Methods for activation and expansion of gamma delta t cells

Abstract

The technology relates in part to methods for activation and expansion of gamma delta (γδ) T cells. In some aspects, the technology relates to methods for activation and expansion of multi-subsets of γδ T cells. In some aspects, the technology relates to a process for effectively expanding polyclonal γδ T cells from human donors and engineering them for therapeutic applications. In some aspects, the technology relates to activated polyclonal γδ T cells produced by methods described herein. In some aspects, the technology relates to methods of using cytokines and / or small molecules to enhance the expansion, purity and cell function of γδ T cells. In some aspects, γδ T cells are gene-edited and engineered with chimeric antigen receptors (CARs) for therapeutic applications. The method described herein has broad applications in immunotherapy, particularly in the treatment of cancer, infectious diseases, and autoimmune disorders.

Description

[0001] PATENT

[0002] POINT-1003PCT

[0003] METHODS FOR ACTIVATION AND EXPANSION OF GAMMA DELTA T CELLS

[0004] Related patent applications

[0005] This patent application claims the benefit of U.S. provisional patent application no. 63 / 714,642 filed on October 31 , 2024, entitled METHODS FOR ACTIVATION AND EXPANSION OF GAMMA DELTA T CELLS, naming Xiaohong WANG, et al. as inventors, and designated by attorney docket no. POINT-1003PROV. The entire content of the foregoing patent application is incorporated herein by reference for all purposes, including all text, tables and drawings.

[0006] Field

[0007] The technology relates in part to methods for activation and expansion of gamma delta (yd) T cells. In some aspects, the technology relates to methods for activation and expansion of multi-subsets of yd T cells. In some aspects, activated and expanded multi-subsets of yd T cells are generated for use in adoptive cell therapy, including the generation of chimeric antigen receptor (CAR)- engineered yd T cells. In some aspects, the technology relates to a process for effectively expanding polyclonal yd T cells from human donors and engineering them for therapeutic applications. In some aspects, the technology relates to activated polyclonal yd T cells produced by methods described herein. In some aspects, the technology relates to methods of using cytokines and / or small molecules to enhance the expansion, purity and cell function of yd T cells. In some aspects, yd T cells are gene-edited and engineered with chimeric antigen receptors (CARs) for therapeutic applications. The methods described herein have broad applications in immunotherapy, particularly in the treatment of cancer, infectious diseases, and autoimmune disorders.

[0008] Background

[0009] Adoptive cell therapy, particularly utilizing CAR (chimeric antigen receptor) T cells, has demonstrated significant clinical efficacy and represents a major advancement in the treatment of hematological malignancies. However, the use of autologous CAR-T cell products is often associated with high costs, extended manufacturing times, and limited patient accessibility.

[0010] Gamma delta (yd) T cells are an innate-like subset of T cells characterized by T cell receptors (TCRs) composed of y and d chains. Unlike conventional alpha beta (a|3) T cells, which primarily recognize peptide antigens presented by major histocompatibility complex (MHO) molecules, yd T cells have a distinctive capacity for antigen recognition. They can identify a wide array of antigens, including stress-induced and non-peptide antigens, in an MHC-independent manner, yd T cells serve as a bridge between innate and adaptive immune responses, playing crucial roles in immune surveillance, host defense, and immunoregulation. They are less likely to induce graft-versus-host disease (GVHD) and are associated with a reduced risk of severe cytokine release syndrome PATENT POINT-1003PCT (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), potentially offering a safer alternative to traditional CAR a|3 T cell therapies.

[0011] Despite these advantages, yd T cells are present at low frequencies in human blood, typically comprising only 1 -5% of T cells. This limited availability presents a significant challenge for their use in adoptive cell therapy. yd T cells encompass diverse subsets with distinct phenotypic and functional properties. Human yd T cells are classified based on their TCRs, which utilize three main Vd genes (Vd1 , Vd2, Vd3) and up to six Vy genes. The two primary yd T cell subsets are Vd1 and Vd2. Vd1 T cells are enriched in peripheral tissues and are involved in various immune functions. Vd2 T cells are more commonly found in blood and secondary lymphoid organs and are noted for their response to certain antigens and stress signals.

[0012] Current methods for expanding yd T cells typically focus on a single subset, such as Vd1 or Vd2, due to limitations with specific activating antibodies or agonists. For example, the expansion of Vd1 T cells often relies on Vd1 TCR activating antibodies, while Vd2 T cells are expanded using agonists like Zoledronate or BTN3A1 / BTN2A1 . This focus on single subsets has resulted in limited clinical responses, highlighting the need for improved expansion techniques and the potential benefits of including multiple yd T cell subsets in therapeutic approaches.

[0013] Provided herein are processes for effective activation and expansion of multi-subsets of polyclonal yd T cells (e.g., from human apheresis products). In one example, a process provided herein begins with collection of apheresis products from healthy donors, followed by depletion of ap T cells to enrich for yd T cells. The resulting immune cells are then cultured with a specific cocktail of activating antibodies and cytokines designed to support preferential expansion of yd T cells while maintaining their viability and function. This process allows for the concurrent expansion of various yd T cell subsets, including Vd1 , Vd2, and Vd1 -Vd2- T cells. During the activation and expansion process, yd T cells can also be gene-edited and engineered with GAR constructs for targeted therapeutic applications.

[0014] The process provides a significant advancement in yd T cell therapy by facilitating the activation and expansion of a diverse range of yd T cell subsets. This approach addresses the limitations of current yd T cell therapies and may enhance therapeutic efficacy, safety, and accessibility. Integrating CAR technology with polyclonal yd T cells could transform the treatment landscape for various malignancies and immune disorders. PATENT

[0015] POINT-1003PCT

[0016] Summary

[0017] Provided in certain aspects are methods for activating and selectively expanding gamma delta (yd) T cells ex vivo, comprising activating gamma delta (yd) T cells and expanding the number of gamma delta (yd) T cells in an originating cell population under activation and expansion conditions, thereby generating an activated and expanded gamma delta (yd) T cell-enriched cell population, where the activation and expansion conditions comprise a) binding agents comprising a cluster of differentiation 3 (CD3) binding agent and a cluster of differentiation 2 (CD2) binding agent; and b) cytokines comprising interleukin 2 (IL-2) and interleukin 4 (IL-4); IL-2 and interleukin 21 (IL-21 ); IL-2, IL-4, and IL-21 ; interleukin 15 (IL-15) and IL-4; IL-15 and IL-21 ; or IL-15, IL-4, and IL-21.

[0018] Also provided in certain aspects are methods for activating and selectively expanding gamma delta (yd) T cells ex vivo, comprising activating and expanding the number of gamma delta (yd) T cells in an originating cell population under activation and expansion conditions, thereby generating an activated and expanded gamma delta (yd) T cell-enriched cell population, where the activation and expansion conditions comprise a) binding agents comprising a cluster of differentiation 3 (CD3) binding agent and a cluster of differentiation 2 (CD2) binding agent; b) cytokines comprising interleukin 2 (IL-2), interleukin 15 (IL-15), or IL-2 and IL-15; and c) one or more media components chosen from one or more salts, one or more sugars, one or more vitamins, and one or more transforming growth factor beta (TGF-beta) inhibitors.

[0019] Also provided in certain aspects are methods for activating and selectively expanding gamma delta (yd) T cells ex vivo, comprising a) activating gamma delta (yd) T cells in an originating cell population under activation conditions, thereby generating an activated gamma delta (yd) T cell population, where the activation conditions comprise binding agents comprising a cluster of differentiation 3 (CD3) binding agent and a cluster of differentiation 2 (CD2) binding agent; and b) expanding the number of gamma delta (yd) T cells in the activated gamma delta (yd) T cell population under expansion conditions, thereby generating an activated and expanded gamma delta (yd) T cell-enriched cell population, where the expansion conditions comprise cytokines comprising interleukin 2 (IL-2) and interleukin 4 (IL-4); IL-2 and interleukin 21 (IL-21 ); IL-2, IL-4, and IL-21 ; interleukin 15 (IL-15) and IL-4; IL-15 and IL-21 ; or IL-15, IL-4, and IL-21.

[0020] Also provided in certain aspects are kits for activating and selectively expanding gamma delta (yd) T cells ex vivo comprising a) binding agents comprising a cluster of differentiation 3 (CD3) binding agent, and a cluster of differentiation 2 (CD2) binding agent; b) cytokines comprising interleukin 2 (IL-2) and interleukin 4 (IL-4); IL-2 and interleukin 21 (IL-21 ); IL-2, IL-4, and IL-21 ; interleukin 15 (IL- 15) and IL-4; IL-15 and IL-21 ; or IL-15, IL-4, and IL-21 ; and c) instructions for use. PATENT

[0021] POINT-1003PCT

[0022] Also provided in certain aspects are methods for preferentially expanding y5 T cells ex vivo, comprising expanding the number of yb T cells in an originating cell population under expansion culture conditions, thereby generating an expanded yb T cell-enriched cell population, where the expansion culture conditions comprise binding agents of: (i) a cluster of differentiation 3 (CD3) binding agent, and (ii) a cluster of differentiation 2 (CD2) binding agent.

[0023] Also provided in certain aspects are kits for selectively expanding yb T cells ex vivo comprising binding agents of: (i) a cluster of differentiation 3 (CD3) binding agent, and (ii) a cluster of differentiation 2 (CD2) binding agent.

[0024] Also provided in certain aspects are kits for selectively expanding yb T cells ex vivo comprising cytokines chosen from two or more of: interleukin-2 (IL-2), interleukin-15 (IL-15), interleukin-4 (IL- 4), interleukin-21 (IL-21 ), and interleukin-10 (IL-10).

[0025] Also provided in certain aspects are media components for selectively expanding yb T cells ex vivo and enhancing the function of expanded cells comprising components chosen from two or more of: L-ascorbic acid, galactose, sodium formate, folic acid, nicotinamide, and TGF-beta receptor inhibitor A83-01 .

[0026] Certain implementations are described further in the following description, examples and claims, and in the drawings.

[0027] Brief Description of the Drawings

[0028] The drawings illustrate certain implementations of the technology and are not limiting. For clarity and ease of illustration, the drawings are not made to scale and, in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular implementations.

[0029] Figs. 1 A-1 G show anti-CD2 agonist antibody enhances the expansion of Vb1 , Vb2, and Vb1 -Vb2- yb T subsets ex vivo. Fig. 1 A shows flow cytometry for a|3 T and yb T cell composition in healthy donor-derived PBMC before and after a|3 T cell depletion on Day 0. Fig. 1 B shows Day 18 culture of a|3 T cell-depleted PBMC activated and expanded by IL-15, OKT3 with or without anti-CD2 Ab. Fig. 1 C shows a change of cell composition in the first week of culture containing anti-CD2 Ab. Fig. 1 D shows total live cell counts over 21 -day culture starting from 1 million of ap T cell-depleted PBMC. Fig. 1 E shows yb T cell fold expansion over 18-day culture. Fig 1 F shows fold expansion of Vb1 +, Vb2+, Vb1 -Vb2- yb T cells from different donors (n=11 ) over 14- to 21 -day culture with OKT3 and anti-CD2 Ab. Fig. 1 G shows a luciferase assay to determine the innate cytotoxicity of expanded yb T cells in 4-day co-culture with tumor cell lines. PATENT

[0030] POINT-1003PCT

[0031] Figs. 2A-2E show IL-2 and IL-15 result in comparable yd T cell expansion efficiency ex vivo. Viability (Fig. 2A), yield (Fig. 2B), fold expansion (Fig. 2C), effector and memory phenotype (Fig. 2D), and inhibitory or exhaustion markers (Fig. 2E) of yd T cells activated and expanded by OKT3, anti-CD2 Ab and IL-2 or IL-15 in 21 -day culture are presented.

[0032] Figs. 3A-3H show small molecules enhance the expansion and transduction of CAR yd T cells. Fold expansion (Fig. 3A) and cell composition (Fig. 3B) of CAR yd T cells following 16-day culture containing IL-2 with or without galactose, sodium formate, folic acid, or A83-01 are presented. Fig 3C shows a tumor killing assay in 48 h coculture of expanded CAR yd T cells with CAR targetpositive TNBC cell line HCC1860. Cell composition (Fig. 3D), fold expansion (Fig. 3E), CAR transduction efficiency as measured by percentage of CAR-positive yd T cells and mean fluorescence intensity (MFI) of CAR expression (Fig. 3F), memory phenotypes (Fig. 3G), and detection of activation and exhaustion markers on CAR yd T cells (Fig. 3H) following 14-day culture containing IL-2 with or without ascorbic acid or nicotinamide are presented.

[0033] Figs. 4A-4E show IL-4 and IL-21 synergistically enhance the expansion and cytolytic activity of gene-edited CAR yd T cells. A representative flow diagram of CAR expression and TGF R2 cell surface expression (Fig. 4A), cell viability (Fig. 4B), and fold expansion (Fig. 4C) of TGFPR2 KO and CAR transduced yd T cells harvested after 14-day culture containing IL-2 with or without IL-4 or / and IL-21 are presented. Cytolytic activity of TGF R2 KO CAR yd T cells was then determined in a 24 h co-culture assay with Luc-GFP-labeled NSCLC cell line NCI-H1975 (Fig. 4D) and TNBC HCC1806 (Fig. 4E).

[0034] Figs. 5A-5C show repeat OKT3 stimulation enhances the expansion of non-Vd2 subsets while maintaining the cytotoxicity of gene-edited CAR yd T cells. Flow cytometry of yd T cell composition (Fig. 5A), fold expansion (Fig. 5B), and cytotoxicity (Fig. 5C) of TGF[3R2 KO CAR yd T cells following a 14-day expansion culture without (control) or with OKT3 restimulation. Lysis of target tumor cell line HCC1806 was measured following 24 h co-culture with gene-edited CAR yd T cells or expanded unmodified yd T cells.

[0035] Figs. 6A-6H show combinations of certain small molecules impact expansion, phenotype, and function of gene-edited CAR yd T cells. Expanded yd T cells were harvested on Day 14 post activation, and CAR transduction (Fig. 6A) and TGF R2 knock-out (Fig. 6B) efficiency, cell composition (Fig. 6C), and yd T cell memory phenotypes (Fig. 6D) were determined by flow cytometry. The cytolytic activity of TGF[3R2 KO CAR yd T cells were measured in a 4-day coculture with 3D tumor spheroids derived from GFP-Luc-labeled target tumor cell lines HCC827 and NCI- H1975 at different E:T ratio (Fig. 6E). The lysis of tumor spheroids was monitored by green fluorescence intensity on BioTek Cytation 5 cell imaging multimode reader. TGFPR2 KO CAR yd T PATENT

[0036] POINT-1003PCT cells were also co-cultured with tumor cell line NCI-H1975 at E:T ratio of 1 :2 (2.5e5 y6 T cells vs 5e5 tumor cells), followed by 2 rounds of repeat fresh NCI-H1975 cells (2.5e5 tumor cells) challenges every 3-4 days. The yd T cell expansion (Fig. 6F), subtype composition (Fig. 6G) and surface CD103 expression (Fig. 6H) following repeat tumor antigen stimulation were determined by flow cytometry. F / A / F represents the combination treatment of formate I A83-01 / folic acid and F / A / G represents formate / A83-01 / galactose.

[0037] Figs. 7A-7D show efficient expansion of gene-edited CAR yO T cells in G-REX static culture and shake flask culture with agitation. A thawed aliquot of a|3 T-depleted apheresis was activated and transduced with CAR retroviral vector in G-REX, followed by electroporation with RNP of TGF|3R2 sgRNA / Cas9 on Day 6 of culture. On day 7, an equal number (5 million) of gene-edited CAR yd T cells were cultured in G-REX 6M or Erlenmeyer Cell Shaker Flask with 150 RMP constant orbital shakes. The final culture was harvested on Day 14 for cell counting and characterization. Cell composition on Day 14 (Fig. 7A), fold expansion of total yd T cells and subtypes over 14-day culture (Fig. 7B), % cells expressing activation (NKG2D), NCRs (NKp30, NKp44, NKp46), and exhaustion (PD1 +TIGIT+, PD1 +TIM3+, PD1 +LAG3+) markers on Day 14 (Fig. 7C) are presented. Fig. 7D shows cytotoxicity of TGF R2 KO CAR yd T cells against target tumor cell lines HCC70 and NCI-H1975 following 24 h co-culture. The tumor-specific killing was determined by luciferase assay.

[0038] Detailed Description

[0039] Provided herein are methods, compositions, and kits for activating and selectively expanding gamma delta (yd) T cells ex vivo. Also provided herein are populations of activated and expanded gamma delta (yd) T cells. In certain embodiments, gamma delta (yd) T cells are modified. For example, modifications may include introduction of a chimeric antigen receptor (CAR) and / or genome editing. A CAR may include one or more antigen binding domains. Genome editing may include a specific gene knock-out.

[0040] Cell culture

[0041] Provided herein are methods and compositions for cell culture. In particular, provided herein are expansion culture conditions (“expansion conditions”). Also provided herein are activation culture conditions (“activation conditions”). Also provided herein are activation and expansion culture conditions (“activation and expansion conditions”). Cell culture, or culture, typically refers to the maintenance of cells in an artificial, in vitro environment, or the maintenance of cells in an external, ex vivo environment (i.e. , outside of an organism). Certain cell culture systems described herein may be an ex vivo environment and / or an in vitro environment. PATENT

[0042] POINT-1003PCT

[0043] Cells may be obtained from a subject and / or a cellular source. Cells obtained from a subject and / or a cellular source may be referred as an originating cell population. An originating cell population is the input population of cells for expansion by culture conditions described herein (e.g., expansion conditions). A cellular source may include a population of circulating blood cells. A cellular source may include a population of peripheral blood mononuclear cells (PBMC). A cellular source may include cord blood cells. A cellular source may include cells isolated from tissue (e.g., skin, tumor). A cellular source may include a population of immune cells. A cellular source may include a population of lymphocytes. A cellular source may include a population of lymphocytes depleted of one or more lymphocyte subtypes. A cellular source may include a population of T cells. A cellular source may include a population of T cells depleted of one or more T cell subtypes. A cellular source may include a population of T cells depleted of alpha beta (a|3) T cells. An originating cell population can be obtained from a subject in a variety of manners (e.g., isolated from circulation, isolated by apheresis, isolated by leukapheresis). In some embodiments, immune cells are isolated from a subject. In some embodiments, lymphocytes cells are isolated from a subject. In some embodiments, T cells are isolated from a subject. In some embodiments, cells may be derived from (e.g., obtained from) a peripheral blood mononuclear cell (PBMC) population isolated from a subject. In some embodiments, cells may be derived from (e.g., obtained from) a blood cell population isolated from cord blood obtained from a subject. In some embodiments, cells may be derived from (e.g., obtained from) an apheresis product. Apheresis generally refers to a process whereby blood of a person is passed through an apparatus that separates one particular constituent and returns the remainder to the circulation. A subject may include any animal, including but not limited to any mammal, such as mouse, rat, canine, feline, bovine, equine, porcine, non-human primate and human. In certain embodiments, a subject is a human. In certain embodiments, a subject is a healthy donor.

[0044] Cell seeding densities may be adjusted according to certain desired culture conditions. For example, an initial seeding density of from about 1 x 103to about 1 -10 x 105cells per cm2may be used. In some embodiments, an initial seeding density of from about 1 -10 to about 1 -10 x 105cells per cm2may be used. In certain instances, 1 x 106cells may be cultured in a 75 cm2culture flask. In certain instances, a cell culture bag is used. In certain instances, a dynamic culture platform (e.g., shaking flask, stirred tank bioreactor, or perfusion bioreactor) is used. Thus, in certain embodiments, the activation and expansion conditions comprise and / or apply to dynamic culture conditions. In certain instances, a static culture platform is used, where cells are grown in a stationary environment, such as a petri dish or T-flask (e.g., without a constant flow of media and / or mechanical stimulation). Thus, in certain embodiments, the activation and expansion conditions comprise and / or apply to static culture conditions. In certain instances, a membrane-based static PATENT

[0045] POINT-1003PCT cell culture system is used (e.g., G-REX (Gas Permeable Rapid Expansion) by SCALEREADY). Cell density may be altered as needed at any passage.

[0046] Cells may be cultivated in a cell incubator at about 37°C at normal atmospheric pressure. The incubator atmosphere may be humidified and may contain from about 3-10% carbon dioxide in the air. In some instances, the incubator atmosphere may contain from about 0.1 -30% oxygen. Temperature, pressure and carbon dioxide and oxygen concentration may be altered as needed. Culture medium pH may be in the range of about 7.1 to about 7.6, or from about 7.1 to about 7.4, or from about 7.1 to about 7.3.

[0047] Cell culture medium may be replaced every 1-2 days or more or less frequently as needed. As the cells approach confluence in the culture vessel, they may be passaged. A cell passage is a splitting or dividing of the cells, and a transferring a portion of the cells into a new culture vessel or culture environment. Cells which are adherent to the cell culture surface may require detachment. Methods of detaching adherent cells from the surface of culture vessels are well known and can include the use of enzymes such as trypsin.

[0048] A single passage refers to a splitting or manual division of the cells one time, and a transfer of a smaller number of cells into a new container or environment. When passaging, the cells can be split into any ratio that allows the cells to attach and grow. For example, at a single passage the cells can be split in a 1 :2 ratio, a 1 :3 ratio, a 1 :4 ratio, a 1 :5 ratio, and so on. In some embodiments, cells are passaged at least about 1 time to at least about 20 times. For example, cells may be passaged at least about 2 times, 3 times, 4 times , 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, or 20 times. In some embodiments, cells are passaged about 1 time to about 20 times. For example, cells may be passaged about 2 times, 3 times, 4 times , 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, or 20 times. In some embodiments, cells are passaged about 10 times.

[0049] Cell growth generally refers to cell division, such that one mother cell divides into two daughter cells. Cell growth may be referred to as cell expansion. Cell growth herein generally does not refer to an increase in the actual size (e.g., diameter, volume) of the cells. Stimulation of cell growth can be assessed by plotting cell populations (e.g., cell population doublings) over time. A cell population with a steeper growth curve generally is considered as growing faster than a cell population with a less steep curve. Growth curves can be compared for various treatments between the same cell types, or growth curves can be compared for different cell types with the same conditions, for example. PATENT

[0050] POINT-1003PCT

[0051] Expanding a population of cells may be expressed as population doubling. A cell population doubling occurs when the cells in culture divide so that the number of cells is doubled. In some instances, cells are counted to determine if a population of cells has doubled, tripled or multiplied by some other factor. The number of population doublings may not be equivalent to the number of times a cell culture is passaged. For example, passaging the cells and splitting them in a 1 :3 ratio for further culturing may not be equivalent to a tripled cell population. A formula that may be used for the calculation of population doublings (PD) is presented in Equation A: n = 3.32 * (log Y - log I) + X Equation A where n = the final PD number of the cell culture when it is harvested or passaged, Y = the cell yield at the time of harvesting or passaging, I = the cell number used as inoculum to begin that cell culture, and X = the PD number of the originating cell culture that is used to initiate the subculture.

[0052] In some embodiments, a method herein comprises expanding a population of cells. Expanding a population of cells may be referred to as proliferating a population of cells. Expanding a population of cells may be expressed as fold increase in cell numbers. A formula that may be used for the calculation of fold increase as a function of population doublings is presented in Equation B:

[0053] F= 2nEquation B where F= the fold increase in cell numbers after n population doublings. For example, after one (1 ) population doubling, the number of cells increases by 2-fold, and after two (2) population doublings, the number of cells increases by 4 (22= 4) fold, and after three (3) population doublings, the number of cells increases by 8 (23= 8) fold, and so on. Hence, after twenty (20) population doublings, the number of cells increases by more than one million-fold (220= 1 ,048,576), and after thirty (30) population doublings, the number of cells increases by more than one billion-fold (230= 1 ,073,741 ,824), and after forty (40) population doublings, the number of cells increases by more than one trillion-fold (240= 1 ,099,51 1 ,627,776), and so on. In some embodiments, a population of cells is expanded, or is capable of being expanded, at least about 2-fold to at least about a trillionfold. For example, a population of cells may be expanded at least about 5-fold, 10-fold, 15-fold, 20- fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 300-fold, 1 ,000-fold, 10,000-fold, 100,000-fold, 1 million-fold, 1 billion-fold, or 1 trillion-fold. A particular fold expansion may occur over a certain period of time in culture such as, for example, 2 days, 3 days, 4 days, 5 days, 10 days, 20 days, 30 days, 40 days, 50 days, 100 days or more.

[0054] In some embodiments, cells are cultures for about 1 hour, 2 hours, 5 hours, 10 hours, 12 hours, 15 hours, 20 hours, or days such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 25, 30, 35, 40, 45, 50, 55, or 60 or more days or weeks such as about 2, 3, 4, 5, 6, 7, 8, 9, or 10 PATENT POINT-1003PCT weeks. In some embodiments, activation conditions described herein are for a period of about 1 hour, 5 hours, 10 hours, 12 hours, 15 hours or 20 hours to about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week. In some embodiments, activation conditions are for a period of between about 12 hours, 24 hours, 36 hours, or 2 days to about 3 days, 4 days, 5 days, 6 days or 1 week, or about 2 days to about 4 days or 5 days, or about 3 days to about 4 days. In some embodiments, expansion conditions described herein are for a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 1 week, 2 weeks, 3, weeks, or more.

[0055] Gamma delta T cell activation

[0056] Provided herein are methods, compositions, and kits for activating a population of gamma delta (yb) T cells. Activation of gamma delta (yb) T cells generally initiates an innate immune response in the cells, which can be used to target a disease, such as a cancer, autoimmune disease, or infectious disease, in a subject in need of treatment for such a disease. Gamma delta (yb) T cells typically are activated via yb T-cell receptors directly or indirectly. Briefly, Vy9b2 T cells can be specifically activated and efficiently expanded with phosphoantigens (e.g. isopentenyl pyrophosphate or analog), or bisphosphonates (e.g. zoledronic acid) in combination with IL-2 and / or other cytokines. Vb1 T cells are specifically activated and expanded with TCRVbl agonist antibodies in combination with IL-2 and / or other cytokines. Alternatively, pan anti-TCR yb agonist antibodies (e.g. clone B1 ) or anti-CD3 agonist antibody (clone OKT3) with a combination of different cytokines with or without artificial antigen presenting cells may activate the cells. Activation of gamma delta (yb) T cells may be assessed, for example, by proliferation (cell division) metrics, expression of surface activation markers such as CD69, CD25, NKG2D, and DNAM-1 , and / or cytolytic activity as indicated by degranulation marker CD107a, inflammatory cytokine interferon gamma ( IFN-y) , cytotoxic granzyme B, and / or perforin upon antigen-exposure.

[0057] In the methods provided herein, an originating cell population can be exposed to activation and expansion conditions simultaneously or sequentially in any order. In certain workflows, gamma delta (yb) T cells are first activated under activation conditions described herein and then are expanded under expansion conditions described herein. In certain workflows, gamma delta (yb) T cells are activated under activation conditions described herein and are simultaneously expanded under expansion conditions described herein. Accordingly, the terms “activation conditions” and “expansion conditions” may be used interchangeably herein. “Activation” conditions described below may also apply to “activation and expansion” conditions.

[0058] In some embodiments, activation conditions comprise one or more agents that bind to one or more antigens. A binding agent in the context of activation condition components herein generally refers to a molecule that is or comprises one or more antibodies, antibody fragments, or antibody PATENT

[0059] POINT-1003PCT derivatives. In some instances, a binding agent may be a ligand. The term antibody generally encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. An antibody fragment generally refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab') 2, and multispecific antibodies formed from antibody fragments. An antibody derivative generally refers to a molecule other than an intact antibody that comprises a portion derived from an intact antibody (or an antigen-binding fragment thereof) and which binds to an antigen to which the intact antibody (or an antigen-binding fragment thereof) binds. Examples of antibody derivatives include, but are not limited to, single chain variable fragments (scFv), VHH fragments, nanobodies, diabodies, triabodies, and the like, aptamers comprising multiple antigenbinding antibody fragments, single chain variable fragments, VHH fragments, nanobodies, diabodies, triabodies, and the like.

[0060] In some embodiments, activation conditions herein comprise one or more agents that bind to cell surface molecules (e.g., immune cell markers). In some embodiments, activation conditions herein comprise one or more cluster of differentiation (CD) binding agents. The cluster of differentiation (also known as cluster of designation or classification determinant) generally are cell surface markers used for immunophenotyping of cells. CD molecules can act as receptors or ligands, often initiating a signal cascade, altering the behavior of the cell. Some CD proteins do not play a role in cell signaling, but have other functions, such as cell adhesion. CD for humans is numbered from 1 to 371 (e.g., CD1 to CD371 ). In some embodiments, activation conditions herein comprise a CD2 binding agent. In some embodiments, a CD2 binding agent is an antibody, antibody fragment, or antibody derivative. Any suitable anti-CD2 antibody, antibody fragment, or antibody derivative may be used in the activation conditions herein, including commercially available anti-CD2 antibodies, antibody fragments, or antibody derivatives. In some embodiments, activation conditions herein comprise a CD3 binding agent. In some embodiments, a CD3 binding agent is an antibody, antibody fragment, or antibody derivative. Any suitable anti-CD3 antibody, antibody fragment, or antibody derivative may be used in the activation conditions herein, including commercially available anti-CD3 antibodies, antibody fragments, or antibody derivatives. In some embodiments, activation conditions comprise binding agents comprising a CD3 binding agent and a CD2 binding agent. In some embodiments, activation conditions comprise binding agents consisting of a CD3 binding agent and a CD2 binding agent.

[0061] In some embodiments, a method herein comprises reactivating a previously activated gamma delta (yd) T cell population. Reactivating may also be referred to herein as restimulating. A method PATENT

[0062] POINT-1003PCT herein may comprise reactivating a gamma delta (yd) T cell population under reactivation conditions. Reactivation conditions may comprise any of the components described herein for an initial activation. For example, reactivation conditions may comprise a cluster of differentiation 3 (CD3) binding agent. In some embodiments, reactivation conditions comprise a cluster of differentiation 2 (CD2) binding agent. In some embodiments, reactivation conditions comprise a cluster of differentiation 2 (CD2) binding agent and a cluster of differentiation 3 (CD3) binding agent. In some embodiments, a gamma delta (yd) T cell population is reactivated after the initial activation and during expansion. In some embodiments, a gamma delta (yd) T cell population is reactivated after the initial activation and after expansion. In some embodiments, a gamma delta (yd) T cell population is reactivated prior to introducing a genetic modification. In some embodiments, a gamma delta (yd) T cell population is reactivated after introducing a genetic modification.

[0063] Gamma delta T cell expansion

[0064] Provided herein are methods, compositions, and kits for expanding a population of gamma delta (yd) T cells. In some embodiments, methods, compositions, and kits are provided for expanding a population of gamma delta (yd) T cells from an originating cell population, where the originating cell population is a heterogeneous population of cells. In some embodiments, the heterogeneous population of cells comprises one or more immune cell types. In some embodiments, the heterogeneous population of cells comprises gamma delta (yd) T cells and one or more other immune cell types. In some embodiments, the heterogeneous population of cells comprises gamma delta (yd) T cells and one or more cell types which may include alpha beta (a(3) T cells, natural killer (NK) cells, monocytes, and / or B cells. In some embodiments, methods, compositions, and kits are provided to selectively expand a population of gamma delta (yd) T cells (i.e. , with minimal corresponding expansion of other immune cells (e.g., alpha beta (a|3) T cells, NK cells, monocytes, and / or B cells)). Selective expansion of gamma delta (yd) T cells produces an expanded cell population enriched with gamma delta (yd) T cells.

[0065] In some embodiments, the number of cells is expanded several-fold in an expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. Cells being expanded a particular fold may refer to total cells in a cell population or may refer to specific cells in a population (e.g., T cells, gamma delta (yd) T cells). In some embodiments, the number of cells is expanded about 100-fold or more in an expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of cells is expanded about 200-fold or more in an expanded gamma delta (yd) T cell-enriched cell population when compared to the number of PATENT

[0066] POINT-1003PCT cells in an originating cell population. In some embodiments, the number of cells is expanded about 300-fold or more in an expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of cells is expanded about 400-fold or more in an expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of cells is expanded about 500-fold or more in an expanded gamma delta (yd) T cell- enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of cells is expanded about 600-fold or more in an expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of cells is expanded about 700-fold or more in an expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of cells is expanded about 800-fold or more in an expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of cells is expanded about 900-fold or more in an expanded gamma delta (yd) T cell- enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of cells is expanded about 1 ,000-fold or more in an expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of cells is expanded about 1500-fold or more in an expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of cells is expanded about 2,000-fold or more in an expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of cells is expanded about 400-fold to about 1 ,000-fold in an expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of cells is expanded about 400-fold to about 800-fold in an expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population.

[0067] A cell population expanded by a method described herein may comprise an enriched gamma delta (yd) T cell population. For example, a cell population expanded by a method described herein may comprise about 80% or more gamma delta (yd) T cells. In some embodiments, a cell population expanded by a method described herein may comprise about 85% or more gamma delta (yd) T cells. In some embodiments, a cell population expanded by a method described herein may comprise about 90% or more gamma delta (yd) T cells. In some embodiments, a cell population expanded by a method described herein may comprise about 95% or more gamma delta (yd) T PATENT POINT-1003PCT cells. In some embodiments, a cell population expanded by a method described herein may comprise about 100% gamma delta (yb) T cells. In some embodiments, a cell population expanded by a method described herein may comprise about 90% to 95% gamma delta (yb) T cells. In some embodiments, a cell population expanded by a method described herein may comprise about 95% to 99% gamma delta (yb) T cells. In some embodiments, a cell population expanded by a method described herein may comprise about 95% to 100% gamma delta (yb) T cells.

[0068] A cell population expanded by a method described herein may be depleted of non-gamma delta (y5) T cells. For example, a cell population expanded by a method described herein may be depleted of natural killer (NK) cells, monocytes, B cells, and / or alpha beta (a|3) T cells. In some embodiments, an expanded gamma delta (yb) T cell-enriched cell population comprises about 20% natural killer (NK) cells or less. In some embodiments, an expanded gamma delta (yb) T cell- enriched cell population comprises about 15% natural killer (NK) cells or less. In some embodiments, an expanded gamma delta (yb) T cell-enriched cell population comprises about 10% natural killer (NK) cells or less. In some embodiments, an expanded gamma delta (yb) T cell- enriched cell population comprises about 5% natural killer (NK) cells or less. In some embodiments, an expanded gamma delta (yb) T cell-enriched cell population comprises about 4% natural killer (NK) cells or less. In some embodiments, an expanded gamma delta (yb) T cell- enriched cell population comprises about 3% natural killer (NK) cells or less. In some embodiments, an expanded gamma delta (yd) T cell-enriched cell population comprises about 2% natural killer (NK) cells or less. In some embodiments, an expanded gamma delta (yb) T cell- enriched cell population comprises about 1% natural killer (NK) cells or less. In some embodiments, an expanded gamma delta (yb) T cell-enriched cell population comprises no detectable natural killer (NK) cells.

[0069] In some embodiments, an expanded gamma delta (yb) T cell-enriched cell population comprises about 5% alpha beta (ap) T cells or less. In some embodiments, an expanded gamma delta (yb) T cell-enriched cell population comprises about 4% alpha beta (ap) T cells or less. In some embodiments, an expanded gamma delta (yb) T cell-enriched cell population comprises about 3% alpha beta (ap) T cells or less. In some embodiments, an expanded gamma delta (yb) T cell- enriched cell population comprises about 2% alpha beta (ap) T cells or less. In some embodiments, an expanded gamma delta (yb) T cell-enriched cell population comprises about 1% alpha beta (ap) T cells or less. For example, an expanded gamma delta (yb) T cell-enriched cell population may comprise about 0.9% alpha beta (ap) T cells or less, about 0.8% alpha beta (ap) T cells or less, about 0.7% alpha beta (ap) T cells or less, about 0.6% alpha beta (ap) T cells or less, about 0.5% alpha beta (ap) T cells or less, about 0.4% alpha beta (op) T cells or less, about 0.3% alpha beta (ap) T cells or less, about 0.2% alpha beta (ap) T cells or less, about 0.1% alpha beta (ap) T cells PATENT

[0070] POINT-1003PCT or less. In some embodiments, an expanded gamma delta (yd) T cell-enriched cell population comprises about 0.5% alpha beta (a[3) T cells or less. In some embodiments, an expanded gamma delta (yd) T cell-enriched cell population comprises about 0.1% alpha beta (ap) T cells or less. In some embodiments, an expanded gamma delta (yd) T cell-enriched cell population comprises no detectable alpha beta (a ) T cells.

[0071] A gamma delta (yd) T cell-enriched cell population expanded by a method described herein may comprise one or more subpopulations of gamma delta (yd) T cells. A subpopulation may comprise Vdeltal (Vd1 ) T cells, Vdelta2 (Vd2) T cells, or non-Vd1 &Vd2 T cells. A gamma delta (yd) T cell- enriched cell population expanded by a method described herein may comprise multi-subsets of gamma delta (yd) T cells. In some embodiments, an expanded gamma delta (yd) T cell population comprises subpopulations of Vdeltal (Vd1 ) T cells and Vdelta2 (Vd2) T cells. In some embodiments, multi-subsets of gamma delta (yd) T cells comprise subpopulations Vdeltal (Vd1 ) T cells, Vdelta2 (Vd2) T cells, and non-Vd1 &Vd2 T cells.

[0072] The fold expansion of each gamma delta (yd) T subpopulation may vary under the expansion conditions described herein. For example, the number of Vd1 T cells may be expanded about 500- fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vd1 T cells may be expanded about 1 ,000-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vd1 T cells may be expanded about 2,000-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vd1 T cells may be expanded about 3,000-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vd1 T cells may be expanded about 4,000-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vd1 T cells may be expanded about 5,000-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vd1 T cells may be expanded about 6,000-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vd1 T cells may be expanded about 7,000-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vd1 T cells may be expanded about 8,000-fold or more in the PATENT

[0073] POINT-1003PCT expanded gamma delta (Yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb1 T cells may be expanded about 9,000-fold or more in the expanded gamma delta (y<5) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb1 T cells may be expanded about 10,000-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb1 T cells may be expanded about 11 ,000-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb1 T cells may be expanded about 12,000-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb1 T cells may be expanded about 13,000-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb1 T cells may be expanded about 14,000-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb1 T cells may be expanded about 15,000-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb1 T cells may be expanded about 6,000-fold to about 12,000-fold in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb1 T cells may be expanded about 8,000-fold to about 12,000-fold in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb1 T cells may be expanded about 1 ,000-fold to about 5,000-fold in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population, the number of Vb1 T cells may be expanded about 1 ,000-fold to about 3,000-fold in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population.

[0074] In another example, the number of Vb2 T cells may be expanded about 100-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb2 T cells may be expanded about 200-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb2 T cells may be expanded about 300-fold or more in the expanded gamma delta (yb) PATENT

[0075] POINT-1003PCT

[0076] T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb2 T cells may be expanded about 400-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb2 T cells may be expanded about 500-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb2 T cells may be expanded about 600-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb2 T cells may be expanded about 700-fold or more in the expanded gamma delta (y<5) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb2 T cells may be expanded about 800-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb2 T cells may be expanded about 850-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb2 T cells may be expanded about 900-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb2 T cells may be expanded about 1 ,000-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb2 T cells may be expanded about 1 ,500-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb2 T cells may be expanded about 2,000-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb2 T cells may be expanded about 2,500-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb2 T cells may be expanded about 3,000-fold or more in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb2 T cells may be expanded about 100-fold to about 3,000-fold in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vb2 T cells may be expanded about 100-fold to about 2,600-fold in the expanded gamma delta (yb) T cell-enriched cell population when compared to the number of PATENT

[0077] POINT-1003PCT cells in an originating cell population. In some embodiments, the number of Vd2 T cells may be expanded about 100-fold to about 1 ,000-fold in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vd2 T cells may be expanded about 100-fold to about 900-fold in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vd2 T cells may be expanded about 100-fold to about 850-fold in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of Vd2 T cells may be expanded about 100-fold to about 800-fold in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population.

[0078] In another example, the number of non-Vd1&Vd2 T cells may be expanded about 100-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 200-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 300-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 400-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 500-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 600-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 700-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 800-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 900-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 1 ,000-fold or more in the expanded gamma delta (yd) T cell-enriched cell PATENT

[0079] POINT-1003PCT population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 1 ,500-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 2,000-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 2,500-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 3,000-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 3,500-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 4,000-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 4,500-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 4,800-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 5,000-fold or more in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 10O-fold to about 4,000-fold in the expanded gamma delta (yd) T cell- enriched cell population when compared to the number of cells in an originating cell population. In some embodiments, the number of non-Vd1 &Vd2 T cells may be expanded about 200-fold to about 3,000-fold in the expanded gamma delta (yd) T cell-enriched cell population when compared to the number of cells in an originating cell population.

[0080] In some embodiments, an expanded gamma delta (yd) T cell population comprises varying amounts of subpopulations. Amounts (e.g., percentages) may refer to the percent of gamma delta (yd) T cells or may refer to the percent of total cells in the population. For example, an expanded gamma delta (yd) T cell population may comprise between about 5-60% Vd1 T cells. In some embodiments, an expanded gamma delta (yd) T cell population comprises between about 5-55% Vd1 T cells. In some embodiments, an expanded gamma delta (yd) T cell population comprises PATENT

[0081] POINT-1003PCT between about 10-60% Vb1 T cells. In some embodiments, an expanded gamma delta (yd) T cell population comprises between about 20-60% Vb1 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 30-60% Vb1 T cells. In some embodiments, an expanded gamma delta (Y<5) T cell population comprises between about 40-60% Vb1 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 50-55% V51 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 53-54% Vb1 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 20-40% V<51 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 25-35% Vb1 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 30% Vb1 T cells.

[0082] In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 10-95% Vb2 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 10-90% Vb2 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 10-85% Vb2 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 15-95% Vb2 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 15-90% Vb2 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 15-85% Vb2 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 25-95% Vb2 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 25-85% Vb2 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 25-75% Vb2 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 25-65% Vb2 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 25-55% Vb2 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 25-45% Vb2 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 25-35% Vb2 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 28-33% Vb2 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 30-31% Vb2 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 50-70% Vb2 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 55-65% Vb2 T cells. In some embodiments, an expanded gamma delta (yb) T cell population comprises between about 60% Vb2 T cells. PATENT

[0083] POINT-1003PCT

[0084] In some embodiments, an expanded gamma delta (yd) T cell population comprises between about 2-30% non-Vd1 &Vd2 T cells. In some embodiments, an expanded gamma delta (yd) T cell population comprises between about 2.5-25% non-Vd1 &Vd2 T cells. In some embodiments, an expanded gamma delta (yd) T cell population comprises between about 5-15% non-Vd1 &Vd2 T cells. In some embodiments, an expanded gamma delta (yd) T cell population comprises between about 6-11 % non-Vd1 &Vd2 T cells. In some embodiments, an expanded gamma delta (yd) T cell population comprises between about 8-9% non-Vd1 &Vd2 T cells. In some embodiments, an expanded gamma delta (yd) T cell population comprises between about 9-1 1% non-Vd1 &Vd2 T cells. In some embodiments, an expanded gamma delta (yd) T cell population comprises between about 10% non-Vd1 &Vd2 T cells.

[0085] Fold expansions of gamma delta (yd) T cells, fold expansion of gamma delta (yd) T cell subpopulations (e.g., Vdeltal (Vd1 ) T cells, Vdelta2 (Vd2) T cells, and non-Vd1 &Vd2 T cells), and / or amounts of gamma delta (yd) T cell subpopulations may be assessed after a particular number of days in culture. In some embodiments, fold expansions of gamma delta (yd) T cells, fold expansions of gamma delta (yd) T cell subpopulations, and / or amounts of gamma delta (yd) T cell subpopulations are assessed after about 10 days to about 25 days in culture. In some embodiments, fold expansions of gamma delta (yd) T cells, fold expansions of gamma delta (yd) T cell subpopulations, and / or amounts of gamma delta (yd) T cell subpopulations are assessed after about 14 days to about 21 days in culture. For example, fold expansions of gamma delta (yd) T cells, fold expansions of gamma delta (yd) T cell subpopulations, and / or amounts of gamma delta (yd) T cell subpopulations may be assessed after about 14 days in culture, after about 15 days in culture, after about 16 days in culture, after about 17 days in culture, after about 18 days in culture, after about 19 days in culture, after about 20 days in culture, or after about 21 days in culture.

[0086] In some embodiments, a method herein comprises isolating the gamma delta (yd) T cells from the activated and expanded gamma delta (yd) T cell-enriched cell population. In some embodiments, a method herein comprises storing the expanded gamma delta (yd) T cell-enriched cell population or isolate thereof in a cell bank. In some embodiments, gamma delta (yd) T cells described herein may provide banks of clinical grade cells that can be used in a number of patients. In some embodiments, a bank is suitably populated with gamma delta (yd) T cells obtained from healthy volunteer donors (e.g., of blood group O). Healthy donors may be selected to maximize the opportunity for Human Leukocyte Antigens (HLA) matching, thereby maximizing the long-term persistence of allograft. Collected and processed gamma delta (yd) T cells described herein may be banked for future use at a cell bank or depository. Accordingly, the cells may be stored in a cryoprotectant such as DMSO or CryoStor™ and subjected to a controlled rate of freezing and PATENT

[0087] POINT-1003PCT storage in liquid nitrogen. Gamma delta (yb) T cells may be stored in a unitized storage of defined units or dosages as required for a single or multiple treatment steps.

[0088] Cytokines

[0089] In some embodiments, activation and / or expansion conditions herein comprise one or more cytokines. Cytokines generally are small proteins (~5-25 kDa) involved in cell signaling. Cytokines typically exert their functions by interacting with specific cytokine receptors on the target cell surface. Cytokines may include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines generally are produced by cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells.

[0090] In some embodiments, activation and / or expansion conditions herein comprise one or more interleukins. Interleukins (ILs) are a group of cytokines that are expressed and secreted by white blood cells (leukocytes) as well as some other body cells. Interleukins may include IL-1 to IL-36. The majority of interleukins are synthesized by CD4 helper T-lymphocytes, as well as through monocytes, macrophages, and endothelial cells. They promote the development and differentiation of T and B lymphocytes, and hematopoietic cells.

[0091] In some embodiments, activation and / or expansion conditions herein comprise interleukin-2 (IL-2). In some embodiments, activation and / or expansion conditions herein comprise a cytokine consisting of IL-2. In some embodiments, activation and / or activation and / or expansion conditions herein comprise an interleukin consisting of IL-2. In some embodiments, activation and / or expansion conditions herein comprise interleukin-15 (IL-15). In some embodiments, activation and / or expansion conditions herein comprise a cytokine consisting of IL-15. In some embodiments, activation and / or expansion conditions herein comprise an interleukin consisting of IL-15. In some embodiments, activation and / or expansion conditions herein comprise interleukin-4 (IL-4). In some embodiments, activation and / or expansion conditions herein comprise a cytokine consisting of IL-4. In some embodiments, activation and / or expansion conditions herein comprise an interleukin consisting of IL-4. In some embodiments, activation and / or expansion conditions herein comprise interleukin-21 (IL-21 ). In some embodiments, activation and / or expansion conditions herein comprise a cytokine consisting of IL-21 . In some embodiments, activation and / or expansion conditions herein comprise an interleukin consisting of IL-21. In some embodiments, activation and / or expansion conditions herein comprise interleukin-10 (IL-10). In some embodiments, activation and / or expansion conditions herein comprise a cytokine consisting of IL-10. In some embodiments, activation and / or expansion conditions herein comprise an interleukin consisting of IL-10. In some embodiments, activation and / or expansion conditions herein comprise interleukin-7 PATENT POINT-1003PCT (IL-7). In some embodiments, activation and / or expansion conditions herein comprise a cytokine consisting of IL-7. In some embodiments, activation and / or expansion conditions herein comprise an interleukin consisting of IL-7. In some embodiments, activation and / or expansion conditions herein comprise interleukin-12 (IL-12). In some embodiments, activation and / or expansion conditions herein comprise a cytokine consisting of IL-12. In some embodiments, activation and / or expansion conditions herein comprise an interleukin consisting of IL-12.

[0092] In some embodiments, activation and / or expansion conditions herein comprise cytokines comprising two or more of IL-2, IL-15, IL-4, IL-21 , IL-10, IL-7, and IL-12. In some embodiments, activation and / or expansion conditions herein comprise cytokines consisting of two of IL-2, IL-15, IL-4, IL-21 , IL-10, IL-7, and IL-12. In some embodiments, activation and / or expansion conditions herein comprise interleukins consisting of two of IL-2, IL-15, IL-4, IL-21 , IL-10, IL-7, and IL-12. In some embodiments, activation and / or expansion conditions herein comprise cytokines comprising three or more of IL-2, IL-15, IL-4, IL-21 , IL-10, IL-7, and IL-12. In some embodiments, activation and / or expansion conditions herein comprise cytokines consisting of three of IL-2, IL-15, IL-4, IL- 21 , IL-10, IL-7, and IL-12. In some embodiments, activation and / or expansion conditions herein comprise interleukins consisting of three of IL-2, IL-15, IL-4, IL-21 , IL-10, IL-7, and IL-12.

[0093] In some embodiments, activation and / or expansion conditions herein comprise cytokines comprising IL-2 and IL-4. In some embodiments, activation and / or expansion conditions herein comprise cytokines consisting of IL-2 and IL-4. In some embodiments, activation and / or expansion conditions herein comprise interleukins consisting of IL-2 and IL-4. In some embodiments, activation and / or expansion conditions herein comprise cytokines comprising IL-2 and IL-21 . In some embodiments, activation and / or expansion conditions herein comprise cytokines consisting of IL-2 and IL-21 . In some embodiments, activation and / or expansion conditions herein comprise interleukins consisting of IL-2 and IL-21 . In some embodiments, activation and / or expansion conditions herein comprise cytokines comprising IL-2, IL-4, and IL-21. In some embodiments, activation and / or expansion conditions herein comprise cytokines consisting of IL-2, IL-4, and IL-21 . In some embodiments, activation and / or expansion conditions herein comprise interleukins consisting of IL-2, IL-4, and IL-21. In some embodiments, activation and / or expansion conditions herein comprise cytokines comprising IL-15 and IL-4. In some embodiments, activation and / or expansion conditions herein comprise cytokines consisting of IL-15 and IL-4. In some embodiments, activation and / or expansion conditions herein comprise interleukins consisting of IL- 15 and IL-4. In some embodiments, activation and / or expansion conditions herein comprise cytokines comprising IL-15 and IL-21 . In some embodiments, activation and / or expansion conditions herein comprise cytokines consisting of IL-15 and IL-21 . In some embodiments, activation and / or expansion conditions herein comprise interleukins consisting of IL-15 and IL-21 . In PATENT

[0094] POINT-1003PCT some embodiments, activation and / or expansion conditions herein comprise cytokines comprising IL-15, IL-4, and IL-21. In some embodiments, activation and / or expansion conditions herein comprise cytokines consisting of IL-15, IL-4, and IL-21. In some embodiments, activation and / or expansion conditions herein comprise interleukins consisting of IL-15, IL-4, and IL-21. In some embodiments, activation and / or expansion conditions herein comprise cytokines comprising IL-2, IL-15, and IL-4. In some embodiments, activation and / or expansion conditions herein comprise cytokines consisting of IL-2, IL-15, and IL-4. In some embodiments, activation and / or expansion conditions herein comprise interleukins consisting of IL-2, IL-15, and IL-4. In some embodiments, activation and / or expansion conditions herein comprise cytokines comprising IL-2, IL-15, and IL-21. In some embodiments, activation and / or expansion conditions herein comprise cytokines consisting of IL-2, IL-15, and IL-21. In some embodiments, activation and / or expansion conditions herein comprise interleukins consisting of IL-2, IL-15, and IL-21. In some embodiments, activation and / or expansion conditions herein comprise cytokines comprising IL-2, IL-15, IL-4, and IL-21 . In some embodiments, activation and / or expansion conditions herein comprise cytokines consisting of IL-2, IL-15, IL-4, and IL-21. In some embodiments, activation and / or expansion conditions herein comprise interleukins consisting of IL-2, IL-15, IL-4, and IL-21 .

[0095] In some embodiments, activation conditions herein comprise cytokines comprising IL-2, IL-4, or IL- 2 and IL-4, and expansion conditions herein comprise cytokines comprising two or more of IL-2, IL- 15, IL-4 and IL-21.

[0096] Media components

[0097] In some embodiments, activation and / or expansion conditions herein comprise one or more media components. Media components may include supplements and / or small molecules such as salts, sugars, vitamins, minerals, amino acids, fatty acids, signaling activators, and signaling inhibitors.

[0098] In some embodiments, activation and / or expansion conditions herein comprise one or more salts (i.e., compounds having ionic bonds). Salts may include simple salts, acidic salts, basic salts, neutral salts, double salts, complex salts, and mixed salts. In some embodiments, activation and / or expansion conditions herein comprise a formate salt. In some embodiments, activation and / or expansion conditions herein comprise sodium formate.

[0099] In some embodiments, activation and / or expansion conditions herein comprise one or more sugars (i.e., molecules made of carbon, hydrogen, and oxygen atoms that can form carbohydrates). Sugars may include simple sugars, also referred to as monosaccharides, which include glucose, fructose, and galactose. Sugars also may include compound sugars, also referred to as disaccharides, which are molecules made of two bonded monosaccharides, which include sucrose (glucose + fructose), lactose (glucose + galactose), and maltose (two molecules of glucose). In PATENT

[0100] POINT-1003PCT some embodiments, activation and / or expansion conditions herein comprise a monosaccharide. In some embodiments, activation and / or expansion conditions herein galactose.

[0101] In some embodiments, activation and / or expansion conditions herein comprise one or more vitamins (i.e., organic compounds which are essential for normal growth and nutrition and are required in small quantities in the diet because they cannot be synthesized by certain organisms). Vitamins may include vitamin A (all-trans-retinols, all-trans-retinyl-esters, all-trans-p-carotene, and other provitamin A carotenoids); B vitamins such as vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin, niacinamide, and nicotinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B7 (biotin), vitamin B9 (folic acid and folates), vitamin B12 (cobalamins); vitamin C (ascorbic acid and ascorbates); vitamin D (calciferols); vitamin E (tocopherols and tocotrienols), and vitamin K (phylloquinones, menaquinones, and menadiones). In some embodiments, activation and / or expansion conditions herein comprise ascorbic acid or a derivative thereof, stereoisomer thereof, salt thereof, or ester thereof (e.g., L-ascorbic acid, ascorbate, sodium ascorbate, calcium ascorbate, potassium ascorbate, dehydroascorbate (DHA), ascorbyl palmitate, erythorbic acid, and sodium erythorbate). In some embodiments, activation and / or expansion conditions herein comprise vitamin C. In some embodiments, activation and / or expansion conditions herein comprise a B vitamin. In some embodiments, activation and / or expansion conditions herein comprise folic acid or folate, a derivative thereof, stereoisomer thereof, salt thereof, or ester thereof. In some embodiments, activation and / or expansion conditions herein comprise nicotinamide, a derivative thereof, stereoisomer thereof, salt thereof, or ester thereof.

[0102] In some embodiments, activation and / or expansion conditions herein comprise one or more signaling inhibitors. In some embodiments, activation and / or expansion conditions herein comprise one or more TGF-beta signaling inhibitors. TGF-beta signaling generally controls proliferation, cellular differentiation, and other functions in a variety of cell types, and can play a role in cell cycle control, regulation of the immune system, and development in certain cell types. Inhibition of TGF- beta signaling may include inhibition of any TGF-beta signaling pathway and / or member of the TGF-beta superfamily including ligands such as TGF-beta1 , TGF-beta2, TGF-beta3, inhibins, activin, anti-mullerian hormone, bone morphogenetic protein (BMP; e.g., BMP1 -7, BMP8a, BMP8b, BMP10, BMP 1 1 , BMP15)), decapentaplegic, nodal, activin, and Vg-1 ; receptors such as TGF-beta superfamily type I receptors, TGF-beta superfamily type II receptors, type I serine / threonine kinase receptors, type II serine / threonine kinase receptors, TGF-beta type I receptor, TGF-beta type II receptor, activin receptor, nodal receptor, activin / nodal receptor, activin receptor-like kinases (ALKs; e.g., ALK1 , ALK2, ALK3, ALK4, ALK5, ALK6, ALK7 and ALK8); and downstream effectors such as R-SMAD and other SMAD proteins (e.g., SMAD1 , SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, SMAD7, SMAD 8 / 9). PATENT

[0103] POINT-1003PCT

[0104] In some embodiments, activation and / or expansion conditions herein comprise one or more activin receptor-like kinase inhibitors. In some embodiments, activation and / or expansion conditions herein comprise one or more TGF-beta receptor inhibitors (e.g., TGF-beta type I receptor inhibitors, type I activin / nodal receptor inhibitors, type I nodal receptor inhibitors, ALK1 inhibitors, ALK2 inhibitors, ALK3 inhibitors, ALK4 inhibitors, ALK5 inhibitors, ALK6 inhibitors, ALK7 inhibitors, and ALK8 inhibitors). In some embodiments, activation and / or expansion conditions herein comprise one or more ALK5, ALK4, and / or ALK7 inhibitors. In some embodiments, activation and / or expansion conditions herein comprise one or more ALK5 inhibitors.

[0105] TGF-beta inhibitors may include one or more small molecule ALK5, ALK4, and / or ALK7 inhibitors. In some embodiments, an ALK5, ALK4, and / or ALK7 inhibitor is an ATP analog. ALK5, ALK4, and / or ALK7 inhibitors may include, for example, A83-01 (3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4- quinoliny I)- 1 H-pyrazole-1 -carbothioamide), GW788388 (4-[4-[3-(2- Pyridinyl)- 1 H-pyrazol-4-yl]-2- pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide), RepSox (2-(3-(6-Methylpyridine-2-yl)-1 H- pyrazol-4-yl)-1 ,5-naphthyridine), and SB 431542 (4-[4-(1 ,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1 H- imidazol-2-yl]benzamide). In some embodiments, the TGF-beta inhibitor is A83-01 .

[0106] Genetic modifications

[0107] In some embodiments, a method herein comprises modifying gamma delta (yd) T cells. In some embodiments, a method herein comprises modifying gamma delta (yd) T cells in an originating cell population (e.g., prior to activation and expansion). In some embodiments, a method herein comprises modifying gamma delta (yd) T cells in an activated gamma delta (yd) T cell-enriched cell population (e.g., during or after activation and prior to expansion). In some embodiments, a method herein comprises modifying gamma delta (yd) T cells in an activated and expanded gamma delta (yd) T cell-enriched cell population (e.g., after activation and during or after expansion). Gamma delta (yd) T cells may be modified (e.g., genetically engineered) by a modification described herein (e.g., introduction of an exogenous nucleic acid (e.g., nucleic acid encoding a CAR), introduction of a genomic modification (e.g., a gene edit, a gene knock-out)).

[0108] In some embodiments, a method herein comprises introducing an exogenous nucleic acid into a population of gamma delta (yd) T cells described herein. An exogenous nucleic acid may be introduced into a population of gamma delta (yd) T cells by a suitable method such as transduction or transfection. An exogenous nucleic acid may encode an exogenous or heterologous protein or polypeptide of interest such as, for example, a chimeric antigen receptor (CAR), a T-cell receptor, a tumor necrosis factor receptor, a myeloid differentiation primary response protein, or an innate immune signal transduction adaptor. An exogenous nucleic acid may include a promoter, enhancer, or other regulator of gene expression. In some embodiments, a method herein PATENT

[0109] POINT-1003PCT comprises introducing an exogenous nucleic acid into a population of gamma delta (yb) T cells such that between about 30% to about 99% or more, or at least about 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,

[0110] 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,

[0111] 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,

[0112] 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% 91%, 92%, 93%,

[0113] 94%, 95%, 96%, 97%, 98%, 99% or fractions thereof, up to 100% of the cells in a population of gamma delta (yb) T cells further comprise a genetic modification comprising an exogenous nucleic acid. The exogenous nucleic acid may be in a retroviral vector or a lentiviral vector and, in certain embodiments, the exogenous nucleic acid is integrated into genomes of one or more cells of the modified cell population. In certain configurations, an exogenous nucleic acid is introduced into a population of gamma delta (yd) T cells by way of an adeno-associated viral vector (AAV), nanoparticle, or mRNA. In certain configurations, an exogenous nucleic acid is introduced into a population of gamma delta (yd) T cells and inserted into the genome by way of genome editing enzymes such as, for example, CRISPR / Cas, or transposon, or retrotransposon.

[0114] In some embodiments, an exogenous nucleic acid may encode a chimeric antigen receptor (CAR) and the cells in the composition comprise a CAR. CARs are recombinant receptors that provide both antigen-binding and T cell activating functions. A CAR generally refers to a chimeric polypeptide which comprises one or more polypeptide components that recognize one or more target antigens (extracellular domain; antigen recognition domains; antigen binding domains) linked to a transmembrane polypeptide and intracellular domain polypeptide selected to activate T cells. When immune cells, such as T cells, are engineered (genetically modified) to express a CAR, it provides the immune cells a new and / or improved ability to target a protein or antigen of interest. The target protein or antigen of interest can, in certain aspects, be a cancer antigen, autoimmune antigen, or an infectious disease antigen, several of which are known and / or identifiable in the art.

[0115] In some embodiments, a method herein comprises introducing one or more genome modifications into a population of gamma delta (yb) T cells described herein. A genome modification may include a disruption of a gene (e.g., a gene edit, a gene knock-out (KO)) introduced by way of a suitable genome editing process (e.g., CRISPR-Cas9; CRISPR-Cas12i). Components for carrying out a genome modification may be introduced into a population of gamma delta (yb) T cells by any suitable method such as electroporation. In some embodiments, a method herein comprises introducing a genome modification into a population of gamma delta (yb) T cells such that between about 30% to about 99% or more, or at least about 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, PATENT

[0116] POINT-1003PCT

[0117] 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or fractions thereof, up to 100% of the cells in a population of gamma delta (yd) T cells further comprise a genome modification.

[0118] Methods of treatment

[0119] Provided herein are methods of treating an infection, autoimmune disease, or cancer in an individual comprising the step of providing said individual with gamma delta (yd) T cells (e.g., activated and expanded gamma delta (yd) T cells; activated, expanded, and modified gamma delta (yd) T cells). In some embodiments, gamma delta (yd) T cells are obtained from a different individual (allogenic treatment). Thus, donor gamma delta (yd) T cells are used for the treatment of an infection, for example, of a virus, bacteria, fungi or protozoa, or for treatment of an autoimmune disease or cancer in a recipient subject where the donor and the recipient are not the same individual. As will be understood, prior to providing the gamma delta (yd) T cells to the second subject, these gamma delta (yd) T cells are activated and selectively expanded according to a method described herein. In some embodiments, gamma delta (yd) T cells are modified as discussed herein, to provide CAR modified gamma delta (yd) T cells. In some embodiments, gamma delta (yd) T cells are further modified to provide gene edited gamma delta (yd) T cells. In some embodiments, gamma delta (yd) T cells are further modified to provide gene knock-out gamma delta (yd) T cells.

[0120] Also provided is a process for providing gamma delta (yd) T cells autologously to a subject comprising the steps of obtaining a sample of gamma delta (yd) T cells from a subject and culturing the gamma delta (yd) T cells to allow them to be administered back to the subject, where the culturing step comprises activating, expanding, and modifying the gamma delta (yd) T cells as described herein.

[0121] A method of administration to provide gamma delta (yd) T cells to a recipient subject may include intravenous, intradermal, intraperitoneal, intrathecal, intratumoral, or subcutaneous injection, for example. Administration may be into an affected area or systemically to the individual. A method of administration can be prophylactic or therapeutic, where a “prophylactically effective amount” or a “therapeutically effective amount” of the gamma delta (yd) T cells which is sufficient to show benefit to the individual, is provided. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g., decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors. PATENT

[0122] POINT-1003PCT

[0123] In some embodiments, gamma delta (yd) T cells described herein are provided for the treatment of a subject with cancer. In some embodiments, cancer is characterized as a hematological cancer (e.g., leukemia, lymphoma, or multiple myeloma). In some embodiments, cancer is characterized as a solid tumor-type cancer. In some embodiments, cancer is characterized as an epithelial tissue cancer. In some embodiments, cancer is characterized as squamous tumor-type cancer. In embodiments, cancer can include but is not limited to pancreatic cancer, gastric cancer, renal cell carcinoma, lung cancer (e.g., non-small cell lung cancer), breast cancer, colon cancer, cervical cancer, ovarian cancer, bladder cancer, prostate cancer (e.g., prostate adenocarcinoma), endometrial endometroid carcinoma, oral squamous cell carcinomas, and papillary thyroid carcinoma.

[0124] In some embodiments, gamma delta (yd) T cells described herein are provided for the treatment of a subject with an autoimmune disease. Autoimmune diseases may include, but are not limited to, Addison disease, celiac disease - sprue (gluten-sensitive enteropathy), dermatomyositis, Graves disease, Hashimoto thyroiditis, inflammatory bowel disease (Crohn’s disease, ulcerative colitis), multiple sclerosis, myasthenia gravis, pernicious anemia, reactive arthritis, rheumatoid arthritis, psoriatic arthritis, Sjogren syndrome, systemic lupus erythematosus (SLE), lupus nephritis, cutaneous lupus erythematosus (CLE), psoriasis, pemphigus, and type I diabetes.

[0125] Also provided herein are pharmaceutical compositions comprising modified gamma delta (yd) T cells described herein. In some embodiments, a pharmaceutical composition comprises a dose of modified gamma delta (yd) T cells suitable to administer to an individual to provide a therapeutic effect. In some embodiments, a pharmaceutical composition further comprises one or more therapeutics chosen from of an antibody immunotherapy, chemotherapeutic agent, biologic, cytokine, or combination thereof.

[0126] Kits

[0127] Provided in certain embodiments are kits. The kits may include any components and compositions described herein useful for performing any of the methods described herein, in any suitable combination. Kits may further include any reagents, media, buffers, or other components useful for carrying out any of the methods described herein. For example, a kit may include cell culture media and supplements suitable for culturing T cells (e.g., GTS™ OpTmizer™ T Cell Expansion media (ThermoFisher Scientific) supplemented with ICTSR (e.g., 2.5%), p / s (e.g., 1%), GLUTAMAX (e.g., 2 mM), and human AB serum (e.g., 2.5-5%). Other cell culture media that may be used include, for example, GTS™ OpTmizer™ Pro (Thermofisher), TEXMACS medium (Mitenyi), LYMOONE medium (Takara), NK MACS medium (Mitenyi), and X-VIVO TM10 medium (Lonza). PATENT

[0128] POINT-1003PCT

[0129] In some embodiments, a kit comprises binding agents comprising (i) a cluster of differentiation 3 (CD3) binding agent (e.g., an antibody or fragment thereof) and (ii) a cluster of differentiation 2 (CD2) binding agent (e.g., an antibody or fragment thereof). In some embodiments, a kit comprises binding agents consisting of a CD3 binding agent and a GD2 binding agent. In some embodiments, a CD3 binding agent is an antibody or fragment thereof. In some embodiments, a CD2 binding agent is an antibody or fragment thereof.

[0130] In some embodiments, a kit comprises one or more cytokines. In some embodiments, a kit comprises cytokines comprising two or more of IL-2, IL-15, IL-4, IL-21 , and IL-10. In some embodiments, a kit comprises cytokines consisting of two of IL-2, IL-15, IL-4, IL-21 , and IL-10. In some embodiments, a kit comprises interleukins consisting of two of IL-2, IL-15, IL-4, IL-21 , and IL- 10. In some embodiments, a kit comprises three or more of IL-2, IL-15, IL-4, IL-21 , and IL-10. In some embodiments, a kit comprises cytokines consisting of three of IL-2, IL-15, IL-4, IL-21 , and IL- 10. In some embodiments, a kit comprises interleukins consisting of three of IL-2, IL-15, IL-4, IL-21 , and IL-10.

[0131] In some embodiments, a kit comprises one or more media components. In some embodiments, a kit comprises one or more salts, one or more sugars, one or more vitamins, and / or one or more signaling inhibitors as described herein. For example, a kit may comprise one or more of sodium formate, galactose, ascorbic acid, folic acid, nicotinamide, and A83-01.

[0132] In some embodiments, a kit comprises cells. In some embodiments, a kit comprises cells derived from a peripheral blood mononuclear cell (PBMC) population. In some embodiments, a kit comprises cells derived from cord blood. In some embodiments, a kit comprises cells that are depleted of alpha beta (ap) T cells.

[0133] Components of a kit may be present in separate containers, or multiple components may be present in a single container. Suitable containers include a single tube (e.g., vial), one or more wells of a plate (e.g., a 96-well plate, a 384-well plate, and the like), and the like.

[0134] Kits may also comprise instructions for performing one or more methods described herein and / or a description of one or more components described herein. For example, a kit may include instructions for activating and / or selectively expanding gamma delta (y5) T cells ex vivo. A kit may include instructions for genetically modifying gamma delta (yd) T cells. Instructions and / or descriptions may be in printed form and may be included in a kit insert. In some embodiments, instructions and / or descriptions are provided as an electronic storage data file present on a suitable computer readable storage medium, e.g., portable flash drive, DVD, CD-ROM, diskette, and the like. A kit also may include a written description of an internet location that provides such instructions or descriptions. PATENT

[0135] POINT-1003PCT

[0136] Certain Implementations

[0137] Following are non-limiting examples of certain implementations of the technology.

[0138] A1 . A method for activating and selectively expanding gamma delta (yO) T cells ex vivo, comprising: activating gamma delta (yd) T cells and expanding the number of gamma delta (yd) T cells in an originating cell population under activation and expansion conditions, thereby generating an activated and expanded gamma delta (yd) T cell-enriched cell population, wherein the activation and expansion conditions comprise a) binding agents comprising a cluster of differentiation 3 (CD3) binding agent and a cluster of differentiation 2 (CD2) binding agent; and b) cytokines comprising interleukin 2 (IL-2) and interleukin 4 (IL-4); IL-2 and interleukin 21 (IL-21 ); IL-2, IL-4, and IL-21 ; interleukin 15 (IL-15) and IL-4; IL-15 and IL-21 ; or IL-15, IL-4, and IL-21 .

[0139] A2. The method of embodiment A1 , wherein the activation and expansion conditions further comprise one or more media components chosen from one or more salts, one or more sugars, one or more vitamins, and one or more transforming growth factor beta (TGF-beta) inhibitors.

[0140] A3. The method of embodiment A2, wherein the one or more salts comprise a formate salt

[0141] A4. The method of embodiment A3, wherein the formate salt is sodium formate.

[0142] A5. The method of any one of embodiments A2-A4, wherein the one or more sugars comprise a monosaccharide.

[0143] A6. The method of embodiment A5, wherein the monosaccharide is galactose.

[0144] A7. The method of any one of embodiments A2-A6, wherein the one or more vitamins comprise ascorbic acid or a derivative thereof, salt thereof, or ester thereof.

[0145] A8. The method of any one of embodiments A2-A7, wherein the one or more vitamins comprise a B vitamin.

[0146] A9. The method of embodiment A8, wherein the B vitamin is folic acid.

[0147] A10. The method of embodiment A8, wherein the B vitamin is nicotinamide.

[0148] A11 . The method of any one of embodiments A2-A10, therein the one or more TGF-beta inhibitors comprise an ALK5 inhibitor.

[0149] A12. The method of embodiment A11 , wherein the ALK5 inhibitor is A83-01 . PATENT

[0150] POINT-1003PCT

[0151] A13. The method of embodiment A1 , wherein the activation and expansion conditions further comprise a salt, a TGF-beta inhibitor, and a vitamin.

[0152] A14. The method of embodiment A13, wherein the salt is a formate salt, the TGF-beta inhibitor is an ALK5 inhibitor, and the vitamin is a B vitamin.

[0153] A15. The method of embodiment A14, wherein the formate salt is sodium formate, the ALK5 inhibitor is A83-01 , and the B vitamin is folic acid.

[0154] A16. The method of embodiment A1 , wherein the activation and expansion conditions further comprise a salt, a TGF-beta inhibitor, and a sugar.

[0155] A17. The method of embodiment A16, wherein the salt is a formate salt, the TGF-beta inhibitor is an ALK5 inhibitor, and the sugar is a monosaccharide.

[0156] A18. The method of embodiment A17, wherein the formate salt is sodium formate, the ALK5 inhibitor is A83-01 , and the monosaccharide is galactose.

[0157] A19. The method of any one of embodiments A1 -A18, further comprising transducing or transfecting the gamma delta (yb) T cells with a nucleic acid.

[0158] A20. The method of embodiment A19, wherein the nucleic acid encodes a chimeric antigen receptor (CAR).

[0159] A21 . The method of any one of embodiments A1 -A20, further comprising introducing a genome modification into the gamma delta (yb) T cells by a genome editing process.

[0160] B1 . A method for activating and selectively expanding gamma delta (yd) T cells ex vivo, comprising: activating and expanding the number of gamma delta (yb) T cells in an originating cell population under activation and expansion conditions, thereby generating an activated and expanded gamma delta (yb) T cell-enriched cell population, wherein the activation and expansion conditions comprise a) binding agents comprising a cluster of differentiation 3 (CD3) binding agent and a cluster of differentiation 2 (CD2) binding agent; b) cytokines comprising interleukin 2 (IL-2), interleukin 15 (IL-15), or IL-2 and IL-15; and c) one or more media components chosen from one or more salts, one or more sugars, one or more vitamins, and one or more transforming growth factor beta (TGF-beta) inhibitors. PATENT

[0161] POINT-1003PCT

[0162] B2. The method of embodiment B1 , wherein the cytokines further comprise interleukin 4 (IL-4), interleukin 21 (IL-21 ), or IL-4 and IL-21.

[0163] B3. The method of embodiment B1 or B2, wherein the one or more salts comprise a formate salt

[0164] B4. The method of embodiment B3, wherein the formate salt is sodium formate.

[0165] B5. The method of any one of embodiments B1 -B4, wherein the one or more sugars comprise a monosaccharide.

[0166] B6. The method of embodiment B5, wherein the monosaccharide is galactose.

[0167] B7. The method of any one of embodiments B1 -B6, wherein the one or more vitamins comprise ascorbic acid or a derivative thereof, salt thereof, or ester thereof.

[0168] B8. The method of any one of embodiments B1 -B7, wherein the one or more vitamins comprise a B vitamin.

[0169] B9. The method of embodiment B8, wherein the B vitamin is folic acid.

[0170] B10. The method of embodiment B8, wherein the B vitamin is nicotinamide.

[0171] B11 . The method of any one of embodiments B1 -B10, therein the one or more TGF-beta inhibitors comprise an ALK5 inhibitor.

[0172] B12. The method of embodiment B11 , wherein the ALK5 inhibitor is A83-01 .

[0173] B13. The method of embodiment B1 , wherein the one or more media components comprise a salt, a TGF-beta inhibitor, and a vitamin.

[0174] B14. The method of embodiment B13, wherein the salt is a formate salt, the TGF-beta inhibitor is an ALK5 inhibitor, and the vitamin is a B vitamin.

[0175] B15. The method of embodiment B14, wherein the formate salt is sodium formate, the ALK5 inhibitor is A83-01 , and the B vitamin is folic acid.

[0176] B16. The method of embodiment B1 , wherein the one or more media components comprise a salt, a TGF-beta inhibitor, and a sugar.

[0177] B17. The method of embodiment B16, wherein the salt is a formate salt, the TGF-beta inhibitor is an ALK5 inhibitor, and the sugar is a monosaccharide.

[0178] B18. The method of embodiment B17, wherein the formate salt is sodium formate, the ALK5 inhibitor is A83-01 , and the monosaccharide is galactose.

[0179] B19. The method of any one of embodiments B1 -B18, further comprising transducing or transfecting the gamma delta (yO) T cells with a nucleic acid. PATENT

[0180] POINT-1003PCT

[0181] B20. The method of embodiment B19, wherein the nucleic acid encodes a chimeric antigen receptor (CAR).

[0182] B21 . The method of any one of embodiments B1 -B20, further comprising introducing a genome modification into the gamma delta (yb) T cells by a genome editing process.

[0183] C1 . A method for activating and selectively expanding gamma delta (y<5) T cells ex vivo, comprising: a) activating gamma delta (yb) T cells in an originating cell population under activation conditions, thereby generating an activated gamma delta (yb) T cell population, wherein the activation conditions comprise binding agents comprising a cluster of differentiation 3 (CD3) binding agent and a cluster of differentiation 2 (CD2) binding agent; and b) expanding the number of gamma delta (yb) T cells in the activated gamma delta (yb) T cell population under expansion conditions, thereby generating an activated and expanded gamma delta (yb) T cell-enriched cell population, wherein the expansion conditions comprise cytokines comprising interleukin 2 (IL-2) and interleukin 4 (IL-4); IL-2 and interleukin 21 (IL-21 ); IL-2, IL-4, and IL-21 ; interleukin 15 (IL-15) and IL-4; IL-15 and IL-21 ; or IL-15, IL-4, and IL-21.

[0184] C2. The method of embodiment C1 , wherein the activation conditions further comprise IL-2.

[0185] C3. The method of embodiment C1 , wherein the activation conditions further comprise IL-4.

[0186] C3.1 The method of embodiment C1 , wherein the activation conditions further comprise IL-15.

[0187] C4. The method of embodiment C1 , wherein the activation conditions further comprise IL-2 and IL- 4.

[0188] C4.1 The method of embodiment C1 , wherein the activation conditions further comprise IL-2, IL-4 and IL-15.

[0189] C5. The method of any one of embodiments C1 -C4.1 , wherein the activation and / or expansion conditions further comprise one or more media components chosen from one or more salts, one or more sugars, one or more vitamins, and one or more transforming growth factor beta (TGF-beta) inhibitors.

[0190] 06. The method of embodiment 05, wherein the one or more salts comprise a formate salt

[0191] 07. The method of embodiment 06, wherein the formate salt is sodium formate.

[0192] 08. The method of any one of embodiments 05-07, wherein the one or more sugars comprise a monosaccharide.

[0193] 09. The method of embodiment 08, wherein the monosaccharide is galactose. PATENT

[0194] POINT-1003PCT

[0195] C10. The method of any one of embodiments C5-C9, wherein the one or more vitamins comprise ascorbic acid or a derivative thereof, salt thereof, or ester thereof.

[0196] C11 . The method of any one of embodiments C5-C10, wherein the one or more vitamins comprise a B vitamin.

[0197] C12. The method of embodiment C11 , wherein the B vitamin is folic acid.

[0198] C13. The method of embodiment C11 , wherein the B vitamin is nicotinamide.

[0199] C14. The method of any one of embodiments C5-C13, therein the one or more TGF-beta inhibitors comprise an ALK5 inhibitor.

[0200] C15. The method of embodiment C14, wherein the ALK5 inhibitor is A83-01 .

[0201] C16. The method of any one of embodiments 01 -04, wherein the activation and / or expansion conditions further comprise a salt, a TGF-beta inhibitor, and a vitamin.

[0202] 017. The method of embodiment 016, wherein the salt is a formate salt, the TGF-beta inhibitor is an ALK5 inhibitor, and the vitamin is a B vitamin.

[0203] 018. The method of embodiment 017, wherein the formate salt is sodium formate, the ALK5 inhibitor is A83-01 , and the B vitamin is folic acid.

[0204] 019. The method of any one of embodiments 01 -04, wherein the activation and / or expansion conditions further comprise a salt, a TGF-beta inhibitor, and a sugar.

[0205] 020. The method of embodiment 019, wherein the salt is a formate salt, the TGF-beta inhibitor is an ALK5 inhibitor, and the sugar is a monosaccharide.

[0206] 021. The method of embodiment 020, wherein the formate salt is sodium formate, the ALK5 inhibitor is A83-01 , and the monosaccharide is galactose.

[0207] 022. The method of any one of embodiments 01 -021 , further comprising reactivating the activated gamma delta (yd) T cell population under reactivation conditions, wherein the reactivation conditions comprise a cluster of differentiation 3 (CD3) binding agent.

[0208] 023. The method of embodiment 022, wherein the activated gamma delta (yd) T cell population is reactivated prior to the expanding in (b).

[0209] 024. The method of embodiment 022, wherein the activated gamma delta (yd) T cell population is reactivated during the expanding in (b).

[0210] 025. The method of any one of embodiments 01 -024, further comprising transducing or transfecting the activated gamma delta (yd) T cells with a nucleic acid. PATENT

[0211] POINT-1003PCT

[0212] C26. The method of embodiment C25, wherein the activated gamma delta (yd) T cells are transduced or transfected with a nucleic acid prior to the expanding in (b).

[0213] C27. The method of embodiment C25 or C26, wherein the nucleic acid encodes a chimeric antigen receptor (CAR).

[0214] C28. The method of any one of embodiments C1 -C27, further comprising introducing a genome modification into the activated gamma delta (yd) T cells by a genome editing process.

[0215] C29. The method of embodiment C28, wherein the genome modification is introduced prior to the expanding in (b).

[0216] D1 . The method of any one of embodiments A1-A21 , B1 -B21 , and C1 -29 wherein the originating cell population is derived from a peripheral blood mononuclear cell (PBMC) population.

[0217] D2. The method of any one of embodiments A1-A21 , B1 -B21 , and C1 -29 wherein the originating cell population is derived from cord blood.

[0218] D3. The method of any one of embodiments A1-A21 , B1 -B21 , C1-29, D1 and D2, wherein the originating cell population is depleted of alpha beta (ap) T cells.

[0219] D4. The method of any one of embodiments A1 -A21 , B1 -B21 , C1 -29, and D1 -D3, wherein the CD3 binding agent is an antibody or fragment thereof.

[0220] D5. The method of any one of embodiments A1 -A21 , B1 -B21 , C1 -29, and D1 -D4, wherein the CD2 binding agent is an antibody or fragment thereof.

[0221] D6. The method of any one of embodiments A1 -A21 , B1 -B21 , C1 -29, and D1 -D5, wherein cells are expanded about 400-fold to about 1 ,000-fold in the activated and expanded gamma delta (yd) T cell-enriched cell population when compared to the originating cell population.

[0222] D7. The method of any one of embodiments A1 -A21 , B1 -B21 , C1 -29, and D1 -D6, wherein the activated and expanded gamma delta (yd) T cell-enriched cell population comprises multi-subsets of gamma delta (yd) T cells.

[0223] D8. The method of embodiment D7, wherein the multi-subsets of gamma delta (yd) T cells comprise Vdeltal (Vd1 ) T cells and Vdelta2 (Vd2) T cells.

[0224] D9. The method of embodiment D7 or D8, wherein the multi-subsets of gamma delta (yd) T cells comprise Vdeltal (Vd1 ) T cells, Vdelta2 (Vd2) T cells, and non-Vd1 &Vd2 T cells.

[0225] D10. The method of embodiment D8 or D9, wherein the Vd1 T cells are expanded about 500-fold to about 11 ,000-fold in the activated and expanded gamma delta (yd) T cell-enriched cell population after about 14-21 days of culture when compared to the originating cell population. PATENT

[0226] POINT-1003PCT

[0227] D10.1 The method of embodiment D8 or D9, wherein the Vd1 T cells are expanded about 1 ,000- fold to about 3,000-fold in the activated and expanded gamma delta (yd) T cell-enriched cell population after about 14-21 days of culture when compared to the originating cell population.

[0228] D11 . The method of any one of embodiments D8-D10.1 , wherein the Vd2 T cells are expanded about 100-fold to about 2,600-fold in the activated and expanded gamma delta (yd) T cell-enriched cell population after about 14-21 days of culture when compared to the originating cell population.

[0229] D11 .1 The method of any one of embodiments D8-D10.1 , wherein the Vd2 T cells are expanded about 100-fold to about 850-fold in the activated and expanded gamma delta (yd) T cell-enriched cell population after about 14-21 days of culture when compared to the originating cell population.

[0230] D12. The method of any one of embodiments D9-D11 .1 , wherein the non-Vd1 &Vd2 T cells are expanded about 200-fold to about 4,800-fold in the activated and expanded gamma delta (yd) T cell-enriched cell population after about 14-21 days of culture when compared to the originating cell population.

[0231] D12.1 The method of any one of embodiments D9-D1 1.1 , wherein the non-Vd1 &Vd2 T cells are expanded about 200-fold to about 3,000-fold in the activated and expanded gamma delta (yd) T cell-enriched cell population after about 14-21 days of culture when compared to the originating cell population.

[0232] D13. The method of any one of embodiments A1 -A21 , B1 -B21 , C1 -29, and D1 -D12.1 , wherein the activated and expanded gamma delta (yd) T cell-enriched cell population comprises about 80% or more gamma delta (yd) T cells.

[0233] D13.1 The method of any one of embodiments A1 -A21 , B1 -B21 , 01 -29, and D1-D12.1 , wherein the activated and expanded gamma delta (yd) T cell-enriched cell population comprises about 90% or more gamma delta (yd) T cells.

[0234] D14. The method of any one of embodiments A1 -A21 , B1 -B21 , C1 -29, and D1 -D12.1 , wherein the activated and expanded gamma delta (yd) T cell-enriched cell population comprises about 95% or more gamma delta (yd) T cells.

[0235] D15. The method of any one of embodiments A1 -A21 , B1 -B21 , C1 -29, and D1 -D14, wherein the activated and expanded gamma delta (yd) T cell-enriched cell population comprises between about 5-55% Vd1 T cells, between about 15-85% Vd2 T cells, and between about 2.5-25% non-Vd1 &Vd2 T cells. PATENT

[0236] POINT-1003PCT

[0237] D16. The method of any one of embodiments A1 -A21 , B1 -B21 , C1 -29, and D1 -D15, wherein the activated and expanded gamma delta (yd) T cell-enriched cell population comprises about 20% natural killer (NK) cells or less.

[0238] D16.1 The method of any one of embodiments A1 -A21 , B1 -B21 , C1 -29, and D1-D15, wherein the activated and expanded gamma delta (yd) T cell-enriched cell population comprises about 10% natural killer (NK) cells or less.

[0239] D16.2 The method of any one of embodiments A1 -A21 , B1 -B21 , 01 -29, and D1-D15, wherein the activated and expanded gamma delta (yd) T cell-enriched cell population comprises about 5% natural killer (NK) cells or less.

[0240] D17. The method of any one of embodiments A1 -A21 , B1 -B21 , C1 -29, and D1 -D15, wherein the activated and expanded gamma delta (yd) T cell-enriched cell population comprises about 1 % natural killer (NK) cells or less.

[0241] D18. The method of any one of embodiments A1 -A21 , B1 -B21 , 01 -29, and D1 -D17, wherein the activated and expanded gamma delta (yd) T cell-enriched cell population comprises about 5% alpha beta (a[3) T cells or less.

[0242] D19. The method of any one of embodiments A1 -A21 , B1 -B21 , 01 -29, and D1 -D17, wherein the activated and expanded gamma delta (yd) T cell-enriched cell population comprises about 1 % alpha beta T (a ) cells or less.

[0243] D19.1 The method of any one of embodiments A1 -A21 , B1 -B21 , 01 -29, and D1 -D17, wherein the activated and expanded gamma delta (yd) T cell-enriched cell population comprises about 0.5% alpha beta T (ap) cells or less.

[0244] D20. The method of any one of embodiments A1 -A21 , B1 -B21 , 01 -29, and D1 -D17, wherein the activated and expanded gamma delta (yd) T cell-enriched cell population comprises about 0.1% alpha beta T (a|3) cells or less.

[0245] D21 . The method of any one of embodiments A1 -A21 , B1 -B21 , 01 -29, and D1 -D20, further comprising storing the activated and expanded gamma delta (yd) T cell-enriched cell population in a cell bank.

[0246] D22. An activated and expanded gamma delta (yd) T cell-enriched cell population produced by the method of any one of embodiments A1 -A21 , B1-B21 , 01-29, and D1 -D21.

[0247] D23. Use of the activated and expanded gamma delta (yd) T cell-enriched cell population of embodiment D22 for production of genetically modified cells. PATENT

[0248] POINT-1003PCT

[0249] D24. Use of the activated and expanded gamma delta (yd) T cell-enriched cell population of embodiment D22 or D23 for treatment of a subject in need thereof.

[0250] D25. The use of embodiment D24, wherein the subject has cancer.

[0251] D28. The use of embodiment D24, wherein the subject has an autoimmune disease.

[0252] D29. The use of embodiment D24, wherein the subject has an infectious disease.

[0253] E1 . A kit for activating and selectively expanding gamma delta (yd) T cells ex vivo comprising: a) binding agents comprising a cluster of differentiation 3 (CD3) binding agent, and a cluster of differentiation 2 (CD2) binding agent; b) cytokines comprising interleukin 2 (IL-2) and interleukin 4 (IL-4); IL-2 and interleukin 21 (IL-21); IL-2, IL-4, and IL-21 ; interleukin 15 (IL-15) and IL-4; IL-15 and IL-21 ; or IL-15, IL-4, and IL- 21 ; and c) instructions for use.

[0254] E2. The kit of embodiment E1 , further comprising one or more media components chosen from one or more salts, one or more sugars, one or more vitamins, and one or more transforming growth factor beta (TGF-beta) inhibitors.

[0255] E3. A kit for activating and selectively expanding gamma delta (yd) T cells ex vivo comprising: a) binding agents comprising a cluster of differentiation 3 (CD3) binding agent, and a cluster of differentiation 2 (CD2) binding agent; b) interleukin 2 (IL-2), interleukin 15 (IL-15), or IL-2 and IL-15; c) one or more media components chosen from one or more salts, one or more sugars, one or more vitamins, and one or more transforming growth factor beta (TGF-beta) inhibitors; and d) instructions for use.

[0256] E4. A kit for activating and selectively expanding gamma delta (yd) T cells ex vivo comprising: a) binding agents comprising a cluster of differentiation 3 (CD3) binding agent, and a cluster of differentiation 2 (CD2) binding agent; b) cytokines comprising interleukin 2 (IL-2) and interleukin 4 (IL-4); IL-2 and interleukin 21 (IL-21); IL-2, IL-4, and IL-21 ; interleukin 15 (IL-15) and IL-4; IL-15 and IL-21 ; or IL-15, IL-4, and IL- 21 ; c) one or more media components chosen from one or more salts, one or more sugars, one or more vitamins, and one or more transforming growth factor beta (TGF-beta) inhibitors; and d) instructions for use.

[0257] E5. The kit of any one of embodiments E2-E4, wherein the one or more salts comprise a formate salt PATENT

[0258] POINT-1003PCT

[0259] E6. The kit of embodiment E5, wherein the formate salt is sodium formate.

[0260] E7. The kit of any one of embodiments E2-E6, wherein the one or more sugars comprise a monosaccharide.

[0261] E8. The kit of embodiment E7, wherein the monosaccharide is galactose.

[0262] E9. The kit of any one of embodiments E2-E8, wherein the one or more vitamins comprise ascorbic acid or a derivative thereof, salt thereof, or ester thereof.

[0263] E10. The kit of any one of embodiments E2-E9, wherein the one or more vitamins comprise a B vitamin.

[0264] E11 . The kit of embodiment E10, wherein the B vitamin is folic acid.

[0265] E12. The kit of embodiment E10, wherein the B vitamin is nicotinamide.

[0266] E13. The kit of any one of embodiments E2-E12, therein the one or more TGF-beta inhibitors comprise an ALK5 inhibitor.

[0267] E14. The kit of embodiment E13, wherein the ALK5 inhibitor is A83-01 .

[0268] E15. The kit of any one of embodiments E2-E4, wherein the one or more media components comprise a salt, a TGF-beta inhibitor, and a vitamin.

[0269] E16. The kit of embodiment E15, wherein the salt is a formate salt, the TGF-beta inhibitor is an ALK5 inhibitor, and the vitamin is a B vitamin.

[0270] E17. The kit of embodiment E16, wherein the formate salt is sodium formate, the ALK5 inhibitor is A83-01 , and the B vitamin is folic acid.

[0271] E18. The kit of any one of embodiments E2-E4, wherein the one or more media components comprise a salt, a TGF-beta inhibitor, and a sugar.

[0272] E19. The kit of embodiment E18, wherein the salt is a formate salt, the TGF-beta inhibitor is an ALK5 inhibitor, and the sugar is a monosaccharide.

[0273] E20. The kit of embodiment E19, wherein the formate salt is sodium formate, the ALK5 inhibitor is A83-01 , and the monosaccharide is galactose.

[0274] E21. The kit of any one of embodiments E1 -E20, further comprising cells derived from a peripheral blood mononuclear cell (PBMC) population.

[0275] E22. The kit of any one of embodiments E1 -E20, further comprising cells derived from cord blood.

[0276] E23. The kit of embodiment E21 or E22, wherein the cells are depleted of alpha beta T cells. PATENT

[0277] POINT-1003PCT

[0278] E24. The kit of any one of embodiments E1 -E23, wherein the CD3 binding agent is an antibody or fragment thereof.

[0279] E25. The kit of any one of embodiments E1 -E24, wherein the CD2 binding agent is an antibody or fragment thereof.

[0280] Examples

[0281] The examples set forth below illustrate certain implementations and do not limit the technology.

[0282] Example 1: Materials and methods

[0283] This Example describes the materials and methods used to produce and analyze the gamma delta (yd) T cells described in subsequent Examples.

[0284] Isolation and ap T cell-depletion of PBMC

[0285] Fresh peripheral blood mononuclear cells (PBMC) were isolated through density gradient centrifugation of diluted whole blood using Ficoll-Paque Plus or collected through apheresis from healthy and consenting donors. a|3 T cells were further depleted using either a human TCRa / p T Cell Depletion Kit (Miltenyi) or using the TCRa / p Product Line (Miltenyi Biotec) according to the manufacturer’s instructions. After confirming the cell numbers, viability, and absence of TCRa / p in the TCRap-depleted PBMCs, cells were immediately cryopreserved in CryoStor® CS10 in multiple vials and subsequently stored in liquid nitrogen until further use.

[0286] Activation and expansion of gamma delta T cell

[0287] Cryopreserved op T cell-depleted PBMCs were thawed at 37°C water bath and diluted with at least 10 times more volume of complete cell culture medium, and spun down at 400 g for 5 min. The cells were washed with cell culture medium for a total of 2 times, ap T-depleted PBMCs were resuspended in CTSTMOpTmizer™ T Cell Expansion media (ThermoFisher Scientific) supplemented with 2.5% ICTSR, 1% p / s, 2 mM GLUTAMAX and 2.5 - 5% human AB serum at 1 million cells per mL. Soluble anti-CD3 (OKT3) and anti-CD2 agonist (Miltenyi) and human interleukin 15 (IL-15) (R&D) were added at the beginning of cell culture. In certain workflow variations, human interleukin 2 (IL-2) may be used in addition to or instead of IL-15. In certain workflow variations, soluble anti-CD3 (OKT3) combined with one or more of NKp44 ligand (PDGF- DD), anti-NKp46, and anti-NKp30 may be used. In certain workflow variations, certain cytokine combinations may be used where the cytokines are chosen from IL-4, IL-2, IL-15, IL-10, and IL-21 , and small molecules such as L-ascorbic acid, galactose, sodium formate, folic acid, A83-01 and / or nicotinamide may be included (Table 1 ). PATENT

[0288] POINT-1003PCT

[0289] The activation reagents were mixed with cell suspension and the cells were transferred into a G- REX well of proper size. Cells were fed with IL-15 and fresh medium at regular intervals until cell harvest between Day 14 to Day 21 . In certain workflow variations, cells may be fed with IL-2 or IL-2 combined with IL-4 or IL-21 or IL-10 and fresh medium at regular intervals until cell harvest on Day PATENT

[0290] POINT-1003PCT

[0291] 14 to Day 21 . Expanded y<5 T cells were phenotyped and cytotoxicity was determined using a coculture assay with GPF-Luc-labeled tumor cell lines.

[0292] CAR transduction

[0293] On Day 4, culture medium in G-REX was partially removed, the cells were resuspended and sampled for cell counting. The remaining cells were transferred into a 50 mL conical tube, and spun down at 400 g for 5 min. The cell pellet was resuspended at 2.5 million cells / mL in CTS complete medium containing 300 lU / mL of human IL-2, 50 mM of L-ascorbic acid, 40 ng / mL of human IL-4, and transduced with CAR retroviral supernatant at a MOI of 2 using a retronectin (Takara) - coated plate. Alternatively, CAR retroviral supernatant (MOI=5) was combined with 20 pg / mL of Vectofusin-1 (Miltenyi) in CTS complete medium, incubated at room temperature for 10 min, then mixed with cell suspension at 1 to 1 ratio, and transferred into G-REX at a final of 0.4mL / cm2. The cells were then incubated at 37°C and 5% CO2 overnight. On the 2ndday (Day 5), 4 volumes of prewarmed CST complete medium containing human IL-2 and L-ascorbic acid, with or without human IL-4 were added to continue the cell culture.

[0294] RNP delivery via electroporation for gene knock-out

[0295] On Day 6, the ribonucleoprotein (RNP) complex was prepared by mixing Cas9 recombinant protein with synthetic single-strand guide RNA (sgRNA) targeting TGF R2 at 1 to 2 molar ratio and incubated for at least 20 min at room temperature. Cells were washed with MAXCYTE buffer and spun down at 180 g for 7 min, and resuspended in MAXCYTE buffer to a final concentration of 100- 200 million cells / mL. RNP was then mixed in at a final concentration of 3 mM. The mixture of T cells and RNP was transferred into an appropriate processing assembly (PA) for electroporation with preloaded program NK-4. After electroporation, the cells were recovered in the incubator for 20 min. After the recovery, the cells were transferred from PA into a proper G-REX with prewarmed CTS complete medium containing human IL-2, L-ascorbic acid, and human IL-4 at a concentration of 1 million cells / mL for continued culture. In certain workflow variation, 7.5 ng / mL of human IL-21 , or 1 mM of Galactose, with or without repeat anti-CD3 (OKT3) stimulation at the concentration of 0.5 pg / mL were added on Day 7, and continued to culture up to Day 21 in G-REX. Human IL-2 or / and human IL-21 was replenished every 2-3 days. yd T cell phenotyping and flow cytometry analysis

[0296] Expanded cell subtype composition was determined via flow cytometry analysis - NK cells (CD3- CD56+), ap T cells (CD3+TCRVa[3+), pan yb T cells (CD3+TCRVY5+) and V61 (CD3+TCRV61 +) and V52 (CD3+TCRV52+) subsets. Activation and inhibitory receptor expression, effector and memory markers, were determined by multichannel FACS analysis. CAR transduction rate was PATENT

[0297] POINT-1003PCT determined via CD34 QBEND / 10 staining, TGF[3R2 knock-out efficiency was determined via antihuman TGFpRII staining. All antibodies were purchased from Biolegend except for TCRVyb (Miltenyi) and CD34 (Abnova).

[0298] Cytotoxicity assay

[0299] Different tumor cell lines including human non-small cell lung cancer (NSCLC) NCI-H1975 and HCC827, pancreatic ductal adenocarcinoma (PDAC) BxPC3, and triple negative breast cancer (TNBC) HCC1806 and HCC70 cell lines that express different levels of CAR targets were engineered to express GFP and firefly luciferase (LUC). Activated and expanded unmodified yb T cells or CAR yb T cells including those with TGF R2 KO were co-cultured with tumor cells at different Effector to Target (E:T) ratios for 24 hours (h), 48 h or 4-5 days (long-term killing). Luciferase activity was quantified using firefly luciferase HTS system (Sigma, St Louis, MO). Tumor-specific lysis was calculated using the formula: 100 x [tumor only - sample) / tumor only.

[0300] Statistical analysis

[0301] Unpaired multiple t-tests were performed in Graphpad Prism Software 9 (San Diego, CA) and the statistical significance is defined as a P value < 0.05.

[0302] Example 2: Anti-CD2 agonist antibody enhanced anti-CD3 antibody OKT3-induced polyclonal yb T cell expansion

[0303] For yb T cell activation and expansion, a vial of cryopreserved a T-depleted healthy donor PBMC was thawed, washed and resuspended in CTS complete medium supplemented with cytokines and agonist antibodies. In the starting culture, yb T cells accounted for 1 -10% of total PBMCs and were slightly enriched in a|3 T cell-depleted PBMCs. The subsets of yb T cells in a|3 T cell-depleted PBMC were 87.8% of Vb2+, 6.3% of Vb1 +, and 5.82% of Vb1 -Vb2- T cells (Fig. 1A). After 18 days of ex vivo culture with anti-CD3 Ab OKT3 and IL-15, yb T cells were enriched to account for approximately 90.4 - 96.2% of the total cell population (Fig. 1 B). The yb T cell subset composition was different in culture activated by OKT3 with anti-CD2 agonist antibody than OKT3 alone, with more Vb1 + T (43.5% vs 28.6%) and Vb2+ T (37.0% vs 18.3%) and less Vb1 -Vb2- T (14.4% vs 52.0%) (Fig. 1 B). The addition of anti-CD2 agonist antibody (0.5 pg / mL, R&D) to the initial cell culture supplemented with 1 pg / mL of OKT3 and human IL-15 (30 ng / mL, R&D), lead to preferential activation and proliferation of yb T cells, which became a predominant cell population in culture by Day 7 (Fig. 1 C), as well as significantly increased expansion of yb T cells compared to those without CD2 agonist Ab (Figs. 1 D-1 E). In general, the activation and expansion using OKT3 and CD2 agonist Ab preferentially enhanced the expansion of Vb1 + and Vb1 -Vb2- T cells than that of Vb2+ T cells (n=11 donors) (Fig. 1 F). However, the innate cytotoxicity of yb T cells activated and expanded with or without anti-CD2 Ab was comparable (Fig. 1 G). PATENT

[0304] POINT-1003PCT

[0305] Example 3: IL-2 and IL- 15 were comparable in expanding polyclonal y T cells

[0306] One million cryopreserved a|3 T-depleted PBMCs were thawed, washed and activated in CTS complete medium containing 2.5-5% of human AB serum, 1 pg / mL of OKT3 (Biolegend), 0.5 pg / mL of anti-CD2 Ab (Miltenyi) with 300 lll / mL of human IL-2 (R&D) or 7.5 ng / mL of human IL-15 (R&D). Medium and cytokine were supplemented every 3 or 4 days throughout the culture. Both cytokines expanded yd T cells efficiently with similar cell viability (Fig. 2A), yield (Fig. 2B), and fold expansion of yd T cells (Fig. 20) over 21 -day of culture. The purity of yd T cells in both culture conditions after 21 -day culture is higher than 95% (data not shown). IL-2 expanded Vd1 -Vd2- yd T cells slightly higher than IL-15 (Fig. 20). In addition, yd T cells cultured with IL-2 show more central memory TCM phenotype whereas yd T cells cultured with IL-15 show more effector memory TEM phenotype (Fig. 2D), indicating IL-2 cultured yd T cells may have better persistence whereas IL-15 cultured yd T cells may have fast onset of activity. Expression of potential exhaustion markers, e.g., PD-1 , TIG IT and LAG3 were similarly low in yd T cells cultured under IL-2 or IL-15. However, IL-15 upregulates TGF3R2 expression (Fig. 2E), which may render ex vivo expanded yd T cells more susceptible to inhibition by TGFp enriched in solid tumor microenvironment.

[0307] Example 4: Small molecules enhanced the expansion and transduction of CAR y5 T cells

[0308] A vial of cryopreserved ap T cell-depleted healthy donor PBMCs or apheresis cells was thawed, washed, and resuspended in CTS complete medium supplemented with OKT3 (1 pg / mL), anti-CD2 Ab (0.5 pg / mL), and IL-2 (300 lU / mL) alone or combined with various small molecules as listed in Table 1. CAR retrovirus transduction was performed on Day 4 in culture, as detailed in the Materials and Methods. IL-2 and small molecules were replenished every 2-3 days.

[0309] Compared to the IL-2 alone group, the addition of galactose, sodium formate, and folic acid enhanced the expansion of CAR yd T cells across different subsets (Fig. 3A). While IL-2 alone achieved a 400-fold expansion of CAR yd T cells, the inclusion of Galactose led to a 780-fold expansion, sodium formate to a 640-fold expansion, and Folic Acid to a 600-fold expansion. The purity of expanded CAR yd T cells were consistently higher than 95%, with similar composition of yd T cell subsets, except in the group treated with TGFp receptor inhibitor A83-01 (Fig. 3B). Notably, A83-01 increased the percentage of Vd1 + (20.8% vs 11 .4%) and Vd1 -Vd2- T cells (28.0% vs 12.4%), at the expense of a reduction in the expansion of Vd2+T cells (50% vs 75%) compared with that treated with IL-2 alone (Fig. 3B and 3A). The CAR yd T cells expanded with galactose, sodium formate, folic acid, and A83-01 showed comparable killing of CAR target-positive HCC1806 tumor cells (Fig. 3C).

[0310] Furthermore, in comparison to the IL-2 only (control) group, the addition of L-ascorbic acid or Nicotinamide resulted in comparably high purity (> 93%) of CD3+ T cells (Fig. 3D), with less than PATENT

[0311] POINT-1003PCT

[0312] 1% of CD3+TCRVap+ T cells (data not shown), and similar composition of yd T cell subsets (Fig. 3D). However, Ascorbic acid significantly enhanced the expansion of yb T cells including Vb1 + (1 ,124-fold vs 385-fold) and Vb2+ T cell subsets (232-fold vs 69-fold) (Fig. 3E), as well as the transduction efficiency of GAR retroviral vector (average 86% vs 76%, average MFI of 337,884 vs 213,404) compared to IL-2 alone (Fig. 3F). yb T cells expanded with IL-2 or additional Ascorbic acid or Nicotinamide showed comparable memory phenotypes (Fig. 3G), high levels (approximate average 91%) of activation receptor NKG2D, and low levels of exhaustion markers PD1 +TIGIT+ (approximate average 14%) and PD1 +TIM3+ (approximate average 20%) (Fig. 3H).

[0313] Example 5: IL-4 and IL-21 synergistically enhanced the expansion and cytotoxicity of gene-edited CAR yd T cells

[0314] A vial of cryopreserved a|3 T cell-depleted PBMCs or apheresis was thawed, washed, and resuspended in GTS completed medium containing OKT3 (1 pg / mL) and anti-CD2 (0.5 pg / mL) supplemented with human IL-2 (300 lU / mL, R&D) to induce yb T cell activation and expansion. CAR retrovirus transduction was conducted on Day 4 after cell activation as described in Example 1 . TGFPR2 KO was conducted 2 days post CAR transduction with electroporation of Cas9 / sgRNA RNP as described in Example 1 . IL-2 was replenished every 2 or 3 days. Human IL-4 (40 ng / mL, Sino Biological) was present in the first 6 days of culture by feeding cells every 3 days. Human IL- 21 (7.5 ng / mL, Sino Biological) was added to the culture post CRISPR / Cas9 KO every 2 or 3 days. As shown in Fig. 4A, CAR transduction was higher in yb T cultured with IL-4 in addition to IL-2 (74.7% vs 55.7% CAR+ cells). CAR-expressing yb T cells were dramatically increased when IL-21 is included in the culture (95.4% CAR+ in culture with IL-21 and 97.6% CAR+ in culture with IL-21 and IL-4). IL-4 and IL-21 also slightly improved the TGF[3R2 KO efficiency in yb T cells. The yb T cell viability was comparably high irrespective of the addition of IL-4 and / or IL-21 (Fig. 4B). The expansion of total gene KO CAR yb T cells, including Vb1 +, Vb2+, and Vb1 -Vb2- T cells was enhanced in the culture with the addition of IL-4 or IL-21 , which was further improved in the presence of both IL-4 and IL-21 (Fig. 4C). Furthermore, IL-21 -expanded gene KO CAR yb T cells showed increased cytotoxicity against target lung and breast tumor cell lines NCI-H1975 and HCC1806 compared to those expanded without IL-21 (Fig. 4D and 4E).

[0315] Example 6: Repeat OKT3 stimulation enhanced the expansion of V52- subsets of genetically modified yd T cells

[0316] In addition to the initial yb T stimulation by anti-CD3 Ab (OKT3, 1 pg / mL), which was present in the cell culture in the first 4 days, a second round of OKT3 stimulation (0.5 pg / mL) of yb T cells was conducted on Day 7 of culture, post CAR transduction and CRISPR / Cas9-mediated gene KO. The repeat OKT3 stimulation led to a more than two-fold increase in the percentage of Vb1 + and Vb1 - PATENT

[0317] POINT-1003PCT

[0318] Vd2- yd T cell population in the final product harvested on Day 14 of culture (Fig. 5A), resulting from significantly enhanced expansion of Vd1 + (average 2,114 vs 955-fold) and Vd1 -Vd2- (average 439 vs 85-fold) yd T cells (Fig. 5B). OKT3 restimulation did not affect CAR expression (data not shown) and the cytolytic activity of gene KO CAR yd T cells against target breast tumor cell line HCC1806 (Fig. 5C). As Vd1 + and Vd1 -Vd2- yd T cells preferentially reside in tissues, the method developed here may enhance therapeutic potential for solid tumors by enhancing product homing to and infiltration of solid tumors.

[0319] Example 7: Impacts of small molecules on the expansion, phenotype, and function of genetically modified y5 T cells ap T cell-depleted PBMCs were activated in CTS complete medium supplemented with OKT3, anti- CD2 Ab, IL-2 and L-ascorbic acid (control), or further supplemented with a small molecule mix of 1 mM of sodium formate, 2 pM of A83-01 , and either 20 pM of folic acid (F / A / F); or 10 mM of galactose (F / A / G). Every 2 or 3 days, the cells were fed with fresh medium and cytokines. On Day 4, cells were transduced with CAR retrovirus, and on Day 6, CAR-transduced yd T cells were electroporated with Cas9 / TGF R2 gRNA RNP for gene KO. From day 6, A83-01 was no longer added in the culture.

[0320] On Day 14, cells were harvested and phenotyped by flow cytometry. In comparison to control culture condition, the addition of a small molecule mix of either F / A / F or F / A / G did not negatively impact the efficiency of CAR transduction or TGF[3R2 gene editing (Figs. 6A and 6B). yd T cells constituted > 95% of the population, with <1% of CD3-CD56+ NK cells (Fig. 6C) and <1% of CD3+TCRap+ T cells (data not shown), yd T cells expanded with the addition of F / A / F or F / A / G had increased Vd1 + T and reduced Vd2+ T cell subsets (Fig. 6C). The small molecules also altered the yd T cell memory phenotypes. As shown in Fig. 6D, both F / A / F and F / A / G increased the population of central memory (TCM) and naive-like (TNAIVE) yd T cells while decreasing the population of effector memory (TEM) yd T cells.

[0321] However, both F / A / F and F / A / G compromised the cytolytic activity of TGF[3R2 KO CAR yd T cells at low E:T ratio (1 :8 - 1 :4) compared to the control, as determined by the lysis of 3D tumor spheroids derived from target tumor cell lines HCC827 and NCI-H1975 following a 4-day coculture. (Fig. 6E). Unmodified yd T cells expanded with the control condition served as a negative control in the killing assay (Fig. 6E).

[0322] TGFpR2 KO CAR yd T cells expanded with the addition of F / A / F or F / A / G showed more robust proliferation upon repeat challenge of tumor cells NCI-H1975 at E:T ratio of 1 :2 every 4 days (Fig. 6F). Following two rounds of tumor antigen stimulations, Vd1 + T and Vd1 -Vd2- T cell populations highly expanded with concurrent reduction of Vd2+ T cell population in both F / A / F and F / A / G PATENT

[0323] POINT-1003PCT groups compared with that in the control group (Fig. 6G), or prior to tumor antigen exposure (Fig. 6C). Furthermore, F / A / G-expanded TGF[3R2 KO CAR yd T cells also displayed significantly higher CD103 expression than that in control or F / A / F culture (Fig. 6H), indicating that galactose may contribute to the upregulation of tissue residence marker CD103 expression in yd T cells.

[0324] Thus, the small molecules of sodium formate, A83-01 , folic acid, and galactose can impact yd T cell expansion, phenotype and function. Both combinations increased Vd1 + T cell subset, nai've-like and central memory yd T cell population, but reduced rapid onset of tumor killing activity at low E:T ratio, while sustaining durable expansion of yd T cells, especially Vd2- T cell subsets, in response to repeat tumor rechallenges.

[0325] Example 8: Efficient expansion of gene-edited CAR yd T cells in both static and dynamic culture

[0326] G-REX® (Gas Permeable Rapid Expansion) is a static culture platform created specifically to produce immune cells and has been applied to CAR T cell manufacturing providing its practical and cost-effective way to scale up and scale out. However, a dynamic culture platform (e.g., stirred tank bioreactor) for manufacturing CAR-T cells has the advantage to efficiently produce large quantities of high-quality T cells with in-process control, which would be more desirable for commercial production.

[0327] To evaluate whether the expansion of gene-edited CAR yd T cells in static culture can be adapted to dynamic culture, TGFPR2 KO CAR yd T cells were produced and an equal number of cells (5 million) was transferred to either static G-REX 6M or Erlenmeyer Cell Shaker Flask under 150 RMP constant orbital shakes in the cell incubator with 5% CO2 and 37°C. For G-REX 6M, fresh cytokines (300 ILI / mL of IL-2 and 7.5 ng / mL of IL-21 ) were added every 2 or 3 days. For Erlenmeyer Cell Shaker Flask, cells were sampled every 24 h for density and viability, and fresh medium with cytokines (300 lU / mL of IL-2 and 7.5 ng / mL of IL-21 ) was added to the culture if the cell density was above 2 million cells / mL. The cells were harvested on Day 14 for cell counting and characterization. The cell viability in both cultures was comparably high (94.6% in G-REX and 94.8% in shake flask). The total live cell numbers in G-REX were 3.34x108, slightly higher than 2.51x108cells in the shake flask, both starting from 5x106cells on Day 7. Both static and dynamic culture generated highly pure yd T cells (98.9% in G-REX and 95.5% in shake flask), whereas the cell culture in the shake flask had a higher proportion of Vd1 + T cells (24%) than that in G-REX (1 1%) (Fig. 7A). Consistently, the fold expansion of V<51 + T cells was higher in shake flask than G- REX (3,903-fold vs 2,469-fold), whereas the fold expansion of V52+ T cells was lower in shake flask than G-REX (764-fold vs 1 ,334-fold) (Fig. 7B). There was no major difference in the expression of activation marker NKG2D, NCRs including NKp30, NKp44, and NKp46, and exhaustion markers PD1 +TIGIT+, PD1 +TIM3+, and PD1 +LAG3+ between the two culture PATENT

[0328] POINT-1003PCT conditions (Fig. 7C). Furthermore, the TGF R2 KO CAR yd T cells expanded in G-REX and shake flask were comparably potent in killing of the two different target tumor cell lines HCC70 and NCI- H1975 in 24 h coculture (Fig. 7D).

[0329] Conclusions

[0330] To enhance the accessibility and therapeutic potential of yb T cells, a highly efficient and scalable process was developed for ex vivo activation, genetic engineering, and expansion of allogeneic, healthy donor-derived yb T cells, resulting in a highly pure, active with minimal exhaustion, and potent modified yb T cell product with desired composition of yb T cell subsets.

[0331] In the feeder cell-free activation process, T cell co-receptor agonist, i.e., anti-CD2 antibody, in combination with T cell receptor agonist (OKT3, anti-CD3 antibody), were highly effective in selectively activating and expanding yb T cells to high purity ex vivo. The cytokine IL-2 or IL-15 was comparable in promoting proliferation and the maintenance of yb T cells in culture, resulting in highly active yb T cells with minimal exhaustion. In addition, small molecules including galactose, formate, folic acid, and ascorbic acid, and cytokines such as IL-4 and IL-21 further enhanced the expansion of yb T cells in culture.

[0332] Since Vb2+ T cell subtype is most dominant in peripheral blood, whereas Vb2- T cell subtypes (Vb1 + T and Vb1 -Vb2- T cells) are naturally tissue resident, the presentation of Vb2- T cell subtypes may improve the product homing and infiltration to diseased tissues in patients with solid tumors, autoimmune or infectious diseases. IL-4, or IL-4 and IL-21 synergistically, or OKT3 restimulation, or the combination of formate, A83-01 , folic acid, or galactose, provided at specific dosing and timing in the process, increased the population of Vb2- T cell subtypes in the final expanded product.

[0333] Additionally, ascorbic acid and IL-21 , and to a lesser extent, IL-4 significantly increased CAR transduction efficiency, CAR expression level, and gene KO efficiency, leading to gene-edited CAR yb T cells with enhanced cytotoxicity.

[0334] Furthermore, expansion of modified yb T cells by methods described herein is applicable to both static culture as well as dynamic culture conditions.

[0335] The entirety of each patent, patent application, publication and document referenced herein is incorporated by reference. Citation of patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission PATENT

[0336] POINT-1003PCT as to the contents or date of these publications or documents. Their citation is not an indication of a search for relevant disclosures. All statements regarding the date(s) or contents of the documents is based on available information and is not an admission as to their accuracy or correctness.

[0337] The technology has been described with reference to specific implementations. The terms and expressions that have been utilized herein to describe the technology are descriptive and not necessarily limiting. Certain modifications made to the disclosed implementations can be considered within the scope of the technology. Certain aspects of the disclosed implementations suitably may be practiced in the presence or absence of certain elements not specifically disclosed herein.

[0338] Each of the terms “comprising,” “consisting essentially of,” and “consisting of” may be replaced with either of the other two terms. The term “a” or “an” can refer to one of or a plurality of the elements it modifies (e.g., “a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described. The term “about” as used herein refers to a value within 10% of the underlying parameter (i.e. , plus or minus 10%; e.g., a weight of “about 100 grams” can include a weight between 90 grams and 110 grams). Use of the term “about” at the beginning of a listing of values modifies each of the values (e.g., “about 1 , 2 and 3” refers to "about 1 , about 2 and about 3"). When a listing of values is described the listing includes all intermediate values and all fractional values thereof (e.g., the listing of values "80%, 85% or 90%" includes the intermediate value 86% and the fractional value 86.4%). When a listing of values is followed by the term "or more," the term "or more" applies to each of the values listed (e.g., the listing of "80%, 90%, 95%, or more" or "80%, 90%, 95% or more" or "80%, 90%, or 95% or more" refers to "80% or more, 90% or more, or 95% or more"). When a listing of values is described, the listing includes all ranges between any two of the values listed (e.g., the listing of "80%, 90% or 95%" includes ranges of "80% to 90%, " "80% to 95%" and "90% to 95%").

[0339] Certain implementations of the technology are set forth in the claim(s) that follow(s).

Claims

PATENTPOINT-1003PCTWhat is claimed is:1 . A method for activating and selectively expanding gamma delta (y5) T cells ex vivo, comprising: activating gamma delta (y6) T cells and expanding the number of gamma delta (yb) T cells in an originating cell population under activation and expansion conditions, thereby generating an activated and expanded gamma delta (y<5) T cell-enriched cell population, wherein the activation and expansion conditions comprise: a) binding agents comprising a cluster of differentiation 3 (CD3) binding agent and a cluster of differentiation 2 (CD2) binding agent; b) cytokines comprising interleukin 2 (IL-2); IL2 and interleukin 4 (IL-4); IL-2 and interleukin 21 (IL-21); IL-2, IL-4, and IL-21 ; interleukin 15 (IL-15); IL15 and IL-4; IL-15 and IL-21 ; or IL-15, IL-4, and IL-21 ; and c) one or more media components chosen from one or more vitamins, one or more salts, one or more sugars, and one or more transforming growth factor beta (TGF- beta) inhibitors.

2. The method of claim 1 , wherein the cytokines comprise IL2 and IL4.

3. The method of claim 1 , wherein the cytokines comprise IL2 and IL21 .

4. The method of claim 1 , wherein the cytokines comprise IL2, IL4, and IL21 .

5. The method of any one of claims 1 -4, wherein the one or more vitamins comprise ascorbic acid or a derivative thereof, salt thereof, or ester thereof.

6. The method of any one of claims 1 -5, wherein the one or more vitamins comprise a B vitamin.

7. The method of claim 6, wherein the B vitamin is folic acid.

8. The method of claim 6, wherein the B vitamin is nicotinamide.

9. The method of any one of claims 1 -8, wherein the one or more salts comprise a formate salt.

10. The method of claim 9, wherein the formate salt is sodium formate.1 1 . The method of any one of claims 1-10, wherein the one or more sugars comprise a monosaccharide.

12. The method of claim 1 1 , wherein the monosaccharide is galactose.

13. The method of any one of claims 1-12, therein the one or more TGF-beta inhibitors comprise an ALK5 inhibitor.PATENTPOINT-1003PCT14. The method of claim 13, wherein the ALK5 inhibitor is A83-01.

15. The method of claim 1 , wherein the cytokines comprise IL2 and IL4, and the one or more media components comprise ascorbic acid or a derivative thereof, salt thereof, or ester thereof.

16. The method of claim 1 , wherein the cytokines comprise IL2 and IL21 , and the one or more media components comprise ascorbic acid or a derivative thereof, salt thereof, or ester thereof.

17. The method of claim 1 , wherein the cytokines comprise IL2, IL4, and IL21 , and the one or more media components comprise ascorbic acid or a derivative thereof, salt thereof, or ester thereof.

18. The method of claim 1 , wherein the cytokines comprise IL2 and IL4, and the one or more media components comprise galactose and ascorbic acid or a derivative thereof, salt thereof, or ester thereof.

19. The method of claim 1 , wherein the cytokines comprise IL2 and IL21 , and the one or more media components comprise galactose and ascorbic acid or a derivative thereof, salt thereof, or ester thereof.

20. The method of claim 1 , wherein the cytokines comprise IL2, IL4, and IL21 , and the one or more media components comprise galactose and ascorbic acid or a derivative thereof, salt thereof, or ester thereof.21 . The method of any one of claims 1-20, wherein the activation and expansion conditions comprise static culture conditions.

22. The method of any one of claims 1-20, wherein the activation and expansion conditions comprise dynamic culture conditions.

23. The method of any one of claims 1-22, wherein the originating cell population comprises a heterogeneous population of cells.

24. The method of claim 23, wherein the heterogeneous population of cells is obtained from an apheresis product from one or more healthy donors.

25. The method of claim 23 or 24, wherein the heterogeneous population of cells comprises gamma delta (yb) T cells and one or more cell types chosen from alpha beta (af>) T cells, natural killer (NK) cells, monocytes, and B cells.

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