells, macrophages, B cells, neutrophils, erythrocytes, monocytes, basophils, neutrophils, mast cells, eosinophils, and any combination thereof.

[0080] Gamma delta (yd) T cells'. Gamma delta (y5) T cells refer to a subset of T cells that express their surface a distinct and defining T cell receptor (TCR). This TCR is composed of one gamma chain (y) and one delta (5) chain. The heterodimer y5 TCR can recognize antigens. y5 T cells can display a pre-activated and memory phenotype and the high frequency of these cells enables rapid responses without the presence of cognate TCR agonists and / or cellular expansion. y5 T cells are mainly from peripheral blood and non-blood tissues such as the skin with a restricted TCR repertoire. In humans, y5 T cells are classified according to their V5 gene segment used. Three V5 genes V51-3; and seven functional Vy gene segments: Vy2-5, Vy8, Vy9, and Vyl 1, exist in human y5 T cells. The y5 TCR repertoire is also associated with the tissue distribution. For example, V51+T cells are abundant in the epithelium in human. In humans and non-human primates, y5 T cells bearing the V52 chain are the main subset present in peripheral blood and this 5 chain is generally associated to the Vy9. Like aP T lymphocytes, the activation of y5 T cells through the TCR requires the participation of accessory molecules. CD3 and CD30 have been identified as coeffectors of the TCR activation.

[0081] Explant: As used herein, the term “explant” refers to a cell, organ, or piece of tissue which has been transferred from animals or plants to a nutrient medium.

[0082] Ex vivo: As used herein, the term “ex vivo" refers to events that occur outside of a living organism, e.g., in tissue taken from an organism in an external environment.

[0083] In vitro'. As used herein, the term “in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.Attorney Docket No. MIL-051WO1

[0084] In vivo'. As used herein, the term “in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cellbased systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0085] Storage'. As used herein, the term “storage” or “storing” refers to the placing of cells under relatively constant and / or ambient conditions for extended periods of time, for example, for at least about one hour, at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about one week, at least about two weeks, at least about four weeks, at least about three months, at least about six months, at least about one year, at least about two years, or even longer in some cases, without making use of their viability, e.g., without rehydrating or otherwise activating the cells. The cells are typically in the dormant, non-physiologically active state during storage (e.g., in a dried state, a frozen state, etc.). Typically, during storage, cells stored under relatively constant and / or ambient conditions are left undisturbed or are only minimally disturbed (e.g., the cells may be moved from one location to another, the temperature may fluctuate around a setpoint, etc.). For example, if the temperature is to be controlled during storage (e.g., at a certain setpoint), the cells may be kept in a liquid nitrogen freezer (e.g., a freezer that is cooled using liquid nitrogen, i.e., at a temperature of about -196° C ), a -80° C. freezer, a conventional freezer (e.g., that maintains an environment of roughly -10° C ), a refrigerator (e.g., about 4° C ), an incubator (e.g., at body temperature, about 37° C ), a (heated) water bath, or the like. Accordingly, A “storage temperature” is the temperature at which the biological material is stored. In certain embodiments, the storage temperature is at or below about 8° C., at or below about 4° C., at or below about 0° C., at or below about -20° C., at or below about -60° C., at or below about -70° C., at or below about -135° C., at or below about -196° C., or in liquid nitrogen.

[0086] Viability. As used herein the term “viability” or “viable” refers to a cell that is capable of normal growth and development after having been cryopreserved and thawed. One of the most crucial factors in any cryobiological procedure is defining the term “viability”. That definition should include one or more critical functions that accurately reflect how the cell normally carries out its biological function. The ultimate test for viability is the reproduction of the cell through two or more generations after freezing. Any methods known to those skilled in the art, such as alamar blue assays, may also be used for cell viability measurements.Attorney Docket No. MIL-051WO1Methods of preparing gamma Delta (y6) T Cells

[0087] Human y8 T-cells use 3 main vS (v81, v82, v83) and at most six Vy region genes to make their TCRs. Among the four subtypes of human y5 T cells defined by the TCR 5 chain, V51 y5 T cells are one of the two subtypes that are the most predominant. V51 y5 T cells, for example, recognize target cells and mediate anti-tumor activity through the direct lysis of transformed cells. In accordance, the present disclosure provides improved methods for expanding V51 y5 T cells.

[0088] In one aspect, the present disclosure provides methods for activating and expanding y5 T cells from a cell population. The cell population may be a mixed cell population comprising y5 T cells prepared from blood, or isolated from a tissue. The cell population comprising y5 T cells is not a pluripotent cell population (e.g., induced pluripotent stem cell (iPSC), or a cell population derived from induced pluripotent stem cells (e.g., iPSCs). In some embodiments, the present disclosure provides methods for activating and expanding V51+ y5 T cells from a cell population. Methods and processes disclosed herein can be used for expanding y5 T cells (e.g., V51+ y5 T cells), as cell therapy. Methods of expanding y5 T cells described herein involve activating gamma delta (y5) T cells in a cell population in the presence of a CD3 agonist and a CD30 agonist prior to expanding y5 T cells. In some embodiments, the methods described herein involve culturing a cell population comprising y5 T cells, with a CD3 agonist and a CD30 agonist for several days, thereby activating y5 T cells within the cell population; the activated y5 T cells are further cultured in a cell culture medium containing a suitable cytokine cocktail that can expand activated y5 T cells within the cell population.

[0089] In various methods and processes disclosed herein, the cells being activated and expanded are cultured in any suitable cell culture medium supplemented with other agents and factors (e.g., cytokines) for cell culture.

[0090] In one aspect, the method disclosed herein is a serial activation and expansion of a population of cells in which a complete population of y5 T cells is activated and expanded. Accordingly, the activation and expansion cycle may be repeated one or more times until a desired amount of y5 T cells is achieved.

[0091] In some embodiments, the activated and expanded y5 T cells, which are the cultured cells, are optionally transfected with e.g., a T cell receptor or a chimeric antigen receptor (CAR) and, in some aspects, can be subjected to a second stimulation and expansion with a different stimulatory molecule that binds to the introduced T cell receptor or the chimeric antigen receptor.Attorney Docket No. MIL-051WO1

[0092] In some embodiments, the activated and expanded yd T cells are cryopreserved and formulated for cell therapy.Source of d T cells

[0093] yd T cells can be obtained from a number of sources including, but not limited to, peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, skin, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. yd T cells may also be obtained from a cultured T cell line.

[0094] In some embodiments the activated and expanded yd T cells are non¬ haem atopoietic tissue-resident y8 T cells, non-haematopoietic tissue-derived, and / or non- haematopoietic tissue-native yd T cells. In these embodiments, the yd T cells were present in a non-haematopoietic tissue at the time the tissue is explanted. In some embodiments, non- haematopoietic tissue-resident yd T cells may be obtained from any suitable human or non¬ human animal non-haematopoietic tissue. In further embodiments, non-haematopoietic tissue are a tissue other than blood or bone marrow. In some embodiments, the yd T cells are not obtained from particular types of samples of biological fluids, such as blood or synovial fluid. Examples of such suitable human or non-human animal non-haematopoietic tissues include skin or a portion thereof (e.g., dermis or epidermis), the gastrointestinal tract (e.g. gastrointestinal epithelium, colon, small intestine, stomach, appendix, cecum, or rectum), mammary gland tissue, lung (preferably wherein the tissue is not obtained by bronchoalveolar lavage), prostate, liver, and pancreas. In some embodiments, non- haematopoietic tissue-resident yd T cells can be derived from a lymphoid tissue, such as thymus, spleen, or tonsil. The yd T cells may also be resident in human cancer tissues, e.g. breast and prostate. In some embodiments, the yd T cells are not obtained from human cancer tissue.

[0095] In some embodiments, a mixed population of cells comprising yd T cells is used in the expanding methods contemplated herein. In other embodiments, an isolated or purified population of yd T cells is used in the expanding methods contemplated herein.

[0096] In some embodiments, the mixed cell population is from a blood sample. In some embodiments, the blood sample is a peripheral blood sample. In other embodiment, the blood sample is a cord blood sample. As a non-limiting example, gamma delta (yd) T cells are active and expanded from peripheral blood mononuclear cells.

[0097] In some embodiments, a population of cells from PBMCs is used in the expanding methods described herein.Attorney Docket No. MIL-051WO1

[0098] In some embodiments, a population of cells from cord blood is used in the expanding methods described herein.

[0099] In some embodiments, the cells are from a human blood sample.

[0100] In some embodiments, a population of cells from a non-hemopoietic tissue sample is used in the expanding methods described herein.

[0101] In some embodiments, a population of cells from a skin sample is used in the expanding methods described herein.

[0102] In some embodiments, the cells are from a human skin explant.

[0103] In some embodiments, the cell population is pretreated prior to activating and expanding y5 T cells within the cell sample.

[0104] In some embodiments, the cell population comprises engineered yd T-cells.

[0105] In some embodiments, the cell population comprises V81+ yd T-cells.

[0106] In some embodiments, the methods of preparing y5 T cells involves a depletion step from the bulk product.Activation o f Gamma Delta (yd) T Cells

[0107] Methods and processes described herein for preparing y5 T cells comprise activating gamma delta (y5) T cells within a cell population in the presence of a CD3 agonist and a CD30 agonist prior to expanding said y5 T cells (e.g., VS I + yd T-cells). The agonists may either be immobilized on a solid support or soluble, whereby the cells are contacted with the agonists. In some embodiments, the CD3 agonist is immobilized on a solid support and the CD30 agonist is soluble. In some embodiments, the CD30 agonist is immobilized on a solid support and the CD3 agonist is soluble. In some embodiments, the CD3 and CD30 agonists are immobilized on a solid support. In some embodiments, the CD3 and CD30 agonists are soluble.

[0108] CD3 is a molecule that is expressed during the maturation process of T cells, forming a larger complex with the (CD3 / TCR complex) with, in this case, the y5 T cell receptor (TCR). The TCR that forms a complex with CD3 is a molecule that transmits a signal into T cells, stimulating proliferation and effector function functions in vivo. CD30 is a molecule known to express on the surface of activated lymphocytes (e.g., T cells), has 6 cysteine-rich pseudo-repeat motifs as extracellular domains, and has, as an intracellular domain, a TNF receptor-related factor (TRAF) binding sequence that stimulates NFKB signal. CD30 can also control the intracellular calcium ion flux through cross-linking with CD3.Attorney Docket No. MIL-051WO1

[0109] In some embodiments, the method described herein comprises contacting y5 T cells within the cell population with a CD3 agonist and a CD30 agonist, for example, culturing the cell population containing y5 T cells in a cell culture medium containing a CD 3 agonist and a CD30 agonist.

[0110] A CD3 agonist can be any molecule that binds to and activates CD3. In some embodiment, the CD3 agonist that specifically binds CD3 is an anti-CD3 agonistic antibody, or a functional fragment thereof (e.g., a divalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody). In some embodiments, the anti-CD3 antibody is, but not limited to, a monoclonal antibody, a F(ab')₂-fragment of a CD3 antibody, a single-chain Fv fragment (scFv), a Fab fragment, a Fab’, an Fv fragment, a single chain antibody, a nanobody, a heavy chain antibody or any functional variant thereof. In some embodiments, the CD3 agonist may be a proteinaceous CD3 binding molecule with antibody-like binding properties.

[0111] Exemplary anti-CD3 agonistic antibodies include the BC3 anti-CD3 antibody, a clone which expresses this antibody being available from the American Type Culture Collection (HB-10166), SP-34, UCHTL OKT3yl (ala-ala), ChAglyCD3 (disclosed in WO 1993 / 19196) or HUM291.

[0112] In one embodiment, the CD3 agonist is Muromonab-CD3 (OKT3).

[0113] A CD30 agonist can be any molecule that binds to and activates CD30. In some embodiment, the CD30 agonist is an anti-CD30 agonistic antibody, or a functional fragment thereof. In some embodiments, the anti-CD30 antibody is, but not limited to, a monoclonal antibody, a scFv, a Fab, a Fab’, a single chain antibody, a nanobody, a heavy chain antibody or any functional variant thereof.

[0114] Exemplary anti-CD30 agonistic antibodies include but are not limited to Brentuximab.

[0115] Without wishing to be bound by any theory, the cell populations including y5 T cells are activated by binding of a CD30 agonist to TCR / CD3 and binding of a CD30 agonist to CD30 on the T cells.

[0116] In various methods for activating and expanding y5 T cells described herein, the CD3 agonist is immobilized on a solid support, or a solid phase surface. For example, the CD3 agonist is coated on the surface of a cell culture container. In some examples, polystyrene beads coated with a CD3 agonist are used. In some embodiments, the CD3 agonist is immobilized on the support or surface covalently or non-covalently. The supportAttorney Docket No. MIL-051WO1can be a magnetic bead, a polymeric bead, a cell culture plate, or a culture flask. For example, a canted neck flask for cell culture may be coated with an anti-CD3 antibody.

[0117] In some embodiments, the agonists are not immobilized to a solid support, i.e., it is present in soluble form or is soluble. In some embodiments, the CD3 and CD30 agonists are directly added to the cell culture medium.

[0118] In some embodiments, the CD3 agonist is immobilized to a solid support and the CD30 agonist is soluble, e.g., present in a cell culture medium. In some examples, the CD3 agonist is immobilized to the surface of a cell culture container. In some examples, the CD3 agonist and fibronectin (RN) or a variant thereof are immobilized to the surface of a cell culture container. In one example, the CD3 agonist and fibronectin (RN) or a variant thereof are immobilized to the surface of a cell culture container and the CD30 agonist is present in a cell culture medium.

[0119] In some embodiments, the culture medium used during cell activation may be further supplemented with at least one cytokine selected from IL-2, IL-7, IL-15, IL-18, IL-2L In some embodiments, the cell culture medium further comprises two cytokines from IL-2, IL-7, IL- 15, IL- 18 and IL-2L In some embodiments, the cell culture medium further comprises three cytokines from IL-2, IL-7, IL- 15, IL- 18 and IL-2L In some embodiments, the cell culture medium further comprises four cytokines from IL-2, IL-7, IL- 15, IL- 18 and IL-2L In some embodiments, the cell culture medium further comprises IL-2, IL-7, IL- 15, IL-18 and IL-2L

[0120] As used herein, “IL-2” refers to native or recombinant IL-2 or a variant thereof. IL-2 can also refer to IL-2 derived from a variety of mammalian species, including, for example, human, simian, bovine, porcine, equine, and murine. Variants may comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics.

[0121] As used herein, “IL- 15” refers to native or recombinant IL- 15 or a variant thereof that acts as an agonist for one or more IL-15 receptor (IL-15R) subunits (e.g., mutants, muteins, analogues, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof). IL-15, like IL-2, is a known T-cell growth factor that can support proliferation of an IL-2-dependent cell line, CTLL- 2. IL-15 can also refer to IL-15 derived from a variety of mammalian species, including, for example, human, simian, bovine, porcine, equine, and murine.Attorney Docket No. MIL-051WO1

[0122] As used herein, “IL-7” refers to native or recombinant IL-7 or a variant thereof that acts as an agonist for one or more IL-7 receptor (IL-7R) subunits (e.g., mutants, muteins, analogues, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof). Mature human IL-7 occurs as a 152 amino acid sequence (less the signal peptide, consisting of an additional 25 N- terminal amino acids). IL-7 can also refer to IL-7 derived from a variety of mammalian species, including, for example, human, simian, bovine, porcine, equine, and murine. Variants may comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics.

[0123] As used herein, “IL-21” refers to native or recombinant IL-21 or a variant thereof that acts as an agonist for one or more IL-21 receptor (IL-21R) subunits (e.g., mutants, muteins, analogues, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof). IL-21 can also refer to IL-21 derived from a variety of mammalian species, including, for example, human, simian, bovine, porcine, equine, and murine.

[0124] As used herein, “IL- 18” refers to native or recombinant IL-21 or a variant thereof. IL-18 can also refer to IL-18 derived from a variety of mammalian species, including, for example, human, simian, bovine, porcine, equine, and murine.

[0125] In addition to CD3 and CD30 agonists, in some examples, one or more additional cytokines are used together with CD3 and CD30 agonists to activate y5 T cells. In some embodiments, the one or more cytokines are chosen from TNFa, IFN-y, IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-18, and IL-21. In some embodiments, the agonist cytokine is TNFa. In some embodiments, the agonist cytokine is IFN-y. In some embodiments, the agonist cytokine is IL-2. In some embodiments, the agonist cytokine is IL-4. In some embodiments, the agonist cytokine is IL-7. In some embodiments, the agonist cytokine is IL-9. In some embodiments, the agonist cytokine is IL-12. In some embodiments, the agonist cytokine is IL-15. In some embodiments, the agonist cytokine is IL-18. In some embodiments, the agonist cytokine is IL-21.

[0126] In some embodiments, the cells are activated for at least one day. In some examples, the cells are activated for 1 to 7 days, or 1 to 4 days, or 2 to 5 days, or 2 to 4 days, or 3 to 4 days, or 3 to 5 days. In some embodiments, the cells are activated 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. In some embodiments, the cells are activated for 1 day. In some embodiments, the cells are activated for 2 days. In some embodiments, the cells are activated for 3 days. In some embodiments, the cells are activated for 4 days. In someAttorney Docket No. MIL-051WO1embodiments, the cells are activated for 5 days. In some embodiments, the cells are activated for 6 days. In some embodiments, the cells are activated for 7 days.Expansion of Gamma Delta (yd) T Cells

[0127] In accordance with methods and processes for preparing y5 T cells, the cell population that has been treated to activate y5 T cells using CD3 and CD30 agonist are further treated to expand activated y5 T cells. In one aspect, the activated cell population is treated for y5 T cells expansion with one or more cytokines. In some embodiments, the activated cell population is cultured in culture medium comprising one or more cytokines for expansion. The expansion stimulates proliferation of y5 T cells and increases the cell number. In some embodiments, the expansion stimulates proliferation of V5 1+ y5 T cells and increases the V5 1+ y5 cell number.

[0128] In some embodiments, the culture medium for expanding activated cells comprises interleukins, such as IL-2, IL-7, IL-15, and / or IL-21. In some embodiments, the culture medium for expanding activated cells may further comprise a CD30 agonist.

[0129] In some embodiments, the activated y5 T cells are further expanded in a medium comprising IL2, IL-7, IL- 15, IL-21, or combination thereof. In some embodiments, the activated y5 T cells are further expanded in a medium comprising IL-7. In some embodiments, the activated y5 T cells are further expanded in a medium comprising IL- 15. In some embodiments, the activated y5 T cells are further expanded in a medium comprising IL-2. In some embodiments, the activated y5 T cells are further expanded in a medium comprising IL-21. In some embodiments, the activated y5 T cells are further expanded in a medium comprising IL-2 and IL-15. In some embodiments, the activated y5 T cells are further expanded in a medium comprising IL-2 and IL-7. In some embodiments, the activated y5 T cells are further expanded in a medium comprising IL-2 and IL-21. In some embodiments, the activated y5 T cells are further expanded in a medium comprising IL-7 and IL-15. In some embodiments, the activated y5 T cells are further expanded in a medium comprising IL-7 and IL-21. In some embodiments, the activated y5 T cells are further expanded in a medium comprising IL- 15 and IL-21. In some embodiments, the activated y5 T cells are further expanded in a medium comprising IL-2, IL-7 and IL-15. In some embodiments, the activated y5 T cells are further expanded in a medium comprising IL-2, IL- 15 and IL-21. In some embodiments, the activated y5 T cells are further expanded in a medium comprising IL-7, IL- 15 and IL-21. In some embodiments, the activated y5 T cells are further expanded in a medium comprising IL-2, IL-7, IL- 15 and IL-21. In someAttorney Docket No. MIL-051WO1embodiments, the activated y5 T cells are further expanded in a medium comprising IL-7, IL-15 and a CD30 agonist. In some embodiments, the activated y5 T cells are further expanded in a medium comprising IL-7, IL-15, IL-21 and a CD30 agonist. In some embodiments, the activated y5 T cells are further expanded in a medium comprising IL-2, IL-7, IL-15, IL-21 and a CD30 agonist.

[0130] In some embodiments, the activated y5 T cells are further treated for 1 to 14 days for expansion. In some embodiments, the activated y5 T cells are further treated for 1 to 10 days, or 2 to 8 days, or 3 to 10 days, 4 to 10 days, or 6 to 14 days, or 7 to 10 days for expansion. In some embodiments, the activated y5 T cells are further expanded for 1 day. In some embodiments, the activated y5 T cells are expanded for 2 days. In some embodiments, the activated y5 T cells are expanded for 3 days. In some embodiments, the activated y5 T cells are expanded for 4 days. In some embodiments, the activated T cells are expanded for 5 days. In some embodiments, the activated y5 T cells are expanded for 6 days. In some embodiments, the activated y5 T cells are expanded for 7 days. In some embodiments, the activated y5 T cells are expanded for 8 days. In some embodiments, the activated y5 T cells are expanded for 9 days. In some embodiments, the y5 T cells are expanded for 10 days.

[0131] In some embodiments, the activated y5 T cells are expanded for 11 days. In some embodiments, the y5 T cells are activated expanded for 12 days. In some embodiments, the activated y5 T cells are expanded for 13 days. In some embodiments, the y5 T cells are activated expanded for 14 days.

[0132] In some embodiments, the activation and expansion periods repeat at least one time. In some embodiments, the activation and expansion periods repeat two times. In some embodiments, the activation and expansion periods repeat three times or more.

[0133] In some examples, the activation process is repeated a second time. In some embodiments, the second activation period is 1 day. In some embodiments, the second activation period is 2 days. In some embodiments, the second activation period is 3 days. In some embodiments, the second activation period is 4 days or more. In some aspects, the expansion process is repeated a second time post-secondary activation. In some embodiments, the y5 T cells are expanded for 1 day post-secondary activation. In some embodiments, the y5 T cells are expanded for 2 days post-secondary activation. In some embodiments, the y5 T cells are expanded for 3 days post-secondary activation. In some embodiments, the y5 T cells are expanded for 4 days post-secondary activation. In some embodiments, the y5 T cells are expanded for 5 days post-secondary activation. In someAttorney Docket No. MIL-051WO1embodiments, the y5 T cells are expanded for 6 days post-secondary activation. In some embodiments, the y5 T cells are expanded for 7 days post-secondary activation. In some embodiments, the y5 T cells are expanded for 8 days post-secondary activation. In some embodiments, the y5 T cells are expanded for 9 days post-secondary activation. In some embodiments, the y5 T cells are expanded for 10 days post-secondary activation. In some embodiments, the y5 T cells are expanded for 11 days post-secondary activation.

[0134] In some embodiments, the activation and expansion processes are repeated two, three, four or more times. The repeats may be determined by the amount of y5 T cells obtained. In some aspects, the activation and expansion process is repeated more than one time to enhance the expansion of the y5 T cells. In some embodiments, the activation and expansion process can be repeated 1 to 10 times to enhance the expansion of the y5 T cells. In some embodiments, the activation and expansion process is repeated 1 time to enhance the expansion of the y5 T cells. In some embodiments, the activation and expansion process is repeated 2 times to enhance the expansion of the y5 T cells. In some embodiments, the activation and expansion process is repeated 3 times to enhance the expansion of the y5 T cells. In some embodiments, the activation and expansion process is repeated 4 times to enhance the expansion of the y5 T cells. In some embodiments, the activation and expansion process is repeated 5 times to enhance the expansion of the y5 T cells. In some embodiments, the activation and expansion process is repeated 6 times to enhance the expansion of the y5 T cells. In some embodiments, the activation and expansion process is repeated 7 times to enhance the expansion of the y5 T cells. In some embodiments, the activation and expansion process is repeated 8 times to enhance the expansion of the y5 T cells. In some embodiments, the activation and expansion process is repeated 8 times to enhance the expansion of the y5 T cells. In some embodiments, the activation and expansion process is repeated 9 times to enhance the expansion of the y5 T cells. In some embodiments, the activation and expansion process is repeated 10 times to enhance the expansion of the y5 T cells.

[0135] In some embodiments, the activation and expansion processes described herein increase the number of y5 T cells (e.g., V5 1+ y5 T cells) within the cell population (e.g., a blood cell population) 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold or more, compared to the number of y5 T cells (e.g., V5 1+ y5 T cells) before the activation and expansion.Attorney Docket No. MIL-051WO1

[0136] Cells can be activated and expanded using any cell culture media. Any commercial cell culture media may be used. A culture medium for cell activation and expansion can be prepared using a basal culture medium. Exemplary media include but are not limited to, IMDM (Iscove's Modified Dulbecco's Medium), RPMI medium (e.g., RPMI- 16 medium), Eagle’s medium (e.g., DMEM, EMEM, BME, and MEM), Ham’s medium (e.g., F10 medium, and F12 medium), and MCDB medium. The culture medium may contain a serum (e.g., FBS, human serum and horse serum). In some embodiments, the culture medium is serum free. In some embodiments, the basal culture medium comprises other factors necessary for cell culture. In one embodiment, the culture medium is IMEM supplemented with 15% fetal bovine serum (FBS), lx gentamycin, ITS medium supplement, and ascorbic acid. In some embodiments, fetal bovine serum (FBS) can be replaced by 5% human platelet lysate (HPL) and 5% OctaPlas to enable a xeno-free approach to expansion of y5 T cells. In some embodiments, replacement of FBS by medium comprising 5% HPL and 5% OctaPlas does not negatively impact expansion or cytotoxicity of y5 T cells.

[0137] In some embodiments, cells may be cultured by adherent culture or suspension culture. Any cell expanding systems can be used for preparing y5 T cells described here.

[0138] In some embodiments, methods and processes described herein are particularly used to prepare V51+ y5 T cells.

[0139] An exemplary process for expanding y5 T cells comprises an activation step that is mediated by a mixture of agonist antibodies and cytokines. Blood or skin explants are depleted of other peripheral mononuclear blood cells (PBMCs), and y5 T cells are selectively seeded in a culture flask in an IMEM medium with 15% FBS. The cells are then transduced with a mixture of cytokines, a caspase inhibitor, and agonist antibodies to expand the y5 T cells. In some embodiments, the depleted cells are cultured in a canted neck flask. In some embodiments, a caspase inhibitor (e.g., ZVAD) is added to the cell culture during the activation phase. The y5 T cells are cultured in the presence of CD3 and CD30 agonists for 4 days. The cells are then allowed to expand to Day 14, wherein the cells are then harvested and cryopreserved. The activation process can be repeated a second time (starting on Day 14) to expand the cells to a higher density by Day 28, wherein the cells are harvested and cryopreserved.Exemplary processes for Activation and Expansion of Gamma Delta (yd) T Cells from BloodAttorney Docket No. MIL-051WO1

[0140] The present invention provides exemplary processes for activating and expanding y5 T cells from blood. In particular, the exemplary processes can be used for activating and expanding V51+ y5 T cells from a blood sample. The blood cells containing y5 T cells can be derived from a peripheral blood sample, or a cord blood sample. In one example, the cells are from peripheral blood.

[0141] In one embodiment, the blood cells are first treated to deplete other cell types in the blood.

[0142] In one embodiment, the method for preparing y5 T cells from blood cells comprises i) culturing / activating the blood cells in a cell culture medium comprising a CD3 agonist and a CD30 agonist, wherein the cell culture medium is further supplemented with cytokines IL-7, IL-15, IL-18, and IL-21, a caspase inhibitor (e.g., ZVAD) and fibronectin (RN); ii) culturing the activated cells in a cell culture medium comprising cytokines IL-7 and IL-15. The CD3 agonist and RN are immobilized to the surface of a culture container. The CD30 agonist is added to the cell culture medium. In some embodiments, the culture medium is IMDM (Iscove's Modified Dulbecco's Medium) supplemented with 15%FBS, lx insulin -transferrin- selenium (G / ITSO and Asc acid base. In some embodiments, the cell are activated for 4 days and the activated cells are then transduced to the culture medium containing IL-7 and IL- 15 for expansion for at least 10 days. The activated and expanded cells are harvested.

[0143] In one embodiment, the method for preparing y5 T cells from blood cells comprises i) culturing / activating the blood cells in a cell culture medium comprising a CD3 agonist and a CD30 agonist, wherein the cell culture medium is further supplemented with cytokines IL-7, IL-15, IL-18, and IL-21, and a caspase inhibitor (e.g., ZVAD) and a RN; ii) culturing the activated cells in a cell culture medium comprising cytokines IL-7 and IL- 15 for one day and continuing to expand the activated cells in a cell culture medium comprising IL-2, IL-7, and IL-15. In some embodiments, the culture medium is IMDM (Iscove's Modified Dulbecco's Medium) supplemented with 15%FBS, lx insulin -transferrin- selenium (G / ITSO and Asc Acid base. The CD3 agonist and RN are immobilized to the surface of a culture container. The CD30 agonist is added to the cell culture medium. In some embodiments, the cell are activated for 4 days and the activated cells are then transduced to the culture medium for expansion for at least 10 days. The activated and expanded cells are harvested.

[0144] In one embodiment, the method for preparing y5 T cells from blood cells comprises i) culturing / activating the blood cells in a cell culture medium comprising a CD3 agonist and a CD30 agonist, wherein the cell culture medium is further supplemented withAttorney Docket No. MIL-051WO1cytokines IL-7, IL-15, IL-18, and IL-21, and a caspase inhibitor (e.g., ZVAD) and a RN; ii) culturing the activated cells in a cell culture medium comprising cytokines IL-7 and IL- 15 for one day and continuing to expand the activated cells in a cell culture medium comprising IL-2, IL-7, IL-15, IL-21 and the CD30 agonist for 9 days. In some embodiments, the culture medium is IMDM (Iscove's Modified Dulbecco's Medium) supplemented with 15%FBS, lx insulin -transferrin- selenium (G / ITSO and Asc acid base. The CD3 agonist and RN are immobilized to the surface of a culture container. The CD30 agonist is added to the cell culture medium. In some embodiments, the cell are activated for 4 days and the activated cells are then transduced to the culture medium for expansion for at least 10 days. The activated and expanded cells are harvested.

[0145] In one embodiment, the method for preparing y5 T cells from blood cells described herein comprises i) culturing / activating the blood cells in a cell culture medium comprising a CD3 agonist and a CD30 agonist, wherein the cell culture medium is further supplemented with cytokines IL-7, IL- 15, IL- 18, and IL-21, and a RN; ii) culturing the activated cells in a cell culture medium comprising cytokines IL-7 and IL- 15 for one day and continuing to expand the activated cells in a cell culture medium comprising IL-2, IL-7, IL-15, IL-21 and the CD30 agonist. The CD3 agonist and RN are immobilized to the surface of a culture container. The CD30 agonist is added to the cell culture medium. In some embodiments, the culture medium is IMDM (Iscove's Modified Dulbecco's Medium) supplemented with 15%FBS, lx insulin -transferrin- selenium (G / ITSO and Asc acid base. In some embodiments, the cell are activated for 4 days and the activated cells are then transduced to the culture medium for expansion for at least 6 days. The activated and expanded cells are harvested.

[0146] In one embodiment, the method for preparing y5 T cells from blood cells described herein comprises i) culturing / activating the blood cells in a cell culture medium comprising a CD3 agonist and a CD30 agonist, wherein the cell culture medium is further supplemented with cytokines IL-7, IL- 15, IL- 18, and IL-21, and a RN; ii) culturing the activated cells in a cell culture medium comprising cytokines IL-7 and IL- 15 for one day and continuing to expand the activated cells in a cell culture medium comprising IL-2, IL-7, IL-15, IL-21 and the CD30 agonist. The CD3 agonist and RN are immobilized to the surface of a culture container. The CD30 agonist is added to the cell culture medium. In some embodiments, the culture medium is IMDM (Iscove's Modified Dulbecco's Medium) supplemented with 15%FBS, lx insulin -transferrin- selenium (G / ITSO and Asc acid base. InAttorney Docket No. MIL-051WO1other embodiments, the cell are activated for 2 days and the activated cells are then transduced to the culture medium for expansion for at least 6 days. The activated and expanded cells are harvested.

[0147] In one embodiment, the method for preparing y5 T cells from blood cells described herein comprises i) culturing / activating the blood cells in a cell culture medium comprising a CD3 agonist and a CD30 agonist, wherein the cell culture medium is further supplemented with cytokines IL-15, IL-18, and IL-21, and a RN; ii) culturing the activated cells in a cell culture medium comprising IL- 15 for one day and continuing to expand the activated cells in a cell culture medium comprising IL-2, IL- 15, IL-21 and the CD30 agonist. The CD3 agonist and RN are immobilized to the surface of a culture container. The CD30 agonist is added to the cell culture medium. In some embodiments, the culture medium is IMDM (Iscove's Modified Dulbecco's Medium) supplemented with 15%FBS, lx insulin -transferrin- selenium (G / ITSO and Asc Acid base culture medium. In some embodiments, the cells are activated for 2 days and the activated cells are then transduced to the culture medium for expansion for at least 8 days. The activated and expanded cells are harvested.

[0148] In various methods described herein, the cells are cultured in a cell culture flask which is coated with the CD3 agonist for y5 T activation. In some embodiments, the activated y5 T cells are transferred to a gas permeable rapid expansion (GREX) device for further expansion.

[0149] Using the protocols laid out above, blood cells containing y5 T cells were cultured by depleting blood of other PBMCs and seeded into a canted neck flask that is coated with anti-CD3 antibody. To activate the y5 cells, a combination of cytokines and anti-CD30 antibody were added exogenously in BM301 complete medium on Day 0. On Day 5, the cells were expanded utilizing a second combination of cytokines until harvest and storage on Day 14. y5 cells that were activated with exogenous anti-CD30 exhibited significant expansion relative to the anti-CD30 isotype control and control without the anti-CD30 antibody.Further, the post-thaw proliferation rates of y5 T cells manufactured in this fashion were significantly increased relative to the anti-CD30 isotype control and control without anti-CD30 antibody. In addition to enhanced proliferation rates post-thaw, y5 T cells manufactured with the addition of anti-CD30 antibody exhibited enhanced cytotoxicity against enhanced cytotoxic activity against target cancer cell lines, wherein the subpopulation of these y5 T cells comprises V51+ y5 T cells, which have been noted to recognize target cancer cells and mediate anti-tumor activity through direct lysis of transformed cells.Attorney Docket No. MIL-051WO1

[0150] In some embodiments, the y5 T cells from blood are seeded into a canted neck flask that is coated with the agonist antibody, anti-CD3. In some embodiments, the y5 T cells are activated with a combination of cytokines.

[0151] In some embodiments, the y5 T cells from blood are expanded using a second combination of cytokines.

[0152] In some embodiments, the cells are activated in the presence of IL-7, IL-15, IL- 18, IL-21, ZVAD, anti-CD30 and (RN / anti-CD3) for 5 days. In some embodiments, the cells are transduced and expanded in the presence of IL-7 and IL-15 for 11 days.

[0153] In some embodiments, the cells are activated in the presence of IL-7, IL-15, IL- 18, IL-21, ZVAD, anti-CD30 and (RN / anti-CD3) for 5 days. In some embodiments, the cells are subsequently transduced with IL-7 and IL-15 for 2 days. In some embodiments, the cells are expanded in the presence of IL-2, IL-7 and IL- 15 for 11 days.

[0154] In some embodiments, the cells are activated in the presence of IL-7, IL-15, IL- 18, IL-21, ZVAD, anti-CD30 and (RN / anti-CD3) for 5 days. In some embodiments, the cells are subsequently transduced with IL-7 and IL-15 for 2 days. In some embodiments, the cells are expanded in the presence of IL-2, IL-7, IL-15, IL-21, and anti-CD30 agonist antibody 11 days.

[0155] In some embodiments, the cells are activated in the presence of IL-7, IL-15, IL- 18, IL-21, anti-CD30 and (RN / anti-CD3) for 5 days. In some embodiments, the cells are subsequently transduced with IL-7 and IL- 15 for 2 days. In some embodiments, the cells are expanded in the presence of IL-2, IL-7, IL-15, IL-21 and anti-CD30 agonist antibody for 6 days.

[0156] In some embodiments, the cells are activated in the presence of IL-7, IL-15, IL- 18, IL-21, anti-CD30 and (RN / anti-CD3) for 3 days. In some embodiments, the cells are subsequently transduced with IL-7 and IL- 15 for 2 days. In some embodiments, the cells are expanded in the presence of IL-2, IL-7, IL-15, IL-21 and anti-CD30 agonist antibody for 6 days.

[0157] In some embodiments, the cells are activated in the presence of IL-15, IL-18, IL-21, anti-CD30 and (RN / anti-CD3) for 3 days. In some embodiments, the cells are subsequently transduced with IL-15 for 2 days. In some embodiments, the cells are expanded in the presence of IL-2, IL-15, IL-21, and anti-CD30 agonist antibody for 8 days.Exemplary processes for Activation and Expansion of Gamma Delta (yd) T Cells from SkinAttorney Docket No. MIL-051WO1

[0158] The present disclosure provides exemplary processes for activating and expanding y5 T cells isolated from a tissue. In some embodiments, the cell population comprising y5 T cells is from a skin explant. In some embodiments, the y5 T cells are derived from human skin explants. In particular, the exemplary processes can be used for activating and expanding V51+ y5 T cells isolated from a skin explant.

[0159] In general, the method for activating and expanding y5 T cells isolated from a skin explant comprising i) culturing and activating y5 T cells in a culture medium comprising a CD3 agonist and a CD30 agonist; and ii) expanding the activated y5 T cells. In some embodiments, the medium used for y5 T cell activation comprises at least one cytokines selected from IL-7, IL-15, IL- 18, and IL-21. In some embodiments, the medium used for y5 T cell activation comprises IL-7. In some embodiments, the medium used for y5 T cell activation comprises IL-15. In some embodiments, the medium used for y5 T cell activation comprises IL-18. In some embodiments, the medium used for y5 T cell activation comprises IL-21. In some embodiments, the medium used for y5 T cell activation comprises IL-7 and IL-15. In some embodiments, the medium used for y5 T cell activation comprises IL-7 and IL-18. In some embodiments, the medium used for y5 T cell activation comprises IL-7 and IL-21. In some embodiments, the medium used for y5 T cell activation comprises IL- 15 and IL-18. In some embodiments, the medium used for y5 T cell activation comprises IL-15 and IL-21. In some embodiments, the medium used for y5 T cell activation comprises IL- 18 and IL-21. In some embodiments, the medium used for y5 T cell activation comprises IL-7, IL- 15 and IL-18. In some embodiments, the medium used for y5 T cell activation comprises IL-7, IL-18 and IL-21. In some embodiments, the medium used for y5 T cell activation comprises IL-15, IL-18 and IL-21. In some embodiments, the medium used for y5 T cell activation comprises IL-7, IL-15, IL-18 and IL-21.

[0160] In some embodiments, the CD3 agonist is immobilized to the surface of a cell culture container (e.g., a plate and a flask). In some embodiments, the CD30 agonist is added to the cell culture medium. In some embodiments, the CD3 agonist is OKT3.

[0161] y5 T cells from a skin explant are treated with the CD3 and CD30 agonists for activation for at least one day. In some embodiments, y5 T cells from a skin explant are treated with the CD3 and CD30 agonists for activation for 1 to 4 days, or 1-2 days, or 1-3 days, or 2-4 days. In some embodiments, y5 T cells from a skin explant are cultured for activation for 1 day. In some embodiments, y5 T cells from a skin explant are cultured for activation for 2 days. In some embodiments, y5 T cells from a skin explant are cultured forAttorney Docket No. MIL-051WO1activation for 3 days. In some embodiments, y5 T cells from a skin explant are cultured for activation for 4 days.

[0162] In some embodiments, the activated y5 T cells are further expanded in a culture medium comprising IL-7, IL- 15 or combination thereof. In some embodiments, the activated y5 T cells are further expanded in a culture medium comprising IL-7. In some embodiments, the activated y5 T cells are further expanded in a culture medium comprising IL-15. In some embodiments, the activated y5 T cells are further expanded in a culture medium comprising IL-7 and IL-15.

[0163] In some embodiments, the y5 T cells derived from skin are cultured in medium comprising fetal bovine serum (FBS). In other embodiments, the culture medium is a xeno-firee medium.

[0164] Using similar protocols to above, y5 T cells from skin explants were seeded into a flask with a combination of cytokines (IL-7, IL-15, IL-18, and IL-21) and agonist antibodies, anti-CD3 (RN / OKT3) and anti-CD30 for activation of y5 T cells on Day 0. The expansion process starts on Day 4 with the addition of IL-7 and IL-15, and spans to Day 14, wherein the y5 T cells are then harvested and cryopreserved.

[0165] In some embodiments, the expansion period spans from Day 5 to Day 14, wherein the y5 T cells are harvested on Day 14. In some embodiments, the expansion period is 1 day. In some embodiments, the expansion period is 2 days. In some embodiments, the expansion period is 3 days. In some embodiments, the expansion period is 4 days. In some embodiments, the expansion period is 5 days. In some embodiments, the expansion period is 6 days. In some embodiments, the expansion period is 7 days. In some embodiments, the expansion period is 8 days. In some embodiments, the expansion period is 9 days. In some embodiments, the expansion period is 10 days.

[0166] In some embodiments, the activation and expansion process may be repeated a second time after reseeding on Day 14, wherein the activation process begins on Day 14 and spans to Day 17. The expansion process then proceeds from Day 17 to Day 28, wherein the y5 T cells are then harvested and cryopreserved.

[0167] In some embodiments, the y5 T cells are harvested after 1 day. In some embodiments, the y5 T cells are harvested after 2 days. In some embodiments, the y5 T cells are harvested after 3 days. In some embodiments, the y5 T cells are harvested after 4 days. In some embodiments, the y5 T cells are harvested after 5 days. In some embodiments, the y5 T cells are harvested after 6 days. In some embodiments, the y5 T cells are harvested after 7Attorney Docket No. MIL-051WO1days. In some embodiments, the y5 T cells are harvested after 8 days. In some embodiments, the y5 T cells are harvested after 9 days. In some embodiments, the y5 T cells are harvested after 10 days.

[0168] In some embodiments, the cells expanded from skin using the anti-CD3 / anti-CD30 activation protocol exhibited a significant increase in total live cell fold expansion over a cytokine-based expansion protocol. In some embodiments the average viability of cells expanded from skin using the anti-CD3 and anti-CD30 antibodies is comparable to a cytokine-based expansion protocol.

[0169] In some embodiments, the average y5 T cell fold expansion using the anti-CD3 / anti-CD30 activation protocol is significantly greater than a cytokine-based expansion protocol. In some embodiments, the average V51 subset fold expansion using the anti-CD3 and anti-CD30 antibodies is significantly greater than a cytokine-based expansion protocol.Cryopreservation and formulations

[0170] In some embodiments of any of the methods provided herein, the method also includes, after said activation and expansion, further cryopreserving y5 T cells harvested. In some embodiments, y5 T cells comprise at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more V51+ y5 T cells. In some embodiments, the prepared y5 T cells contain greater than 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90% of V51+y5 T cell subset as a percentage of the total T cells in the formulation or the total cells in the formulation.

[0171] In some embodiments, the isolated y5 cells are cryopreserved in a cryoformulation medium. In some embodiments, the isolated V51+y5 cells are cryopreserved in a cryoformulation medium.

[0172] Various cryoformulation media for preserving gamma delta T cells are known in the art, as well as methods thereof. In some embodiments, the cryoformulation medium comprises CS10. CryoStor® CS10 is a uniquely formulated cryopreservation medium containing 10% dimethyl sulfoxide (DMSO). In some embodiments, the cryo-formulation medium comprises CS10.

[0173] In some embodiments, suitable freezing solutions contain DMSO and other suitable media supplements, such as human serum albumin, dextran, dextrose, NaCl, Hespan or PlasmaLyte A. Cells then are frozen to a temperature of about -80° C to about -200°C, such as about -80° C to about -135°C. In some embodiments, cryopreservation is accomplished by placing vials in a freezing container and then storing in a -80° C freezer, for example for 1-3 days, followed by transfer to the vapor phase of a liquid nitrogen storageAttorney Docket No. MIL-051WO1system. In some embodiments, the isolated gamma delta T cells are frozen in a controlled rate freezer.

[0174] In some embodiments, after cryopreservation, cells may be thawed (i.e., defrosted), for example in a 37°C water bath. The thawed cells may be subsequently used in an expansion method. Methods of expansion may comprise any of the methods described herein, or as described in the art, for example see WO2017072367 and W02018202808.

[0175] In some embodiments, the present disclosure provides y5 T cells. In some embodiments, the present disclosure provides V51+ y5 T cells. In some embodiments, the activated and expanded y5 T cells prepared by a method described herein are functionally equivalent to naturally isolated y5 T cells.

[0176] In some embodiments, y5 T cells provided herein are cytotoxic.

[0177] In some embodiments, y5 T cells provided herein can be further engineered to express one or more therapeutics. In one example, y5 T cells provided here are further modified to express a chimeric antigen receptorViability and Activity of Activated and Expanded d T Cells

[0178] In some embodiments, the y5 T cells manufactured through activation by addition of anti-CD3 and anti-CD30 agonist antibodies exhibit greater proliferation rates post-thaw as compared to y5 T cells manufactured using anti-CD30 isotype control and without anti-CD30 antibody.

[0179] In some embodiments, the y5 T cells manufactured through activation by addition of cytokines with anti-CD3 and anti-CD30 agonist antibodies exhibit greater cytotoxic activity against target cells as compared to y5 T cells manufactured using anti-CD30 isotype control and without anti-CD30 antibody.

[0180] In some embodiments, the average viability of y5 T cells prepared with methods described herein is comparable to y5 T cells prepared using a cytokine-based control expansion protocol. In some embodiments, the average viability of V51+ y5 T cells prepared with methods described herein is comparable to V51+ y5 T cells prepared using a cytokinebased control expansion protocol.

[0181] In some embodiments, y5 T cells expanded using the agonist anti-CD3 and anti-CD30 antibodies exhibit higher overnight cytotoxicity as compared to y5 T cells expanded using the cytokine-based control protocol.Attorney Docket No. MIL-051WO1

[0182] In some embodiments, the activated and expanded y5 T cells have a viability of 50%, 60%, 70%, 80%, 90%, 95% or greater. In some embodiments, the activated and expanded y5 T cells have a viability of 50% or greater. In some embodiments, the activated and expanded y5 T cells have a viability of 60% or greater. In some embodiments, the activated and expanded y5 T cells have a viability of 70% or greater. In some embodiments, the activated and expanded y5 T cells have a viability of 80% or greater. In some embodiments, the activated and expanded y5 T cells have a viability of 90% or greater. In some embodiments, the activated and expanded y5 T cells have a viability of 95% or greater. In some embodiments, the activated and expanded y5 T cells have a viability of 96% or greater. In some embodiments, the activated and expanded y5 T cells have a viability of 97% or greater. In some embodiments, the activated and expanded y5 T cells have a viability of 98% or greater. In some embodiments, the activated and expanded y5 T cells have a viability of 99% or greater.

[0183] In some embodiments, the activated and expanded V51+ y5 T cells have a viability of 50%, 60%, 70%, 80%, 90%, 95% or greater.Compositions

[0184] In another aspect, the present disclosure provides a pharmaceutical composition comprising y5 T cells manufactured by methods and processes described herein. In some embodiments, the pharmaceutical composition comprises V51+ y5 T cells manufactured by methods and processes described herein. The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A “pharmaceutically acceptable carrier’’ refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular cell and / or by the method of administration. Accordingly, there are a variety of suitable formulations.

[0185] In some embodiments, the pharmaceutical composition comprises y5 T cells (e.g., V51+ y5 T cells) in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects.Attorney Docket No. MIL-051WO1

[0186] In some embodiments, the composition comprises a desired cell dosage. The desired cell dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition. The cells may be administered using standard administration techniques, formulations, and / or devices. Provided are formulations and devices, such as syringes and vials, for storage and administration of the compositions. Cells can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).

[0187] In some embodiments, compositions comprising cells are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

[0188] In some embodiments, the pharmaceutical composition comprises y5 T cells, wherein the y5 T cells are predominantly V51+ y5 T cells.

[0189] In some embodiments, the pharmaceutical composition comprises y5 T cells at a pre-determined dose.

[0190] In some embodiments, the pharmaceutical composition comprises y5 T cells of between 105to 1014cells. In some embodiments, the pharmaceutical composition comprises 105y5 T cells. In some embodiments, the pharmaceutical composition comprises 106y5 T cells. In some embodiments, the pharmaceutical composition comprises 107y5 T cells. In some embodiments, the pharmaceutical composition comprises 108y5 T cells. In some embodiments, the pharmaceutical composition comprises 109y5 T cells. In some embodiments, the pharmaceutical composition comprises 1010y5 T cells. In some embodiments, the pharmaceutical composition comprises 1011y5 T cells. In some embodiments, the pharmaceutical composition comprises 1012y5 T cells. In some embodiments, the pharmaceutical composition comprises 1013y5 T cells. In some embodiments, the pharmaceutical composition comprises 1014y5 T cells.Attorney Docket No. MIL-051WO1

[0191] In some embodiments, the pharmaceutical composition comprises V51+ y5 T cells of between 105to 1014cells. In some embodiments, the pharmaceutical composition comprises 1X105, IX 106, 1X107, IX 108’ 1X109, IX IO10, 1X1011, IX 1012, 1X10°, or IX 1014V51+ y5 T cells.

[0192] In some embodiments, the pharmaceutical composition comprises a buffer, a salt, and an excipient. In some embodiments, the pharmaceutical composition comprises a buffer. In some embodiments, the pharmaceutical composition comprises a salt. In some embodiments, the pharmaceutical composition comprises an excipient.

[0193] In some embodiments, the y5 T cells derived from blood or skin are reconstituted in a pharmaceutically acceptable carrier. In some embodiments, the y5 T cells derived from blood are reconstituted in a pharmaceutically acceptable carrier. In some embodiments, the y5 T cells derived from skin are reconstituted in a pharmaceutically acceptable carrier.Methods of use of y6 T Cells

[0194] In another aspect, the present disclosure provides methods of use of y5 T cells (e.g., V51+ y5 T cells) prepared by a method described herein.Cell Therapy

[0195] In accordance with the present disclosure, y5 T cells prepared by a method described herein may be used for applications in allogeneic cell therapy. Allogeneic cell therapy is a type of immunotherapy in which T cells are given to a patient to help the immune system fight diseases such as cancers. T cells for adoptive cell therapy can be isolated from the blood and / or tissues of the patient to be treated, grown in vitro / ex vivo at large numbers and optionally modified to enhance targeting of cancers, then reintroduced at therapeutically effective doses into the patient. T cells engineered to express a CAR (CAR-T) or a TCR, and tumor infiltrating lymphocyte (TIL) therapies fall under the umbrella of allogeneic T cell therapy. Reintroduction of these T cells into a subject in need thereof is critical for augmenting the immune response against diseases.

[0196] In some embodiments, the y5 T cells prepared by a method described herein for use are V51+ y5 T cells.

[0197] In some embodiments, V51+ y5 T cells are derived from a donor and prepared for allogeneic cell therapy for treating one or more patients.

[0198] In some embodiments, y5 T cells may be used for treatment of a disease, wherein the disease is an autoimmune / rheumatic disease, cancer and / or other malignancy.Attorney Docket No. MIL-051WO1

[0199] In some embodiments, the autoimmune / rheumatic disease can include, but is not limited to rheumatic arthritis, systemic lupus erythematosus, systemic sclerosis, ankylosing spondylitis, and juvenile idiopathic arthritis.

[0200] Examples of cancers include, but are not limited to, carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, or lymphoid malignancies. More particular examples of such cancers are noted below and include: squamous cell cancer (e.g., epithelial squamous cell cancer), Ewing sarcoma, Wilms tumor, astrocytomas, glioblastomas, lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrine tumors, medullary thyroid cancer, differentiated thyroid carcinoma, breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer, anal carcinoma, penile carcinoma, as well as head-and-neck cancer. The term “cancer” includes primary malignant cells or tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original malignancy or tumor) and secondary malignant cells or tumors (e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor).

[0201] Other examples of cancers or malignancies include, but are not limited to: Acute Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the Renal Pelvis and Ureter, Central Nervous System (Primary) Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, Childhood Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood HypothalamicAttorney Docket No. MIL-051WO1and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders, Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple Myeloma / Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo- / Malignant Fibrous Sarcoma, Osteosarcoma / Malignant Fibrous Histiocytoma, Osteosarcoma / Malignant Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Paraproteinemias, Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Transitional RenalAttorney Docket No. MIL-051WO1Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.

[0202] In some embodiments, the y5 T cells for use in the treatment of a disease are V51+ y5 T cells.

[0203] In some embodiments, the V51 y5 T cells are administered at a dose of 105to 1014cells. In some embodiments, the V51 y5 T cells are administered at a dose of 105cells. In some embodiments, the V51 y5 T cells are administered at a dose of 106cells. In some embodiments, the V51 y5 T cells are administered at a dose of 107cells. In some embodiments, the V51 y5 T cells are administered at a dose of 108cells. In some embodiments, the V51 y5 T cells are administered at a dose of 109cells. In some embodiments, the V51 y5 T cells are administered at a dose of 1010cells. In some embodiments, the V51 y5 T cells are administered at a dose of 1011cells. In some embodiments, the V51 y5 T cells are administered at a dose of 1012cells. In some embodiments, the V51 y5 T cells are administered at a dose of 1013cells. In some embodiments, the V51 y5 T cells are administered at a dose of 1014cells.

[0204] In some embodiments, V51 y5 T cells are administered intravenously, intramuscularly, intraci sternal magna, intrathecally, intraperitoneally, intradermally or subcutaneously. In some embodiments, V51 y5 T cells are administered intravenously. In some embodiments, V51 y5 T cells are administered intramuscularly. In some embodiments, V51 y5 T cells are administered intracistemal magna. In some embodiments, V51 y5 T cells are administered intrathecally. In some embodiments, V51 y5 T cells are administered intraperitoneally. In some embodiments, V51 y5 T cells are administered intradermally. In some embodiments, V51 y5 T cells are administered subcutaneously.

[0205] The composition comprising y5 T cells can be administered to a patient in need thereof one or more times per day, week, month (e.g., one or more times every 2 weeks), or year depending on the severity of the disease and change in disease state of the patient during the treatment. Generally, it is expected that a typical dosage would include 5 x 105to 5 x 1012(e.g., 5 x 105, 5 x 106, 5 x 107, 5 x 108, 5 x 109, 5 x 1010, 5 x 1011or 5 x 1012) T cells. Severity of symptoms, change in symptoms, patient response to treatment, any adverse effects of treatment, and / or effect of any additional treatment(s), can be used to determine theAttorney Docket No. MIL-051WO1frequency of treatment and the dosage, i.e., the number of T cells to be administered to a patient.

[0206] The composition comprising V51+ y5 T cells can be administered in any medically useful form.EXAMPLES

[0207] Various aspects of the invention are described in further detail in the following examples. The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that these examples are for illustrative purposes only and are not meant to limit the scope of the invention.Example 1. Overview of Methods of Cellular Activation and Expansion

[0208] This example outlines the exemplary protocols and conditions used for the expansion of immune cells of interest.

[0209] The process begins with the depletion of specific peripheral mononuclear blood cells (PBMCs; e.g., aP T cells and NK cells) and subsequent seeding of the cells of interest in a canted neck tissue culture flask in IMEM medium with a mixture of cytokines (e.g., IL-7, IL-15 and IL-21), antibodies, and a caspase inhibitor (e.g. ZVAD) on Day 0. Following this step, the cells are transduced with the cytokines IL-7 and IL-15 on Day 4.The cells are then grown and harvested and cryopreserved on Day 14. FIGs. 1A - IB outline exemplary conditions for the optimization of the expansion protocol.Example 2. Activation, Expansion, Viability, and Activity of y6 T Cells Derived from Blood

[0210] This example herein shows the results of the in vitro expansion, viability, and activity of exemplary y5 T cells harvested from blood.

[0211] Briefly, the y5 T cells were first cultured in a canted neck flask coated with anti-CD3 antibody in BM301 media with a combo-1 of cytokines and an anti-CD30 antibody on Day 0. On Day 5, the y5 T cells were reseeded in BM301 media with combo-2 of cytokines and an anti-CD30 antibody; this was then allowed to expand to Day 14, when the cells were harvested and cryopreserved.

[0212] The above protocol for the expansion of y5 T cells was tested from two individual donors, Donor- 1 and Donor-2. As compared to the population of y5 T cells expanded withoutAttorney Docket No. MIL-051WO1anti-CD30 antibody and the anti-CD30 isotype control, both populations of y5 T cells exhibited significantly enhanced expansion, about 210-fold from Donor- 1 (FIG. 2A) and nearly 1600-fold from Donor-2 (FIG. 2B). Further, the addition of anti-CD30 promoted the post-thaw proliferation rates of y5 T cells by nearly 6-fold as compared to the populations of y5 T cell products not previously expanded with anti-CD30 antibody and the isotype control from both donors (FIGs. 3A - 3B).

[0213] In addition to enhanced proliferative properties, the y5 T cells manufactured with the anti-CD30 from Donor- 1 and Donor-2 exhibited enhanced cytotoxicity against the GSU (gastric carcinoma) cancer cell line upon repeated challenge every 3 to 4 days relative to cells manufactured without the anti-CD30 antibody and the isotype control (FIGs. 4A - 4B). Further, it is noted that upon repeated antigen stimulation with GSU cells, y5 T cells manufactured with the anti-CD30 antibody showed a near 60-fold increase of the V51 subpopulation in the total population at Day 14, as measured by flow cytometry (FIGs. 5A -5B)Example 3. Activation, Expansion, Viability, and Activity of y6 T Cells Derived from Skin

[0214] This example herein shows the results of the ex vivo expansion, viability, and activity of exemplary y5 T cells harvested and expanded from skin explants of healthy donors.

[0215] FIGs. 6A - 6B outline the exemplary protocols for ex vivo activation and expansion of y5 T cells using similar protocols to above. Briefly, the activation of the cells occurs after seeding on Day 0 by adding IL-7, IL-15, IL-18, IL-21, anti-CD30 antibody, and anti-CD3 antibody (RN / OKT3), and expansion starts on Day 4 with the addition of IL-7 and IL-15. The y5 T cells are then harvested on Day 14 (FIG. 6A). This process can also be repeated a second time (i.e., to Day 28) to expand to a greater number of y5 T cells (FIG. 6B)

[0216] In using the protocol outlined above, cells cultured from skin explants had a significant total live cell fold expansion relative to the cytokine-based expansion control (FIG. 7A) and comparable viability at harvest (FIG. 7B). The same trend was observed in y5 T cells and the V51 subpopulation (FIGs. 8A-8D).

[0217] In using the expansive 28-day protocol shown in FIG. 6B, a near 600-fold total cell expansion was observed with respect to the cytokine-based control expansion protocol and an expansion protocol without using anti-CD3 and anti-CD30 antibodies (FIG. 9).Attorney Docket No. MIL-051WO1Further, it is noted that for optimal total cell expansion, both anti-CD3 and anti-CD30 antibodies are required (FIG. 10A), and that the anti-CD30 isotype control does not maintain high cell expansion using the same protocol (FIG. 10B).

[0218] y5 T cells that were expanded using the protocol involving the anti-CD3 and anti-CD30 antibodies exhibited better cytotoxic activity against OVCAR-3 (ovarian adenocarcinoma) cells at various effector: target (E: T) ratios as compared to the cytokinebased control protocol (FIG. 11A). Further, upon repeat antigen stimulation using OVCAR-3 cells at an E: T ratio of 5: 1, y5 T cells that were expanded using the protocol involving the anti-CD3 and anti-CD30 antibodies were able to kill more effectively relative to the y5 T cells that were expanded using the control protocol (FIG. 11B).

[0219] To enable a xeno-free process without impacting expansion, the inventors tried replacing the fetal bovine serum (FBS) that was used in the protocol seen in FIG. 6A with 5% human platelet lysate (HPL) and 5% OctaPlas. It is noted that in using the expansion protocol with anti-CD3 and anti-CD30 that total live cell expansion was not significantly affected (FIG. 12A). y5 T cells that were expanded using the protocol involving the anti-CD3 and anti-CD30 antibodies in 5% HPL and 5% OctaPlas exhibited better cytotoxic activity against OVCAR-3 cells at various E: T ratios as compared to the same protocol in FBS (FIG.12B)EQUIVALENTS AND SCOPE

[0220] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of examples only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed.

Claims

Attorney Docket No. MIL-051WO1CLAIMS1. A method of expanding gamma delta (y5) T cells comprising activating gamma delta (y5) T cells in a mixed cell population in the presence of a CD3 agonist and a CD30 agonist prior to expanding said gamma delta (y5) T cells,wherein the mixed cell population is not a pluripotent cell population (, or a cell population derived from the pluripotent cell population.

2. The method of claim 1, wherein the mixed cell population is from a blood sample, or a non- hematopoietic tissue.

3. The method of claim 1 or 2, wherein the gamma delta (y5) T cells are V51+ y5 T cells.

4. The method of any one of claims 1 to 3, wherein the CD3 agonist is an anti-CD3 antibody, or a functional fragment thereof; and wherein the CD30 agonist is an anti-CD30 antibody, or a functional fragment thereof.

5. The method of any one of claims 1 to 4, wherein the y5 T cells are activated in the presence of the CD3 agonist and the CD30 agonist for a period of 1 to 5 days.

6. The method of any one of claims 1 to 5, wherein the mixed cell population is cultured in a cell culture medium comprising one or more cytokines from IL-7, IL- 15, IL- 18, IL-21 and combinations thereof, for activating the y5 T cells.

7. The method of any one of claims 1 to 6, comprising expanding the activated gammadelta T cells in the presence of one or more cytokines from IL-2, IL-7, IL- 15, IL- 18 and IL-21.

8. The method of claim 6, wherein the mixed cell population is from a blood sample.

9. The method of claim 8, wherein the blood sample is a peripheral blood sample, or a cord blood sample.

10. The method of claim 8 or 9, wherein the gamma-delta T cells is activated in the culture medium comprising IL-18 and IL-21.Attorney Docket No. MIL-051WO111. The method of claim 10, wherein the activated gamma-delta T cells are expanded in the culture medium comprising the CD30 agonist, IL-2, IL- 15 and IL-21, or in the culture medium comprising IL-2, IL-7, IL- 15, IL-21 and the CD30 agonist.

12. The method of claim 6, wherein the mixed cell population is from a non-hematopoietic tissue.

13. The method of claim 12, wherein the non- hematopoietic tissue is a skin tissue.

14. The method of claim 12 or 13, wherein the gamma-delta T cells is activated in the culture medium comprising IL-7, IL-15, IL-18 and IL-21.

15. The method of claim 14, wherein the activated gamma-delta T cells are expanded in the culture medium comprising IL-7 and IL-15.

16. The method of any one of the preceding claims, wherein the expanded gamma-delta T cells are harvested on Day 14.

17. The method of any one of the preceding claims, wherein the CD3 agonist is present at a concentration ranging from 0.1 - 3000 ng / mL and the CD30 agonist is present at a concentration ranging from 0.1 - 300 ng / mL, and the IL-2 is present at a concentration ranging from 0.1-100 ng / mL, and the IL-7 is present at a concentration ranging from 0.1-10 ng / mL, and the IL-15 is present at a concentration ranging from 0.1-10 ng / mL, and the IL-18 is present at a concentration ranging from 0.1-50 ng / mL, and the IL-21 is present at a concentration ranging from 0.1-20 ng / mL.

18. The method of any one of the preceding claims, wherein the gamma-delta T cells are expanded at least 10-fold.

19. The method of any one of the preceding claims, wherein the gamma-delta T cells have increased post-thaw survival rate.

20. The method of any one of the preceding claims, wherein the gamma-delta T cells have increased cytotoxicity.

21. A method of expanding y5 T cells from a mixed cell population derived from a non-hematopoietic tissue comprising activating y5 T cells in the mixed cell population in the presence of a CD3 agonist and a CD30 agonist, in a culture medium comprising one or moreAttorney Docket No. MIL-051WO1cytokines selected from IL-7, IL- 15, IL- 18 IL-21 and combinations thereof; and expanding the activated y5 T cells in a culture medium comprising IL-7 and IL-15.

22. The method of claim 21, wherein the non-hematopoietic tissue is a skin tissue.

23. The method of claim 21 or 22, wherein the culture medium for activating the y5 T cells comprises cytokines IL-7, IL- 15, IL- 18 and IL-21.

24. The method of any one of claims 21 to 23, wherein the y5 T cells are activated for 1 to 3 days.

25. The method of claim 24, wherein the y5 T cells are activated for 3 days.

26. The method of claim 25, wherein the activated y5 T cells are expanded in the cell culture medium comprising IL-7 and IL- 15 for 11 days.

27. The method of any one of claims 21 to 26, wherein the method comprises repeating the activation and expansion steps to further expand the y5 T cells.

28. The method of any one of claims 21 to 27, where the y5 T cells are expanded by at least 500-fold.

29. A method of expanding y5 T cells from a mixed cell population derived from a blood sample comprising activating y5 T cells in the mixed cell population in the presence of a CD3 agonist and a CD30 agonist, in a culture medium comprising one or more cytokines selected from IL-7, IL- 15, IL- 18 and IL-21; and expanding the activated y5 T cells in a cell culture medium comprising the CD30 agonist and one or more cytokines selected from IL-2, IL-7, IL-15 IL-21 and combinations thereof.

30. The method of claim 29, wherein the blood sample is a peripheral blood sample, or a cord blood sample.

31. The method of claim 29 or 30, wherein the blood sample is a human blood sample.

32. The method of any one of claims 29 to 31, wherein the cell culture medium for activating the y5 T cells comprises cytokines IL-7, IL- 15, IL- 18 and IL-21.

33. The method of any one of claims 29 to 31, wherein the cell culture medium for activating the y5 T cells comprises cytokines IL- 15, IL- 18 and IL-21.Attorney Docket No. MIL-051WO134. The method of any one of claims 29 to 33, wherein the activated y5 T cells are expanded in the cell culture medium comprising cytokines IL-7 and IL-15.

35. The method of any one of claims 29 to 33, wherein the activated y5 T cells are expanded in the cell culture medium comprising cytokines IL-2, IL-7 and IL-15.

36. The method of any one of claims 29 to 33, wherein the activated y5 T cells are expanded in the cell culture medium comprising cytokines IL-2, IL-7, IL- 15 and IL-21.

37. The method of any one of claims 29 to 33, wherein the activated y5 T cells are expanded in the cell culture medium comprising cytokines IL-2, IL- 15 and IL-21.

38. The method of any one of claims 29 to 37, wherein the total fold expansion of gamma delta T cells is between 200 to 1600-fold.

39. The method of claims 21 to 38, wherein the CD3 agonist is an anti-CD3 antibody, or a functional fragment thereof; and wherein the CD30 agonist is an anti-CD30 antibody, or a functional fragment thereof.

40. The method of any one of claims 21 to 39, wherein the y5 T cells are V51+ y5 T cells.

41. The method of any one of the preceding claims, further comprising depleting other non- y5 T cells from the mixed cell population prior to activating and expanding the y5 T cells.

42. The method of any one of the preceding claims, wherein the cell culture medium comprises 5% human platelet lysate (HPL) and 5% OctaPlas.

43. The method of any one of claims 21 to 42, wherein the V51+ y5 T cells have increased cytotoxicity.

44. A composition comprising the y5 T cells manufactured by the method of any one of the preceding claims.

45. The composition of claim 42, wherein the y5 T cells are for allogeneic cell therapy.

46. Use of the y5 T cells produced by the method of any one of claims 1 to 43, for treatment of a disease.Attorney Docket No. MIL-051WO147. The use of claim 46, wherein the cells are used for allogeneic cell therapy.

48. A method of treating a disease, the method comprising administering to a subject in need thereof, a composition comprising the y5 T cells manufactured by the method of any one of claims 1 to 43.

49. The method of claim 48, wherein the disease is a cancer, an infectious disease, an autoimmune disease, or an inflammatory disease.

50. The method of any of the preceding methods, wherein the method does not comprise any IL-12.

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