WO2025199494A1

WO2025199494A1 - Methods of generating populations of therapeutic cells

Info

Publication number: WO2025199494A1
Application number: PCT/US2025/021010
Authority: WO
Inventors: Leslie JONART; Marissa TROJE; Jeff LITER
Original assignee: Luminary Therapeutics Inc
Current assignee: Luminary Therapeutics Inc
Priority date: 2024-03-22
Filing date: 2025-03-21
Publication date: 2025-09-25
Anticipated expiration: 2026-09-22

Abstract

Provided herein are compositions and methods for improved generation of expanded populations of polyclonal gamma delta T cells suitable for clinical applications. For example, methods disclosed herein can generate a high fold-expansion of polyclonal populations of engineered (e.g., transposon-engineered CAR-expressing) gamma delta T cells. Populations of gamma delta T cells provided by methods disclosed herein can have advantageous functional properties, for example, associated with polyclonality of gdTCR subunit expression, lack of expression of exhaustion markers, and other functional attributes such as cancer cell killing, activation, expansion, cytokine production, and durable responses upon repeat exposure to target cells.

Description

METHODS OF GENERATING POPULATIONS OF THERAPEUTIC CELLS

CROSS REFERENCE

[0001] This Application claims priority to and the benefit of United States Provisional Patent Application No. 63/568,877, filed March 22, 2024, which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Unlike alpha beta (ab) T cells, which natively rely on antigen presentation via MHC/HLA molecules, gamma delta T cells can natively recognize stress-induced molecules expressed by tumor cells. Certain gamma delta T cell subsets can also recognize phosphoantigens and other non-peptide antigens overexpressed by transformed cells. This innate recognition enables gamma delta T cells to target a broad range of tumors without the need for specific tumor antigens or prior sensitization, or HLA-dependent activation cues.

[0003] Additionally, gamma delta T cells exhibit a broad spectrum of tumor reactivity, by recognizing a variety of tumor types including both hematological and solid tumors. This broad reactivity can be attributed to the diversity of gamma delta T cell subsets expressing different T cell receptor (TCR) variable delta (Vd or V8) and variable gamma (Vg, Vy, or Vy) chains, each with distinct tissue tropism and antigen specificities.

SUMMARY

[0004] Disclosed herein, in some aspects is a method of generating an expanded population of therapeutic cells, the method comprising: seeding at least 8 xl0^A5 gamma delta T cells per square cm in a culture vessel comprising a gas permeable membrane submerged under a column of medium; and incubating the gamma delta T cells in an expansion culture medium comprising a gamma delta T cell receptor (gdTCR) stimulating agent.

[0005] In some embodiments, the culture vessel is a Gas permeable Rapid Expansion (GREX®) cell culture vessel. In some embodiments, at least 1 xlO^A6 of the gamma delta T cells are seeded per square cm. In some embodiments, 1-1.5 xlO^A6 of the gamma delta T cells are seeded per square cm. In some embodiments, the gamma delta T cells expand at least 2,000- fold. In some embodiments, the gamma delta T cells expand at least 5,000-fold. In some embodiments, the gdTCR stimulating agent comprises an anti-gdTCR antibody or antigenbinding fragment thereof. In some embodiments, the gdTCR stimulating agent comprises a pan anti-gdTCR antibody or antigen-binding fragment thereof. In some embodiments, the anti- gdTCR antibody is bound to a surface of the culture vessel. In some embodiments, the expansion culture medium further comprises an anti-CD28 antibody or antigen-binding fragment thereof. In some embodiments, the expansion culture medium and the culture vessel lack an anti-CD3 antibody or antigen-binding fragment thereof. In some embodiments, after the incubating, the population of gamma delta T cells comprises at least 10% Vd 1+ cells and at least 10% Vd2+ cells. In some embodiments, after the incubating, the gamma delta T cells comprise at least 1% Vdl- Vd2- gamma delta T cells. In some embodiments, after the incubating, the gamma delta T cells comprise at least 11% Vd3+ cells. In some embodiments, the method further comprises electroporating the gamma delta T cells to introduce a transgene encoding a chimeric antigen receptor into the gamma delta T cells. In some embodiments, the method further comprises (i) following the electroporating, incubating the gamma delta T cells in recovery medium for about 1 -2 hours, and (iii) after the incubating in the recovery medium for about 1-2 hours, adjusting the cell concentration to about 1-1.5 x 10^A6 cells per mL. In some embodiments, the method further comprises, prior to the electroporating, incubating the gamma delta T cells in the expansion culture medium at a volume of about 300-400 pL per square cm. In some embodiments, comprising assaying the population of gamma delta T cells to determine viability. In some embodiments, comprising assaying the population of gamma delta T cells to determine polyclonal gdTCR phenotype. In some embodiments, the method further comprises assaying the population of gamma delta T cells to determine expression of exhaustion markers. In some embodiments, the incubating is at about 37°C. In some embodiments, the method further comprises enriching the gamma delta T cells to reduce the frequency of non-gamma delta T cells. In some embodiments, gamma delta T cells are incubated in the culture vessel comprising a gas permeable membrane submerged under a column of medium for at least 20 days. In some embodiments, the gamma delta T cells are incubated in the culture vessel comprising a gas permeable membrane submerged under a column of medium for about 2-25 days. In some embodiments, the method further comprises dislodging adherent gamma delta T cells from the culture vessel. In some embodiments, the method further comprises harvesting the gamma delta T cells after culturing for about 21 days in the expansion culture medium after the electroporating. In some embodiments, the method further comprises quantifying glucose and lactate after a period of incubation of the population of polyclonal gamma delta T cells in the expansion culture medium. In some embodiments, the gamma delta T cells retain cytotoxic capacity after three rounds of rechallenge with target cells. In some embodiments, the method provides at least 1 x 10^A8 gamma delta T cells. In some embodiments, the method provides at least 1 x 10^A9 gamma delta T cells. In some embodiments, the method provides at least 1 x 10^Al 0 gamma delta T cells. In some embodiments, the method provides at least 100 patient doses of a cell therapy product. In some embodiments, the method provides at least 200 patient doses of a cell therapy product. In some embodiments, the method provides at least 500 patient doses of a cell therapy product.

[0006] Disclosed herein, in some aspects is a method of generating an expanded population of therapeutic cells, the method comprising: incubating a population of engineered polyclonal gamma delta T cells in a culture vessel in an expansion culture medium comprising a gamma delta T cell receptor (gdTCR) stimulating agent; and treating the population of engineered polyclonal gamma delta T cells in the expansion culture medium every 2-3 days with: (i) IL-2 freshly added to a final concentration of at least 1000 lU/mL, (ii) IL-7 freshly added to a final concentration of at least 5 ng/mL, and (iii) IL-15 freshly added to a final concentration of at least 5 ng/mL.

[0007] In some embodiments, the culture vessel is a Gas permeable Rapid Expansion (GREX®) cell culture vessel. In some embodiments, at least 1 xl 0^A6 of the gamma delta T cells are seeded per square cm of the GREX cell culture vessel. In some embodiments, 1-1.5 xlO⁷^ of the gamma delta T cells are seeded per square cm of the GREX cell culture vessel. In some embodiments, the population of engineered polyclonal gamma delta T cells expands at least 2,000-fold. In some embodiments, the population of engineered polyclonal gamma delta T cells expands at least 5,000-fold. In some embodiments, the gdTCR stimulating agent comprises an anti-gdTCR antibody or antigen-binding fragment thereof. In some embodiments, the gdTCR stimulating agent comprises a pan anti-gdTCR antibody or antigen-binding fragment thereof. In some embodiments, the anti-gdTCR antibody is bound to a surface of the culture vessel. In some embodiments, the expansion culture medium further comprises an anti-CD28 antibody or antigen-binding fragment thereof. In some embodiments, the expansion culture medium and the culture vessel lack an anti-CD3 antibody or antigen-binding fragment thereof. In some embodiments, after the incubating, the population of polyclonal gamma delta T cells comprises at least 10% Vdl+ cells and at least 10% Vd2+ cells. In some embodiments, after the incubating, the population of polyclonal gamma delta T cells comprises at least 1% Vdl- Vd2- gamma delta T cells. In some embodiments, after the incubating, the population of polyclonal gamma delta T cells comprises at least 11% Vd3+ cells. In some embodiments, the method further comprises electroporating to introduce a transgene encoding a chimeric antigen receptor into a population of polyclonal gamma delta T cells, thereby generating the population of engineered polyclonal gamma delta T cells. In some embodiments, following the electroporating, the method comprises incubating the engineered polyclonal gamma delta T cells in recovery medium for about 1-2 hours, and (iii) after the incubating in the recovery medium for about 1-2 hours, adjusting the cell concentration to about 1-1.5 x 10^A6 cells per mL. In some embodiments, the method further comprises, prior to the electroporating, incubating the polyclonal gamma delta T cells in the expansion culture medium at a volume of about 300-400 pL per square cm. In some embodiments, viral vectors are not used to generate the engineered polyclonal gamma delta T cells. In some embodiments, the method further comprises genomically integrating a transgene encoding a chimeric antigen receptor. In some embodiments, the genomically integrating utilizes a transposon-based system. In some embodiments, the method further comprises assaying the population of engineered polyclonal gamma delta T cells to determine viability. In some embodiments, the method further comprises assaying the population of engineered polyclonal gamma delta T cells to determine polyclonal gdTCR phenotype. In some embodiments, the method further comprises assaying the population of engineered polyclonal gamma delta T cells to determine expression of exhaustion markers. In some embodiments, the incubating is at about 37°C. In some embodiments, the method further comprises enriching the polyclonal gamma delta T cells to reduce the frequency of non-gamma delta T cells. In some embodiments, the method further comprises dislodging adherent gamma delta T cells from the culture vessel. In some embodiments, the method further comprises harvesting cells after culturing for about 21 days in the expansion culture medium after the electroporating. In some embodiments, the method further comprises quantifying glucose and lactate after a period of incubation of the population of engineered polyclonal gamma delta T cells in the expansion culture medium. In some embodiments, the engineered polyclonal gamma delta T cells retain cytotoxic capacity after three rounds of rechallenge with target cells. In some embodiments, the engineered polyclonal gamma delta T cells exhibit at least 50% higher killing of target cells compared to a control population of cells that is not polyclonal. In some embodiments, the engineered polyclonal gamma delta T cells exhibit at least 2-fold increased proliferation in response to target cells compared to a control population of cells that is not polyclonal. In some embodiments, the engineered polyclonal gamma delta T cells exhibit at least 10% reduced exhaustion compared to a control population of cells that is not polyclonal. In some embodiments, the method provides at least 1 x 10^A8 engineered polyclonal gamma delta T cells. In some embodiments, the method provides at least 1 x 10^A9 engineered polyclonal gamma delta T cells. In some embodiments, the method provides at least 1 x 10^Al 0 engineered polyclonal gamma delta T cells. In some embodiments, the method provides at least 100 patient doses of a cell therapy product. In some embodiments, the method provides at least 200 patient doses of a cell therapy product. In some embodiments, the method provides at least 500 patient doses of a cell therapy product.

[0008] Disclosed herein, in some aspects, is a method of generating an expanded population of therapeutic cells, the method comprising: engineering a population of polyclonal gamma delta T cells to express a chimeric antigen receptor, thereby providing a population of engineered polyclonal gamma delta T cells; and incubating the population of engineered polyclonal gamma delta T cells in a culture vessel in an expansion culture medium comprising a gamma delta T cell receptor (gdTCR) stimulating agent; wherein the population of engineered polyclonal gamma delta T cells expands at least 1,000-fold.

[0009] In some embodiments, the culture vessel is a Gas permeable Rapid Expansion (GREX®) cell culture vessel. In some embodiments, at least 1 xlO^A6 of the engineered polyclonal gamma delta T cells are seeded per square cm of the GREX cell culture vessel. In some embodiments, 1-1.5 xlO^A6 of the gamma delta T cells are seeded per square cm of the GREX cell culture vessel. In some embodiments, the population of engineered polyclonal gamma delta T cells expands at least 2,000-fold. In some embodiments, the population of engineered polyclonal gamma delta T cells expands at least 5,000-fold. In some embodiments, the gdTCR stimulating agent comprises an anti-gdTCR antibody or antigen-binding fragment thereof. In some embodiments, the gdTCR stimulating agent comprises a pan anti-gdTCR antibody or antigen-binding fragment thereof. In some embodiments, the anti-gdTCR antibody is bound to a surface of the culture vessel. In some embodiments, the expansion culture medium further comprises an anti-CD28 antibody or antigen-binding fragment thereof. In some embodiments, the expansion culture medium and the culture vessel lack an anti-CD3 antibody or antigen-binding fragment thereof. In some embodiments, after the incubating, the population of engineered polyclonal gamma delta T cells comprisesat least 10% Vdl+ cells and at least 10% Vd2+ cells. In some embodiments, after the incubating, the population of engineered polyclonal gamma delta T cells comprises at least 1% Vdl- Vd2- gamma delta T cells. In some embodiments, after the incubating, the population of polyclonal gamma delta T cells comprises at least 11% Vd3+ cells. In some embodiments, the method further comprises electroporating the polyclonal gamma delta T cells to introduce a transgene encoding the chimeric antigen receptor. In some embodiments, the method further comprises: (i) following the electroporating, incubating the engineered polyclonal gamma delta T cells in recovery medium for about 1-2 hours, and (iii) after the incubating in the recovery medium for about 1-2 hours, adjusting the cell concentration to about 1-1.5 x 10^A6 cells per m . In some embodiments, prior to the electroporating, the method comprises incubating the polyclonal gamma delta T cells in the expansion culture medium at a volume of about 300-400 L per square cm. In some embodiments, viral vectors are not used to generate the engineered polyclonal gamma delta T cells. In some embodiments, the method further comprises genomically integrating the transgene encoding the chimeric antigen receptor. In some embodiments, the genomically integrating utilizes a transposon-based system. In some embodiments, the method further comprises assaying the population of engineered polyclonal gamma delta T cells to determine viability. In some embodiments, the method further comprises assaying the population of engineered polyclonal gamma delta T cells to determine polyclonal gdTCR phenotype. In some embodiments, the method further comprises assaying the population of engineered polyclonal gamma delta T cells to determine expression of exhaustion markers. In some embodiments, the incubating is at about 37°C. In some embodiments, the method further comprises enriching the polyclonal gamma delta T cells to reduce the frequency of non-gamma delta T cells. In some embodiments, the population of polyclonal gamma delta T cells are incubated in the GREX culture vessel for at least 20 days. In some embodiments, the population of polyclonal gamma delta T cells are incubated in the GREX culture vessel for about 2-25 days. In some embodiments, the method further comprises dislodging adherent gamma delta T cells from the culture vessel. In some embodiments, the method further comprises harvesting cells after culturing for about 21 days in the expansion culture medium after the electroporating. In some embodiments, the method further comprises quantifying glucose and lactate after a period of incubation of the population of polyclonal gamma delta T cells in the expansion culture medium. In some embodiments, the engineered polyclonal gamma delta T cells retain cytotoxic capacity after three rounds of rechallenge with target cells. In some embodiments, the engineered polyclonal gamma delta T cells exhibit at least 50% higher killing of target cells compared to a control population of cells that is not polyclonal. In some embodiments, the engineered polyclonal gamma delta T cells exhibit at least 2-fold increased proliferation in response to target cells compared to a control population of cells that is not polyclonal. In some embodiments, the engineered polyclonal gamma delta T cells exhibit at least 10% reduced exhaustion compared to a control population of cells that is not polyclonal. In some embodiments, the method provides at least 1 x 10^A8 engineered polyclonal gamma delta T cells. In some embodiments, the method provides at least 1 x 10^A9 engineered polyclonal gamma delta T cells. In some embodiments, the method provides at least 1 x 10^Al 0 engineered polyclonal gamma delta T cells. In some embodiments, the method provides at least 100 patient doses of a cell therapy product. In some embodiments, the method provides at least 200 patient doses of a cell therapy product. In some embodiments, the method provides at least 500 patient doses of a cell therapy product.

INCORPORATION BY REFERENCE

[0010] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The novel features of the invention are set forth with particularity in the appended claims. Abetter understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0012] FIG. 1 shows fold expansion results, viability, percentage of cells expressing CAR, percentage of cells expressing the Vdl gdTCR subunit, and the percentage of cells expressing the Vd2 gdTCR subunit for populations of polyclonal gamma delta T cells produced by methods disclosed herein from two donors.

[0013] FIG. 2A shows killing of mantle cell target cells by gamma delta T cells produced by a method disclosed herein and engineered to express a BAFF CAR (BAFF) or without expression of a CAR (pulsed).

[0014] FIG.2B shows killing of multiple myeloma cell target cells by gamma delta T cells produced by a method disclosed herein and engineered to express a BAFF CAR (BAFF) or without expression of a CAR (pulsed).

[0015] FIG. 3 shows fold expansion, percentage of cells expressing the Vdl gdTCR subunit, and the percentage of cells expressing the Vd2 gdTCR subunit for gamma delta T cells produced by a method disclosed herein in which culturing in G-rex culture vessels began on day 12.

[0016] FIG. 4 A shows fold expansion, percentage of cells expressing CAR, percentage of cells expressingthe Vdl gdTCR subunit, and the percentage of cells expressingthe Vd2 gdTCR subunit for gamma delta T cells produced by a method disclosed herein.

[0017] FIG. 4B shows complete killing at 3 :1 E:T over 48 hours of Mantle Cell (Jeko-1) and Multiple Myeloma (IM-9) target cells by gamma delta T cells after a 23 day expansion protocol disclosed herein, with (BAFF) or without (pulse) expression of a BAFF CAR. [0018] FIG. 5A shows fold expansion results, viability, percentage of cells expressing CAR, percentage of cells expressing the Vdl gdTCR subunit, and the percentage of cells expressing the Vd2 gdTCR subunit for a population of polyclonal gamma delta T cells produced by methods disclosed herein.

[0019] FIG. 5B shows killing of esophageal cancer target cells by gamma delta T cells produced by a method disclosed herein and engineered to express a split CAR (split co-stim) or without expression of a CAR (pulsed).

[0020] FIG. 5C shows killing of cholangiocarcinoma target cells by gamma delta T cells produced by a method disclosed herein and engineered to express a split CAR (split co-stim) or without expression of a CAR (pulsed).

[0021] FIG. 6A shows killing of cholangiocarcinoma target cells by gamma delta T cells expanded by a method disclosed herein and engineered to express a split CAR (split co-stim).

[0022] FIG. 6B shows killing of esophageal cancer target cells by gamma delta T cells expanded by a method disclosed herein and engineered to express a split CAR (split co-stim).

[0023] FIG. 6C shows killing of renal carcinoma target cells by gamma delta T cells expanded by a method disclosed herein and engineered to express a split CAR (split co-stim).

[0024] FIG. 7A shows the number of effector T cells (ab = alpha beta, gd = gamma delta) either engineered to express a split CAR, or non-engineered (pulsed), after co-culture with target cells at a 1 :5 E:T ratio, and five rounds of rechallenge with fresh target cells every 2 days.

[0025] FIG. 7B shows the ability of effector T cells (ab = alpha beta, gd = gamma delta) either engineered to express a split CAR, or non-engineered (pulsed), to kill FLO-1 target cells after five rounds of rechallenge with fresh target cells every 2 days.

[0026] FIG. 8A shows fold expansion results, viability, percentage of cells expressing CAR, percentage of cells expressing the Vdl gdTCR subunit, and the percentage of cells expressing the Vd2 gdTCR subunit for populations of polyclonal gamma delta T cells produced by methods disclosed herein from two donors.

[0027] FIG. 8B shows killing of glioblastoma target cells by gamma delta T cells produced by a method disclosed herein and engineered to express a split CAR (split co-stim) or without expression of a CAR (pulsed).

[0028] FIG. 9 shows fold expansion results, viability, percentage of cells expressing CAR, percentage of cells expressing the Vdl gdTCR subunit, and the percentage of cells expressing the Vd2 gdTCR subunit for a population of polyclonal gamma delta T cells produced by methods disclosed herein. [0029] FIG. 10A shows that -98.39% of an expanded population of cells generated by methods disclosed herein were CD3+.

[0030] FIG. 10B shows that -95.52% of T cells in an expanded population of cells generated by methods disclosed herein were negative for expression of alpha/beta TCR.

[0031] FIG. 10C shows that -64.85% of an expanded population of cells generated by methods disclosed herein were positive for expression of a BAFF CAR.

[0032] FIG. 10D shows gamma delta TCR poly clonality of an expanded population of cells generated by methods disclosed herein. -73.4% were Vdl+, -12.9% were Vd2+, and -13.5% were negative for Vdl and Vd2.

[0033] FIG. 11A shows proportions of Vdl+, Vd2+, and Vdl-Vd2- cells in a polyclonal condition of an experiment at seeding and after two re-challenges.

[0034] FIG. 11B shows proportions of Vdl+, Vd2+, and Vdl-Vd2- cells in a Vdl+/Vdl- Vd2- condition of an experiment at seeding and after two re-challenges.

[0035] FIG. 11C shows proportions of Vdl+, Vd2+, and Vdl-Vd2- cells in a Vd2+ condition of an experiment at seeding and after two re-challenges.

[0036] FIG. 12A shows the percentage of target versus effector cells at indicated time points for a polyclonal condition of a co-culture experiment.

[0037] FIG. 12B shows the percentage of target versus effector cells at indicated time points for a Vdl+/Vdl-Vd2- condition of a co-culture experiment.

[0038] FIG. 12C shows the percentage oftarget versus effector cells at indicated time points for a Vd2+ condition of a co-culture experiment.

[0039] FIG. 13A provides representative scatterplots showing the proportions of CAR+ cells at 24 and 48 hours in a co-culture experiment using indicated populations of engineered gamma delta T cells.

[0040] FIG. 13B provides representative scatterplots showing the proportions of CAR+ cells after rechallenges in a co-culture experiment using indicated populations of engineered gamma delta T cells.

[0041] FIG. 14 shows the percentage of CAR+ gamma delta T cells upon co-culture and rechallenge with target cells.

[0042] FIG. 15A provides scatter plots showing changes in populations of CD 19+ target cells (upper left quadrants) and CD3+ effector cells (lower right quadrants) at indicated time points for a polyclonal gamma delta T cell effector population (upper panels) and a Vdl+/Vdl- Vd2- effector population (lower panels). [0043] FIG. 15B provides scatter plots showing changes in populations of CD 19+ target cells (upper left quadrants) and CD3+ effector cells (lower right quadrants) at indicated time points for a Vd2+ effector population.

[0044] FIG. 16A shows the concentration of remaining target cells at indicated time points after co-culture and re-challenge of indicated engineered gamma delta T cell populations with target cells.

[0045] FIG. 16B shows the percentage of remaining CD 19+ target cells at indicated time points after co-culture and re-challenge of indicated engineered gamma delta T cell populations with target cells.

[0046] FIG. 17 shows levels of co-expression of TIM-3 and LAG-3 on populations of engineered gamma delta T cells after co-culture and re-challenge.

[0047] FIG. 18 shows percentages of memory phenotypes within the Vdl+, Vd2+, and Vdl-/Vd2- (shown as Vd3) subsets of a polyclonal population of engineered gamma delta T cells after three rounds of killing.

DETAILED DESCRIPTION

[0048] Provided herein are compositions and methods for improved generation of expanded populations of polyclonal gamma delta T cells suitable for clinical applications.

[0049] Gamma delta (y8, gd, or yd) T cells hold significant promise as an innovative approach for cancer therapy due to their unique features andpotential advantages, for example, over more common alpha beta (a or ab) T cell-based therapies. Several aspects contribute to the potential utility of y8 T cells in cancer treatment. Unlike ab T cells, which natively rely on antigen presentation via MHC/HLA molecules, gamma delta T cells can natively recognize stress-induced molecules expressed by tumor cells, including heat shock proteins, MICA, MICB, and ULBP ligands. Certain gamma delta T cell subsets can also recognize phosphoantigens and other non-peptide antigens overexpressed by transformed cells. This innate recognition enables gamma delta T cells to target a broad range of tumors without the need for specific tumor antigens or prior sensitization, or HLA-dependent activation cues.

[0050] Additionally, gamma delta T cells exhibit a broad spectrum of tumor reactivity, by recognizing a variety of tumor types including both hematological and solid tumors. This broad reactivity can be attributed to the diversity of gamma delta T cell subsets expressing different T cell receptor (TCR) variable delta (Vd or V8) and variable gamma (Vg, Vy, or Vy) chains, each with distinct tissue tropism and antigen specificities. [0051] Gamma delta T cells can recognize and kill cancer stem cells that are resistant to conventional therapies and are directly implicated in minimal residual disease and tumor recurrence. Moreover, gamma delta T cells possess potent cytotoxic capabilities - they are able of killing tumor cells through a diversity of mechanisms, including the release of cytotoxic molecules such as granzymes and perforin, through the engagement of death receptors (e.g., Fas/FasL pathway), and the production of pro-apoptotic cytokines (e.g., TNF-a, IFN-g).

[0052] Because gamma delta T cells can eliminate tumor cells independent of antigen processing and presentation, these cells are particularly effective against tumor cells with defects in antigen processing or MHC expression. Gamma Delta T cells are also an attractive candidate for cancer immunotherapy due to their natural propensity to reside in epithelia tissues and mucosal surfaces where many tumors originate. This tissue-resident population of gamma delta T cells can infiltrate tumors and exert local anti -tumor effects, contributing to tumor immune surveillance and containment. Strategies to enhance recruitment of infiltrating gamma delta T cells into tumors, such as cytokine stimulation or chemokine targeting, may further augment their therapeutic efficacy.

[0053] Gamma delta T cells can interact synergistically with other immune effector cells, including ab T cells, natural killer (NK) cells, dendritic cells, and macrophages, to enhance antitumor immune responses. For example, gamma delta T cells can promote dendritic cell maturation and antigen presentation leading to the activation of ab T cells and the generation of a long-lasting antitumor immunity. Additionally, gamma delta T cells can collaborate with NK cells to enhance tumor cell killing and cytokine production to further amplify the antitumor immune response.

[0054] Given the unique qualities of this subset of T cells, strategies aimed at harnessing and potentiating the antitumor activity of gamma delta T cells hold promise for the development of innovative and effective treatments for various malignancies. Gamma delta T cells can be attractive either as standalone therapies, with or without cell engineering (e.g., introduction of a chimeric antigen receptor), or in combination with existing treatment modalities such as chemotherapy and immune checkpoint blockade. However, their clinical application requires efficient ex vivo expansion protocols in order to generate therapeutically relevant numbers of functional gamma delta T cells. Protocols have been developed to efficiently expand and activate y8 T cells while maintaining their cytotoxic and functional properties, yet many challenges and limitations remain.

[0055] Stimulation with phosphoantigens (e.g., isopentenyl pyrophosphate, IPP) or aminobisphosphonates (e.g., zoledronic acid) activates gamma delta T cells via the TCR, leading to proliferation and effector differentiation. One of the limitations for these protocols is that phosphoantigens can specifically or preferentially activate a subset of y8 T cells that express specific Vy9V82 T cell receptors. This limits the applicability of phosphoantigens to a subset of y8 T cells, restricting their potential therapeutic efficacy and reducing the benefits associated with polyclonal gamma delta T cell populations. Gamma delta T cells are heterogeneous, comprising different subsets with diverse functional properties. Phosphoantigen-mediated expansion can preferentially amplify certain subsets over others, potentially impacting the overall therapeutic outcome. Additionally, the safety profile of phosphoantigens and expanded y8 T cells can require careful evaluation, particularly concerning potential autoimmune reactions or cytokine release syndrome. Prolonged activation of y8 T cells by phosphoantigens can, in some embodiments, induce T cell exhaustion, characterized by reduced effector function and proliferative capacity, ultimately compromising their ability to eliminate target cells.

[0056] Another expansion protocol applies co-culture with autologous or allogeneic antigen presenting cells (APCs), such as dendritic cells which provide additional co-stimulatory signals and enhance gamma delta T cell activation and expansion. While dendritic cells (DCs) are commonly used due to their potent antigen-presenting capacity, obtaining sufficient quantities of functional DCs for clinical applications canbe difficult and costly. Moreover, APC populations are heterogeneous, comprising various subsets with distinct phenotypic and functional properties. Selecting the most appropriate APC subset(s) for y8 T cell expansion and standardizing culture protocols across different APC populations can be challenging. The maturation and activation status of APCs can significantly impact their ability to stimulate y8 T cell expansion and function. A repeatable and optimal balance between APC maturation and activation to avoid premature exhaustion or overstimulation of y8 T cells can be difficult to achieve. Consequently, scaling up APC-based y8 T cell expansion for clinical applications presents logistical and manufacturing challenges. Standardizing production processes, ensuring quality control, and meeting regulatory requirements for clinical -grade cell therapy products can be important for successful translation into clinical practice.

[0057] Protocols disclosed herein provide solutions to many of the current challenges and limitations associated with expanding populations of gamma delta T cells in order to enhance expansion, improve manufacturing processes, and advance the clinical translation of potent and durable y8 T cell-based therapies. For example, in some embodiments expansion protocols disclosed herein can generate 2,000-5,000 fold expansion of polyclonal populations of engineered (e.g., transposon-engineered CAR-expressing) gamma delta T cells and greater than 10,000 fold expansion of unmodified gamma delta T cells. In some embodiments, populations of gamma delta T cells provided by methods disclosed herein can have advantageous functional properties, for example, associated with polyclonality of gdTCR subunit expression, lack of expression of exhaustion markers, and other functional attributes such as cancer cell killing, activation, expansion, cytokine production, and durable responses upon repeat exposure to target cells.

I. CELLS

[0058] Compositions and methods disclosed herein can comprise or utilize cells and/or populations thereof, for example, immune cells, such as T cells or gamma delta (y8, gd, or yd) T cells. A population of cells disclosed herein can comprise gamma delta T cells. Gamma delta T cells can be T cells (e.g., CD3+) that express a T cell receptor (TCR) comprising gamma and delta chains.

[0059] Compositions and methods disclosed herein can comprise or utilize one or more polyclonal populations of gamma delta T cells. A polyclonal population of gamma delta T cells can describe a diverse mixture of gamma delta T cells with various TCR gamma & delta variable subunits, specificities, and functional properties. The diversity and functional plasticity of a polyclonal population of gamma delta T cells can provide several advantages in terms of antigen recognition, immune responsiveness, and adaptability to the tumor microenvironment, making them promising cell populations for immunotherapy against cancer and other (e.g., autoimmune) diseases.

[0060] Polyclonal gamma delta T cells populations can collectively recognize a wider range of antigens which allows them to target various pathogens, cancer cells, and infected cells, thereby enhancing the versatility and efficacy of gamma delta T cell based immunotherapy. Additionally, different subsets of gamma delta T cells within a polyclonal population can exhibit distinct effector functions which allows for complementary immune responses against different types of tumor cells, improving the overall effectiveness of gamma delta mediated immune response. A heterogeneous population of gamma delta T cells with diverse TCR specificities also reduces the likelihood of immune escape by tumor cells through antigen loss or downregulation. Even if target antigens are no longer recognized by a subset of gamma delta T cells (or, e.g., expression of a CAR target antigen is lost), other clones within the polyclonal population can still exert immune surveillance and a potent cytotoxic response against target cells.

[0061] Interactions between subsets of gamma delta T cells and other immune cells such as aP T cells, NK cells, dendritic cell and macrophages can also promote a synergistic immune response. And given the dynamic and heterogenous complexity of the tumor microenvironment, a diverse polyclonal population of gamma delta T cells may exhibit greater adaptability allowing subsets with distinct functional properties to overcome immune suppression.

[0062] In some embodiments, utilizing a polyclonal gamma delta T cell population can reduce the risk of autoimmune responses compared to monoclonal therapies targeting specific antigens. In some embodiments, the presence of multiple TCR specificities can mean that gamma delta T cells are less likely to recognize self antigens and induce autoimmune reactions.

[0063] A polyclonal population of gamma delta T cells can exhibit advantageous properties over, for example, a population of gamma delta T cells with less diversity of TCR gamma/delta variable subunits.

[0064] A cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise any suitable combination of gamma and delta chains, “y” “gamma” “g” and “y” can be used interchangeably herein referring to gamma, for example, “Vy2” “Vg2” V gamma 2” and “Vy2) can all refer to the same gamma chain or gamma chain variable domain. “5” “delta” and “d” can all be used interchangeably herein to refer to delta, for example, “V81” “Vdl” and “V delta 1” can all refer to the same delta chain or delta chain variable domain.

[0065] A cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a TCR gamma chain, for example, with a Vy2, Vy3, Vy4, Vy5, Vy8, or Vy9 gamma chain variable domain. In some embodiments, the TCR gamma chain is a Vyl, Vy6, Vy7, VylO, Vyl 1, Vyl2, Vyl3, or Vyl4 gamma chain variable domain.

[0066] A cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a TCR delta chain, for example, with a V81, V82, or V83 delta chain variable domain. In some embodiments, the delta chain is a V84, V85, V86, V87, or V88 delta chain variable domain.

[0067] A cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a TCR comprising (a) a gamma chain comprising a Vy2, Vy3, Vy4, Vy5, Vy8, or Vy9 gamma chain variable domain, and (b) a delta chain comprising a V81, V82, or V33 delta chain variable domain.

[0068] A cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a TCR comprising (a) a gamma chain comprising a Vyl, Vy6, Vy7, VylO, Vyl l, Vyl2, Vyl3, Vyl4, Vy2, Vy3, Vy4, Vy5, Vy8, or Vy9 gamma chain variable domain, and (b) a delta chain comprising a V84, V85, V86, V87, V88, V81, V82, or V83 delta chain variable domain. [0069] In some embodiments, the y-chain comprises a Vy2 variable domain and the 8-chain comprises a V81 variable domain. In some embodiments, the y-chain comprises a Vy3 variable domain and the 8-chain comprises a V81 variable domain. In some embodiments, the y-chain comprises a Vy4 variable domain and the 8-chain comprises a V81 variable domain. In some embodiments, the y-chain comprises a Vy5 variable domain and the 8-chain comprises a V81 variable domain. In some embodiments, the y-chain comprises a Vy8 variable domain and the 8- chain comprises a V81 variable domain. In some embodiments, the y-chain comprises a Vy9 variable domain and the 8-chain comprises a V81 variable domain.

[0070] In some embodiments, the y-chain comprises a Vy2 variable domain and the 8-chain comprises a V82 variable domain. In some embodiments, the y-chain comprises a Vy3 variable domain and the 8-chain comprises a V82 variable domain. In some embodiments, the y-chain comprises a Vy4 variable domain and the 8-chain comprises a V82 variable domain. In some embodiments, the y-chain comprises a Vy5 variable domain and the 8-chain comprises a V82 variable domain. In some embodiments, the y-chain comprises a Vy8 variable domain and the 8- chain comprises a V82 variable domain. In some embodiments, the y-chain comprises a Vy9 variable domain and the 8-chain comprises a V82 variable domain.

[0071] In some embodiments, the y-chain comprises a Vy2 variable domain and the 8-chain comprises a V83 variable domain. In some embodiments, the y-chain comprises a Vy3 variable domain and the 8-chain comprises a V83 variable domain. In some embodiments, the y-chain comprises a Vy4 variable domain and the 8-chain comprises a V83 variable domain. In some embodiments, the y-chain comprises a Vy5 variable domain and the 8-chain comprises a V83 variable domain. In some embodiments, the y-chain comprises a Vy8 variable domain and the 8- chain comprises a V83 variable domain. In some embodiments, the y-chain comprises a Vy9 variable domain and the 8-chain comprises a V83 variable domain.

[0072] Methods disclosed herein can comprise enriching, selecting, and/or expanding gamma delta T cells from a heterogeneous population of cells in a starting material. Methods disclosed herein can comprise reducing the number or proportion of non-gamma delta T cells, for example, reducing the number or proportion of alpha beta T cells in a population.

[0073] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1 %, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of cells express a TCR gamma chain, for example, as determined by flow cytometry.

[0074] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1 %, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of cells express a TCR delta chain, for example, as determined by flow cytometry.

[0075] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of cells express a TCR gamma chain and a TCR delta chain, for example, as determined by flow cytometry.

[0076] In some embodiments, of a population of cells disclosed herein or a subpopulation thereof, at most about 1%, at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, or at most about 80% of the cells are not gamma delta T cells.

[0077] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at most about 1%, at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, or at most about 80% of the cells are alpha beta T cells.

[0078] In some embodiments, of a population of T cells disclosed herein, at most about 1%, at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, or at most about 80% of the cells are alpha beta T cells.

[0079] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1 %, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, or at least about 95% of cells express a TCR comprising a TCR gamma or delta chain variable domain disclosed herein, for example, Vyl, Vy2, Vy3, Vy4, Vy5, Vy6, Vy7, Vy8, Vy9, VylO, Vyl 1, Vyl2, Vyl3, Vyl4, V81, V82, V83, V84, V85, V86, V87, or V88 variable domain.

[0080] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1 %, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, or at least about 95% of cells express a TCR comprising Vyl, for example, as determined by flow cytometry.

[0081] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1 %, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, or at least about 95% of cells express a TCR comprising Vy2, for example, as determined by flow cytometry.

[0082] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, or at least about 95% of cells express a TCR comprising Vy3, for example, as determined by flow cytometry.

[0083] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1 %, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, or at least about 95% of cells express a TCR comprising Vy4, for example, as determined by flow cytometry.

[0084] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of cells express a TCR comprising Vy5, for example, as determined by flow cytometry.

[0085] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1 %, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, or at least about 95% of cells express a TCR comprising Vy8, for example, as determined by flow cytometry.

[0086] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, or at least about 95% of cells express a TCR comprising Vy9, for example, as determined by flow cytometry.

[0087] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, or at least about 95% of cells express a TCR comprising V51, for example, as determined by flow cytometry. [0088] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1 %, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, or at least about 95% of cells express a TCR comprising V82, for example, as determined by flow cytometry.

[0089] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 0.1%, atleast about 0.5%, at least about 1%, atleast about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, or at least about 95% of cells express a TCR comprising V83, for example, as determined by flow cytometry.

[0090] In some embodiments, of a population of gamma delta T cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) disclosed herein, at least about 0.1%, at least about 0.5%, atleast about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, or at least about 70% of cells are V81 negative and V82 negative, e.g., as determined by flow cytometry.

[0091] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1% of cells express a TCR comprising V81, and at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of cells express a TCR comprising V82, for example, as determined by flow cytometry.

[0092] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 5% of cells express a TCR comprising V81 , and at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of cells express a TCR comprising V82, for example, as determined by flow cytometry.

[0093] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 10% of cells express a TCR comprising V81, and at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% of cells express a TCR comprising V82, for example, as determined by flow cytometry.

[0094] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 20% of cells express a TCR comprising V81, and at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of cells express a TCR comprising V82, for example, as determined by flow cytometry.

[0095] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 50% of cells express a TCR comprising V81, and at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% of cells express a TCR comprising V82, for example, as determined by flow cytometry.

[0096] In some embodiments, a cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a TCR comprising a constant domain from a y-chain and/or a constant domain from a 8-chain. Constant domains can be indicated by a C preceding the y-chain and 8-chain designations, e.g., Cy2, Cy3, Cy4, Cy5, Cy8, Cy9, Cyl l, C81, C82, C83, and C85.

[0097] In some embodiments, a cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a Vy9V82 TCR or functional fragment thereof. [0098] In some embodiments, a cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a gdTCR capable of recognizing an EPCR protein on a cell surface of a target cell. In some embodiments, a cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a gdTCR capable of recognizing annexin A2 on a cell surface of a target cell. In some embodiments, a cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a gdTCR capable of recognizing aberrant HLA protein expression on a cell surface of a target cell. In some embodiments, a cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a gdTCR capable of recognizing cancers in an MHC/HLA-unrestricted manner.

[0099] In some embodiments, a cell, population of cells, or subpopulation comprises, consists essentially of, or consists of a clonal or monoclonal population of gamma delta T cells.

[0100] Cells can be selected or enriched for having or not having one or more given factors (e.g., cells may be separated based on the presence or absence ofone or more factors). Selection techniques include positive selection and negative selection techniques, e.g., fluorescent activated cell sorting (FACS) or magnetic activated cell sorting (MACS). In some cases, cells can be selected before engineering, for example, to enrich for a population of cells disclosed herein (e.g., immune cells, such as T cells or a T cell subset disclosed herein, such as gamma delta T cells or alpha beta T cells). The cells can also be selected independent of engineering, e.g., the cells are not subject to engineering modifications before or after sorting and other methods disclosed herein. Cells can be selected after engineering, for example, to enrich for a population of cells disclosed herein (e.g., engineered cells that express a polypeptide (e.g., CAR) or additional polypeptide). Engineered cells can be selected or enriched based on a tag or marker, such as an epitope tag. The tag or marker can be appended to the polypeptide (e.g., CAR). In some embodiments, the tag or marker is not appended to the polypeptide (e.g., CAR). The tag or marker can be co-expressed with the polypeptide (e.g., CAR) as disclosed herein. The tag or marker can comprise a reporter gene, such as a fluorescent protein. In some embodiments, no enrichment of gamma delta T cells is conducted (for example, other than activation with a gamma delta TCR stimulating agent disclosed herein).

[0101] Heterogeneous populations of cells (e.g., peripheral blood mononuclear cells (PBMCs) or leukapheresis products) can be sorted to enrich or select for gamma delta T cells, e.g., using magnetic and/or fluorescent activated cell sorting. Illustrative products and protocols that can be used include Miltenyi manual and CliniMACs purification of gamma delta T cells. In some embodiments, an anti-TCRap agent (e.g., antibody) is used to negatively select or reduce the proportion of alpha beta T cells in a population of cells. In some embodiments, an anti-CD14 agent (e.g., antibody) is used to negatively select or reduce the proportion of myeloid cells or monocytes in a population of cells. In some embodiments, an anti-CD19 agent (e.g., antibody) is used to negatively select or reduce the proportion of B cells in a population of cells. In some embodiments, an anti-gdTCR agent (e.g., antibody) is used to positively select or increase the proportion of gamma delta T cells in a population of cells. In some embodiments, a combination of agents is used to positively and/or negatively select, for example, an anti-TCRaP agent (e.g., antibody) can be used to negatively select or reduce the proportion of alpha beta T cells and an anti-CD14 agent (e.g., antibody) can be used to negatively select or reduce the proportion of myeloid cells or monocytes in a population of cells. The anti-TCRaP agent, anti-CD14, antiCD 19, or anti-gdTCR agent can be coupled to an agent suitable for negative or positive selection as appropriate, e.g., via direct or indirect magnetic binding, or fluorescent sorting.

[0102] In some embodiments, selected cells can be expanded ex vivo and/or in vitro before gene editing or delivery of a nucleic acid molecule, after gene editing or delivery of a nucleic acid molecule, before selection, after selection, before expansion, after expansion, or a combination thereof. In some embodiments, selected cells can be expanded ex vivo and/or in vitro before gene editing or delivery of a nucleic acid molecule. In some embodiments, selected cells can be expanded ex vivo and/or in vitro after gene editing or delivery of a nucleic acid molecule. In some embodiments, selected cells can be expanded ex vivo and/or in vitro before selection and/or enrichment. In some embodiments, selected cells can be expanded ex vivo and/or in vitro after selection and/or enrichment. In some embodiments, selected cells can be expanded ex vivo and/or in vitro before expansion. In some embodiments, selected cells can be expanded ex vivo and/or in vitro after expansion.

[0103] Cells can be selected, enriched, or expanded on the basis of being positive or negative for a given factor. In some embodiments, cells are selected, enriched, or expanded on the basis of being positive for two or more factors. In some embodiments, cells can be selected, enriched, or expanded on the basis of being positive for one or more factors, and negative for one or more factors.

[0104] In some embodiments, cells are rested between steps of a method disclosed herein, e.g. in basal or complete medium at 37°C and 5% CO₂, e.g., after thawing and before enriching gamma delta T cells, after enriching for gamma delta T cells and before stimulation, or after engineering and before expansion.

[0105] Cells and populations disclosed herein can be characterized by various assays to determine percentage viability, or demonstrate the number or frequency of cells of a given type, phenotype, population/subpopulation, etc. In some embodiments, a sample of cells from a population is stained with labelled antibodies and analyzed by flow cytometry. Illustrative markers stained for can include, but are not limited to, FVD, CD3, CD4, CD8, Vdl, Vd2, abTCR, CD19, CD14, CD16, CD25, CD27, CD28, CD56, CD45RA, CD45RO, CD62L, CD69, CD70, CD107a, gdTCR (e.g., any gdTCR, orgdTCR comprising a one or more variable regions disclosed herein), abTCR, LAG3, PD-1, TIM-3, TIGIT, and a heterologous immune receptor introduced into the cells (e.g., a CAR). In some embodiments, a sample of cells from a population is stained with labelled antibodies to assess polyclonal phenotype, e.g., to identify cells expressing Vdl, Vd2, or another gamma delta TCR chain or variable domain disclosed herein. In some embodiments, a sample of cells from a population is stained with labelled antibodies to assess purity of gamma delta T cells, a level of activation, or a level of contamination of non-gamma delta T cells in a population of cells. In some embodiments, a sample of cells from a population is stained with labelled antibodies to assess exhaustion, differentiation, and/or a functional phenotype. In some embodiments, a sample of cells from a population is stained with labelled antibodies and analyzed by flow cytometry at appropriate steps of a protocol, for example, after thawing, after gamma delta TCR enrichment, before or following a first stimulation (e.g., about day 0-2), during an expansion incubation (e.g., about day 7 or 8), before second stimulation (e.g., about day 12) and/or at harvest (e.g., about day 22- 23).

[0106] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof disclosed herein retains cytotoxic capacity (for example, an ability to kill target cells in a coincubation) after 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, atleast2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 rounds of re-challenge.

[0107] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof disclosed herein exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold increased expansion compared to a control population of cells generated by a control method, or compared to a control population of cells that is not polyclonal or is less polyclonal.

[0108] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof disclosed herein exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold increased cytotoxicity compared to a control population of cells generated by a control method, or compared to a control population of cells that is not polyclonal or is less polyclonal.

[0109] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof disclosed herein exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold increased survival or persistence compared to a control population of cells generated by a control method, or compared to a control population of cells that is not polyclonal or is less polyclonal.

[0110] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof disclosed herein exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold increased proliferation in response to target cells compared to a control population of cells generated by a control method, or compared to a control population of cells that is not polyclonal or is less polyclonal.

[oni] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof disclosed herein exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold increased effector function (e.g., cytolytic activity or cytokine production) compared to a control population of cells generated by a control method, or compared to a control population of cells that is not polyclonal or is less polyclonal. [0112] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof disclosed herein exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold reduced exhaustion compared to a control population of cells generated by a control method, or compared to a control population of cells that is not polyclonal or less polyclonal. The exhaustion can be a percentage of the cells co-expressing, e.g., two, three, or more markers of exhaustion, such as after several rounds of rechallenge.

[0113] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof disclosed herein exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold increased expression of a memory phenotype, or retention of a memory phenotype, compared to a control population of cells generated by a control method. In some embodiments, of the retention in memory phenotype is after several (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20) rounds of rechallenge, e.g., in co-culture assays with target cells.

[0114] A cell in a composition or method disclosed herein can be a mammalian cell. A cell in a composition or method disclosed herein can be a human cell. A cell in a composition or method disclosed herein can be a non-rodent cell. A cell in a composition or method disclosed herein can be a primate cell, e.g., human or non-human primate. In some cases, a cell is a primary cell. In some cases, a cell is not a primary cell. A cell can be a therapeutic cell, for example, suitable for use in a therapeutic application in a suitable subject, such as a human.

[0115] A population of cells disclosed herein can comprise, consist essentially of, or consist of mammalian cells. A population of cells disclosed herein can comprise, consist essentially of, or consist of human cells. A population of cells disclosed herein can comprise, consist essentially of, or consist of non-rodent cells. A population of cells disclosed herein can comprise, consist essentially of, or consist of primate cells, e.g., human or non-human primate cells. A population of cells disclosed herein can comprise, consist essentially of, or consist of primary cells, or in some embodiments, non-primary cells (e.g., a cell line). A population of cells can be a population of therapeutic cells, for example, suitable for use in a therapeutic application in s suitable subject, such as a human. A population of therapeutic cells can comprise any cell type(s) or combinations thereof disclosed herein, and in some embodiments comprises a population of polyclonal gamma delta T cells (e.g., engineered to express a CAR/transgene, or non-engineered).

[0116] A cell in a composition, population, or method disclosed herein can be an immune cell. A cell in a composition or method disclosed herein can be a lymphocyte, T cell, alpha-beta T cell, gamma-delta T cell, CD4+ T cell, CD8+ T cell, a T effector cell, naive T cell, memory T cell (e.g., central memory, effector memory, or resident memory), lymphoid cell, innate lymphoid cell (ILC), a regulatory T-cell, a thymocyte, or any mixture or combination of cells thereof.

[0117] In some embodiments, a cell in a composition or method disclosed herein, or in a population of cells disclosed herein, comprises a dendritic cell, an eosinophil, a granulocyte, a Langerhans cell, a macrophage, a neutrophil, a mast cell, a megakaryocyte, a monocyte, a myeloid cell, a plasma cell, B cell, an NK cell, an NKT cell or any mixture or combination of cells thereof.

[0118] In some embodiments, a cell in a composition or method disclosed herein, or in a population of cells disclosed herein, comprises a precursor of an immune cell. In some embodiments, a cell in a composition or method disclosed herein, or in a population of cells disclosed herein, comprises or is derived from a stem cell, e.g., an iPSC or hematopoietic stem cell.

[0119] A cell in a composition, population, or method disclosed herein can be engineered, for example, modified to comprise a transgene, expression construct, nucleotide, and/or genomic alteration (e.g., insertion, deletion, knockout, translocation) as compared to a native cell. In some embodiments, a cell in a composition, population, or method disclosed herein can be nonengineered, for example, lack an artificially introduced transgene, expression construct, nucleotide, and/or genomic alteration, or can be non-genetically or genomically modified.

[0120] In some embodiments, an engineered cell comprises a disruption or deletion of one or more TCR-encoding genes, such as TRAC, TRB, TRG, and/or TRD. In some embodiments, an engineered cell comprises a disruption or deletion of a variable region of one or more TCR- encoding genes, such as a disruption or deletion in TRAC, TRB, TRG, and/or TRD.

[0121] In some embodiments, an engineered cell comprises a disruption or deletion of TRAC and/or TRB, and comprises an exogenously introduced gamma delta TCR and/or CAR. II. CULTURE & EXPANSION CONDITIONS

[0122] Culture conditions disclosed herein can contribute to advantageous aspects of cell expansion protocols, for example, the ability to generate expanded populations of polyclonal gamma delta T cells with desirable functional properties at a large scale.

[0123] Compositions and methods disclosed herein can utilize suitable culture vessels, for example, at one or more steps of a method disclosed herein. In some embodiments, standard tissue culture vessels are used, for example, 48 well, 24 well, 12 well, or 6 well tissue culture plates, T25, T75, T182, or T300 tissue culture flasks. In some embodiments, standard tissue culture vessels (e.g., plates or flasks) are used for a first and/or second stimulation step of a method, e.g., in which the cells are incubated in complete medium with anti-gamma delta TCR antibody, anti-CD28 antibody, and/or anti-CD3 antibody.

[0124] In some embodiments, a step of a method disclosed herein (e.g., a stimulation step) comprises incubating the population of cells in medium (e.g., an expansion culture medium) at a volume per square cm of the tissue culture well/flask surface area that facilitates improved T cell activation and viability, e.g., based on improved gas exchange. The surface area can be the surface area of the lower/inferior surface of the vessel (e.g., well, flask) that the cells rest on. In some embodiments, the step (e.g., stimulation step) comprises incubating the population of cells in medium (e.g., an expansion culture medium) at a volume of 300-400 pL per square cm of the tissue culture well/flask surface area. In some embodiments, the step (e.g., stimulation step) comprises incubating the population of cells in medium (e.g., an expansion culture medium) at a volume of at least 150, at least 200, at least 250, at least 300, at least 350, or at least 400 pL per square cm of the tissue culture well/flask surface area. In some embodiments, the step (e.g., stimulation step) comprises incubating the population of cells in medium (e.g., an expansion culture medium) at a volume of at most 700, at most 600, at most 550, at most 500, at most 450, at most 400, at most 350, or at most 300 pL per square cm of the tissue culture well/flask surface area. In some embodiments, the step (e.g., stimulation step) comprises incubating the population of cells in medium (e.g., an expansion culture medium) at a volume of 100-500, 100-400, 100- 300, 200-500, 200-400, 200-300, 300-500, or 300-400 pL per square cm of the tissue culture well/flask surface area.

[0125] Standard tissue culture vessels (e.g., plates or flasks) can be used for a stimulation step of a cell expansion method disclosed herein. The stimulation step can comprise seeding a population of cells (e.g., a population of gamma delta T cells, or a population of cells comprising a population of gamma delta T cells) at a concentration of about 500-800 cells/cm2 (per square centimeter), about 500-800 gamma delta T cells/cm2, about 5 x!0^A5 to 8 x!0^A5 cells/cm2, about 5 xlO^A5 to 8 xlO^A5 gamma delta T cells/cm2, about 5 xlO^A7 to 8 xlO^A7 cells/cm2, or about 5 xlO^A7 to 8 xlO^A7 gamma delta T cells/cm2.

[0126] In some embodiments, rather than standard tissue culture vessels (e.g., plates or flasks), culture vessels are used that comprise a gas permeable membrane submerged under a column of medium, for example, at one or more steps of a method disclosed herein, such as an expansion step. Exemplary culture vessels that utilize a gas permeable membrane submerged under a column of medium include Gas permeable Rapid Expansion (GREX®) cell culture vessels. Culture vessels that comprise a gas permeable membrane submerged under a column of medium can be designed to utilize convection to provide access to nutrients, allowing a greater height of liquid culture medium than in standard tissue culture vessels, and providing improved access to oxygen and nutrients. Culture vessels that utilize a gas permeable membrane submerged under a column of medium are described in Bajgain et al. "Optimizing the production of suspension cells using the G-Rex “M” series." Molecular Therapy Methods & Clinical Development 1 (2014); Gotti et al. "Optimization of therapeutic T cell expansion in G- Rex device and applicability to large-scale production for clinical use." Cytotherapy 24.3 (2022): 334-343; Palmerini et al. "A serum-free protocol for the ex vivo expansion of Cytokine- Induced Killer cells using gas-permeable static culture flasks." Cytotherapy 22.9 (2020): 511- 518; Xiao et al. "Large-scale expansion of Vy9V82 T cells with engineered K562 feeder cells in G-Rex vessels and their use as chimeric antigen receptor-modified effector cells." Cytotherapy 20.3 (2018): 420-435; and Forget et al. "The beneficial effects of a gas-permeable flask for expansion of Tumor-Infiltrating lymphocytes as reflected in their mitochondrial function and respiration capacity." Oncoimmunology 5.2 (2016): el057386; each of which is incorporated herein by reference in its entirety.

[0127] Illustrative capacities for expansion of populations of gamma delta T cells in culture vessels that utilize a gas permeable membrane submerged under a column of medium (e.g.,

GREX®) include those provided in TABLE 1.

[0128] The type of tissue culture vessel used at a given step of a method can be determined based on a number of cells, e.g., gamma delta T cells. In some embodiments, standard tissue culture plates are used for the initial stimulation and/or expansion phases of a method if less than about 2 x 10^A6 viable gamma delta T cells are seeded or available. In some embodiments, standard tissue culture plates are used for the initial stimulation and/or expansion phases of a method until about 2 x 10^A6 viable gamma delta T cells are seeded or available. In some embodiments, culture vessels that comprise a gas permeable membrane submerged under a column of medium (e.g., GREX) are used for an early expansion phase of a method (e.g., following an initial two day stimulation and/or engineering/electroporation) if more than about 2 x 10^A6 viable gamma delta T cells are seeded or available. In some embodiments, cells cultured in culture vessels that comprise a gas permeable membrane submerged under a column of medium (e.g., GREX) directly following an initial two day stimulation and/or electroporation (e.g., on day 2), are not mixed, sampled, or expanded until day 7 or day 8, however media can be doubled on day 5 and cells are mixed and split on day 7 or 8 with media doubled again.

[0129] Cells can be seeded in cell/tissue culture vessels at an appropriate density per surface area (e.g., of the lower/inferior surface of the vessel that the cells settle on or rest on). In some embodiments, an appropriate density of cells per surface area contributes to advantageous aspects of cell expansion protocols, for example, the ability to generate expanded populations of polyclonal gamma delta T cells with desirable functional properties at a large scale. For example, in some embodiments cells are seeded at a density per surface area and/or per volume that is contrary to conventional teachings, industry standards, or manufacturer’s recommended protocols, but surprisingly, the higher initial density contributes to superior results (e.g., improved fold expansion and/of functional attributes) of resulting expanded populations of gamma delta T cells.

[0130] In some embodiments, during a step of a method disclosed herein, cells (e.g., gamma delta T cells, or all cells present) are seeded or cultured at a density of at least 5 x10^A4, at least 1 xl0^A5, atleast 2 xl0^A5, atleast 3 xl0^A5, at least 4 xl0^A5, at least 5 xl0^A5, at least 6 xl0^A5, at least 7 xl0^A5, at least 8 xl0^A5, at least 9 xl0^A5, at least 1 xlO^A6, at least 2 xlO^A6, at least 3 xlO^A6, at least 4 xlO^A6, atleast 5 xlO^A6, at least 6 xlO^A6, at least 7 xlO^A6, at least 8 xlO^A6, at least 9 xlO^A6, at least 1 xlO^A7, at least 2 xlO^A7, at least 3 xlO^A7, at least 4 xlO^A7, at least 5 xlO^A7, atleast 6 xlO^A7, atleast 7 xlO^A7, at least 8 xlO^A7, at least 9 xlO^A7, at least 1 xl0^A8, at least2 xl0^A8, atleast 5 xl0^A8, or atleast 6 xl0^A8 cells per square centimeter. The step can be, for example, a recovery, stimulation, expansion (e.g., first expansion or second expansion), or restimulation (e.g., first stimulation or second stimulation) step. In some embodiments, if gd T cells are seeded at a density of less than about 1 xlO^A6 cells/cm2 or 8 xlO^A5/cm2, they will crash and will not reach >800 fold expansion. [0131] In some embodiments, during a step of a method disclosed herein, cells (e.g., gamma delta T cells, or all cells) are seeded or cultured ata density of at most 3 xl0^A5, at most 4 xl0^A5, at most 5 xl0^A5, at most 6 xl0^A5, at most 7 xl0^A5, at most 8 xl0^A5, at most 9 xl0^A5, at most 1 xlO^A6, at most 2 xlO^A6, at most 3 xlO^A6, at most 4 xlO^A6, at most 5 xlO^A6, at most 6 xlO^A6, at most 7 x10^A6, at most 8 x10^A6, at most 9 x10^A6, at most 1 x10^A7, at most 2 x10^A7, at most 3 xlO^A7, at most 4 xlO^A7, at most 5 xlO^A7, at most 6 xlO^A7, at most 7 xlO^A7, at most 8 xlO^A7, at most 9 xlO^A7, at most 1 xl0^A8, at most 2 xl0^A8, at most 5 xl0^A8, at most 6 xl0^A8, or at most 1 xlO^A9 cells per square centimeter. The step can be, for example, a recovery, stimulation, expansion (e.g., first expansion or second expansion), or restimulation (e.g., first stimulation or second stimulation) step.

[0132] In some embodiments, during a step of a method disclosed herein, cells (e.g., gamma delta T cells, or all cells) are seeded or cultured at a density of about 3 xl0^A5, about 4 xl0^A5, about 5 xl0^A5, about 6 xl0^A5, about 7 xl0^A5, about 8 xl0^A5, about 9 xl0^A5, about 1 xlO^A6, about2 xlO^A6, about 3 xlO^A6, about 4 xlO^A6, about 5 xlO^A6, about 6 xlO^A6, about 7 xlO^A6, about 8 xlO^A6, about 9 xlO^A6, about 1 xlO^A7, about 2 xlO^A7, about 3 xlO^A7, about 4 xlO^A7, about 5 xlO^A7, about 6 xlO^A7, about 7 xlO^A7, about 8 xlO^A7, about 9 xlO^A7, about 1 xl0^A8, about 2 xl0^A8, about 5 xl0^A8, about 6 xl0^A8, or about 1 xlO^A9 cells per square centimeter. The step can be, for example, a recovery, stimulation, expansion (e.g., first expansion or second expansion), or restimulation (e.g., first stimulation or second stimulation) step.

[0133] In some embodiments, during a step of a method disclosed herein, cells (e.g., gamma delta T cells, or all cells) are seeded or cultured at a density ofabout 3 xl0^A5 to about 1 xlO^A9, about 3 xl0^A5 to about 1 xl0^A8, about 3 xl0^A5 to about 8 xlO^A7, about 3 xl0^A5 to about 5 xlO^A7, about 3 xl0^A5 to about 1 xlO^A7, about 3 xl0^A5 to about 5 xlO^A6, about 3 xl0^A5 to about 3 xlO^A6, about 3 xl0^A5 to about 2 xlO^A6, about 3 xl0^A5 to about 1 xlO^A6, about 3 xl0^A5 to about 1.5 xlO^A6, about 3 xl0^A5 to about 5 xl0^A5, about 5 xl0^A5 to about 1 xlO^A9, about 5 xl0^A5 to about 1 xl0^A8, about 5 xl0^A5 to about 8 xlO^A7, about 5 xl0^A5 to about 5 xlO^A7, about 5 xl0^A5 to about 1 xlO^A7, about 5 xl0^A5 to about 5 xlO^A6, about 5 xl0^A5 to about 3 xlO^A6, about 5 xl0^A5 to about 2 xlO^A6, about 5 xl0^A5 to about 1 xlO^A6, about 5 xl0^A5 to about 1.5 xlO^A6, about 5 xl0^A5 to about 8 xl0^A5, about 1 xlO^A6to about 1 xlO^A9, about 1 xlO^A6 to about 1 xl0^A8, about 1 xlO^A6 to about 8 xlO^A7, about 1 xlO^A6 to about 5 xlO^A7, about 1 xlO^A6 to about 1 xlO^A7, about 1 xlO^A6to about 5 xlO^A6, about 1 xlO^A6 to about3 xlO^A6, about 1 xlO^A6 to about 2 xlO^A6, 1 xlO^A7 to about 1 xlO^A9, 1 xlO^A7 to about 1 xl0^A8, 1 xlO^A7 to about 8 xlO^A7, 5 xlO^A7 to about 1 xlO^A9, 5 xlO^A7 to about 1 xl0^A8, or 5 xlO^A7 to about 8 xlO^A7 cells per square centimeter. The step can be, for example, a recovery, stimulation, expansion (e.g., first expansion or second expansion), or restimulation (e.g., first stimulation or second stimulation) step.

[0134] In some embodiments, during a step of a method disclosed herein, cells (e.g., gamma delta T cells, or all cells) are seeded or cultured at a density of about 1 xl0^A5 to about 5 xlO^A6, about 1 xl0^A5 to about 1 xlO^A6, about 1 xl0^A5 to about 6 xl0^A5, about 3 xl0^A5 to about 5 xlO^A6, about 3 xl0^A5 to about 1 xlO^A6, or about 3 xl0^A5 to about 6 xl0^A5 cells per square centimeter. The step can be, for example, an expansion step (e.g., first incubation or second incubation) in a tissue culture plate or flask.

[0135] In some embodiments, during a step of a method disclosed herein, cells (e.g., gamma delta T cells, or all cells) are seeded or cultured at a density of about 1 xl0^A5 to about 1 xlO^A7, about 1 xl0^A5 to about 5 xlO^A6, about 1 xl0^A5 to about 3 xlO^A6, about 1 xl0^A5 to about 2 xlO^A6, about 1 xl0^A5 to about 1.5 xl O^A6, about 5 xl0^A5 to about 1 xlO^A7, about 5 xl0^A5 to about 5 xlO^A6, about 5 xl0^A5 to about 3 xlO^A6, about 5 xl0^A5 to about 2 xlO^A6, about 5 xl0^A5 to about 1.5 xlO^A6, about 5 xl0^A5 to about 8 xl 0^A5, about 1 xlO^A6 to about 1 xlO^A7, about 1 xlO^A6 to about 5 xlO^A6, about 1 xlO^A6 to about 3 xlO^A6, about 1 xlO^A6 to about 2 xlO^A6, or about 1 xlO^A6 to about 1.5 xlO^A6 cells per square centimeter. The step can be, for example, a recovery or expansion step (e.g., first expansion or second expansion) in a culture vessel comprising a gas permeable membrane submerged under a column of medium (e.g., GREX culture vessel).

[0136] In some embodiments, during a step of a method disclosed herein, cells (e.g., gamma delta T cells, or all cells) are seeded or cultured at a density of about 5 xl0^A5 to about 3 xlO^A6, about 5 xl0^A5 to about 2 xlO^A6, about 5 xl0^A5 to about 1.5 xlO^A6, about 5 xl0^A5 to about 8 xl0^A5, 1 xlO^A6 to about 1 xl0^A8, about 1 xlO^A7 to about 1 xl0^A8, about 3 xlO^A7 to about 1 xl0^A8, about 5 xlO^A7 to about 1 xl0^A8, about 1 xlO^A6to about 8 xlO^A7, about 1 xlO^A7 to about 8 xlO^A7, about 3 xlO^A7 to about 8 xlO^A7, about 5 xlO^A7 to about 8 xlO^A7 cells per square centimeter. The step can be, for example, a stimulation step (e.g., first or second stimulation step) in which the cells are activated with, e.g., an anti-gdTCR agent, an anti-CD3 agent, an anti- CD28 agent, or a combination thereof.

[0137] Cells can be seeded in cell/tissue culture vessels at an appropriate density per volume of medium. In some embodiments, an appropriate density of cells per volume of medium contributes to advantageous aspects of cell expansion protocols, for example, the ability to generate expanded populations of polyclonal gamma delta T cells with desirable functional properties at a large scale. [0138] In some embodiments, during a step of a method disclosed herein, cells (e.g., gamma delta T cells, or all cells) are seeded or cultured at a density of at least 1 xl0^A5, at least 5 xl0^A5, at least ? xl0^A5, atleast 8 xl0^A5, at least 9 xl0^A5, atleast 1 xlO^A6, atleast 1.5 xlO^A6, or at least 2 xl O^A6, cells per mL. The step can be, for example, a recovery (e.g., after electroporation), stimulation (e.g., first or second stimulation,) or expansion (e.g., first or second expansion) step. In some embodiments, the cells are in a culture vessel comprising a gas permeable membrane submerged under a column of medium (e.g., GREX culture vessel).

[0139] In some embodiments, during a step of a method disclosed herein, cells (e.g., gamma delta T cells, or all cells) are seeded or cultured at a density of at most 1 xlO^A6, at most 1.5 xlO^A6, at most 2 xlO^A6, at most 3 xlO^A6, at most 5 xlO^A6, at most 5 xlO^A6, or at most 1 xlO^A7 cells per mL. The step can be, for example, a recovery (e.g., after electroporation), stimulation (e.g., first or second stimulation,) or expansion (e.g., first or second expansion) step. In some embodiments, the cells are in a culture vessel comprising a gas permeable membrane submerged under a column of medium (e.g., GREX culture vessel).

[0140] In some embodiments, during a step of a method disclosed herein, cells (e.g., gamma delta T cells, or all cells) are seeded or cultured at a density of about 1 xl0^A5, about 5 xl0^A5, about 7 xl0^A5, about 8 xl0^A5, about 9 xl0^A5, about 1 xlO^A6, about 1.5 xlO^A6, about 2 xlO^A6, about 3 xlO^A6, about4 xl O^A6, or about 5 xlO^A6 cells per mL. The step can be, for example, a recovery (e.g., after electroporation), stimulation (e.g., first or second stimulation,) or expansion (e.g., first or second expansion) step. In some embodiments, the cells are in a culture vessel comprising a gas permeable membrane submerged under a column of medium (e.g., GREX culture vessel).

[0141] In some embodiments, during a step of a method disclosed herein, cells (e.g., gamma delta T cells, or all cells) are seeded or cultured at a density of about 1 xl0^A5 to about 1 xlO^A7, about 1 xl0^A5 to about 5 xlO^A6, about 1 xl0^A5 to about 3 xlO^A6, about 1 xl0^A5 to about 2 xlO^A6, about 1 xl0^A5 to about 1.5 xlO^A6, about 5 xl0^A5 to about 1 xlO^A7, about 5 xl0^A5 to about 5 xlO^A6, about 5 xl 0^A5 to about 3 xlO^A6, about 5 xl0^A5 to about 2 xlO^A6, about 5 xl0^A5 to about 1.5 xlO^A6, about 1 xlO^A6 to about 1 xlO^A7, about 1 xlO^A6 to about 5 xlO^A6, about 1 xlO^A6 to about 3 xlO^A6, about 1 xlO^A6to about 2 xlO^A6, or about 1 xlO^A6 to about 1.5 xlO^A6 cells permL. The step can be, for example, a recovery (e.g., after electroporation), stimulation (e.g., first or second stimulation,) or expansion (e.g., first or second expansion) step. In some embodiments, the cells are in a culture vessel comprising a gas permeable membrane submerged under a column of medium (e.g., GREX culture vessel). [0142] Compositions and methods disclosed herein can comprise an expansion culture medium that can be suitable for expanding a population of therapeutic cells, for example, gamma delta T cells or polyclonal gamma delta T cells.

[0143] An expansion culture medium can comprise a suitable basal component for culturing T cells, for example, OpTimizer T-cell expansion Basal Medium, TheraPEAK T-Vivo, AIM-V, X-VIVO15, TexMACS, or RPMI.

[0144] An expansion culture medium can comprise one or more antibiotics, for example, penicillin and streptomycin. In some embodiments, an expansion culture medium used in one or more steps of a method disclosed herein does not contain antibiotics, for example, lacks penicillin and streptomycin.

[0145] An expansion culture medium can comprise one or more cell culture supplements, for example, L-glutamine, Glutamax, non-essential amino acids, HEPES, 2-mercaptoethanol, Sodium Bicarbonate (NaHCO3), trace elements, vitamins, inorganic salts, and the like.

[0146] An expansion culture medium can comprise serum, for example, human AB seium or fetal bovine serum. In some embodiments an expansion culture medium is serum-free, for example, comprises a serum substitute such as Physiologix™ Cell-Vive™ CTS™ Immune Cell SR, Proliferum LSR, or other available serum substitutes. In some embodiments an expansion culture medium is a xeno-free formulation.

[0147] An expansion culture medium can be chemically defined, non-animal origin (NAO), and/or serum-free.

[0148] An expansion culture medium can comprise one or more cytokines or growth factors, for example, interleukin 2 (IL-2), interleukin 7 (IL-7), and/or interleukin 15 (IL-15). In some embodiments, an expansion culture medium comprises IL-2. In some embodiments, an expansion culture medium comprises IL-7. In some embodiments, an expansion culture medium comprises IL-15. In some embodiments, an expansion culture medium comprises IL-2 and IL-7. In some embodiments, an expansion culture medium comprises IL-2 and IL-15. In some embodiments, an expansion culture medium comprises IL-7 and IL-15. In some embodiments, an expansion culture medium comprises IL-2, IL-7, and IL-15. In some embodiments, an expansion culture medium lacks IL-2, IL-7, and/or IL-15.

[0149] Treating cells with appropriate concentrations of cytokines at suitable intervals can contribute to advantageous aspects of cell expansion protocols, for example, the ability to generate expanded populations of polyclonal gamma delta T cells with desirable functional properties at a large scale. Concentrations of cytokines added to populations of cells as disclosed herein can be calculated accounting for the full final volume of medium after a given step. For example, when feeding cells it can be common to replace half the volume of “spent” media with fresh media, e.g., every two days. If a protocol calls for a maintaining or treating cells with IL-2 at a concentration of 1000 international units (IU)/mL, it can be common to add IL-2 to the fresh media at a concentration of 1000 lU/mL, such that the final concentration is about 500 lU/mL once a 1 :1 ratio of fresh media is added to spent media. Methods disclosed herein can comprise adding a sufficient quantity of cytokines (e.g., each time the cells are fed or each time cytokines are added) based on a calculation of the final volume rather than a calculation of the fresh media volume. For example, in the above example 2000 lU/mL (2X the final desired concentration) of IL-2 can be included in fresh media added 1 : 1 to spent media, such that the final concentration is 1000 lU/mL. In some embodiments, concentrations of cytokines added to populations of cells are calculated accounting for the full final volume of medium when the cells are cultured in a culture vessel comprising a gas permeable membrane submerged under a column of medium (e.g., GREX culture vessel). In some embodiments, concentrations of cytokines added to populations of cells are calculated accounting for the full final volume of medium starting day 7 or day 8 of a protocol disclosed herein.

[0150] In some embodiments, expansion culture medium comprises IL-2. The expansion culture medium can comprise IL-2 at a concentration of 1000 lU/mL. The expansion culture medium can comprise IL-2 at a final concentration of at least 1 , at least 10, at least 100, at least 200, at least 300, atleast400, atleast 500, at least 600, at least 700, at least 800, at least 900, at least 950, at least 1000, at least 1250, at least 1500, or at least 2000 lU/mL. In some embodiments, the expansion culture medium comprises IL-2 at a final concentration of at most 500, at most 600, at most 700, at most 800, at most 900, at most 950, at most 1000, at most 1250, at most 1500, at most 2000, at most 3000, at most 5000, or at most 10,000 lU/mL. In some embodiments, the expansion culture medium comprises the IL-2 at a final concentration of about 10-5000, 10-1000, 100-5000, 100-3000, 100-2500, 100-2000, 100-1500, 100-1000, 500- 5000, 500-3000, 500-2500, 500-2000, 500-1500, 500-1000, 750-5000, 750-3000, 750-2500, 750-2000, 750-1500, 750-1000, 750-1250 lU/mL.

[0151] The IL-2 can be freshly added at a suitable frequency, for example, every 1, 2, 3, 4, or 5 days. In some embodiments the IL-2 is freshly added every 2 days. In some embodiments the IL-2 is freshly added every 3 days. In some embodiments the IL-2 is freshly added every 2-3 days. The IL-2 can be added to achieve the final concentration based on a calculation accounting for the relative volume of the fresh media and the spent media as disclosed herein.

[0152] In some embodiments, expansion culture medium comprises IL-7. The expansion culture medium can comprise IL-7 at a concentration of about 5 ng/mL. The expansion culture medium can comprise IL-7 at a final concentration of at least 0.5, at least 1, at least 2.5, at least 5, at least 7.5, or at least 10 ng/mL. In some embodiments, the expansion culture medium comprises IL-7 at a final concentration of at most 1, at most 2.5, at most 5, at most 7.5, at most 10, at most 25, or at most 50 ng/mL. In some embodiments, the expansion culture medium comprises the IL-7 at a final concentration of about 0.5-50, 1-50, 2.5-50, 5-50, 0.5-25, 1-25, 2.5- 25, 5-25, 0.5-10, 1-10, 2.5-10, 5-10, 0.5-7.5, 1-7.5, 2.5-7.5, 5-7.5, 0.5-5, 1-5, or 2.5-5 ng/mL.

[0153] The IL-7 can be freshly added at a suitable frequency, for example, every 1, 2, 3, 4, or 5 days. In some embodiments the IL-7 is freshly added every 2 days. In some embodiments the IL-7 is freshly added every 3 days. In some embodiments the IL-7 is freshly added every 2-3 days. The IL-7 can be added to achieve the final concentration based on a calculation accounting for the relative volume of the fresh media and the spent media as disclosed herein.

[0154] In some embodiments, expansion culture medium comprises IL-15. The expansion culture medium can comprise IL-15 at a concentration of about 5 ng/mL. The expansion culture medium can comprise IL-15 at a final concentration of at least 0.5, at least 1, at least 2.5, at least 5, at least 7.5, or at least 10 ng/mL. In some embodiments, the expansion culture medium comprises IL-15 at a final concentration of atmost 1, atmost2.5, at most 5, atmost 7.5, at most 10, at most 25, or at most 50 ng/mL. In some embodiments, the expansion culture medium comprises the IL- 15 at a final concentration of about 0.5-50, 1-50, 2.5-50, 5-50, 0.5-25, 1-25, 2.5-25, 5-25, 0.5-10, 1-10, 2.5-10, 5-10, 0.5-15.5, 1-15.5, 2.5-15.5, 5-15.5, 0.5-5, 1-5, or 2.5-5 ng/mL.

[0155] The IL- 15 can be freshly added at a suitable frequency, for example, every 1, 2, 3, 4, or 5 days. In some embodiments the IL-15 is freshly added every 2 days. In some embodiments the IL-15 is freshly added every 3 days. In some embodiments the IL-15 is freshly added every 2-3 days. The IL-15 can be added to achieve the final concentration based on a calculation accounting for the relative volume of the fresh media and the spent media as disclosed herein.

[0156] An expansion culture medium can comprise one or more T cell and/or gamma delta T cell stimulating agents, for example, a gamma delta T cell receptor (gdTCR) stimulating agent, a CD3 stimulating agent, or a CD28 stimulating agent. The one or more T cell and/or gamma delta T cell stimulating agents can be coated to a surface of a culture vessel, and/or not coated onto the surface of the culture vessel. In some embodiments, one or more of the T cell and/or gamma delta T cell stimulating agents is coated to a surface of a culture vessel, and one or more of the T cell and/or gamma delta T cell stimulating agents is not coated to a surface of a culture vessel, e.g., is present in liquid medium in a free or soluble form. In some embodiments, one or more of the T cell and/or gamma delta T cell stimulating agents is coated to a surface of a bead, for example, a microbead or nanobead. In some embodiments, antigen-specific activation is used to activate engineered gamma delta T cells, for example, with an antigen recognized via a CAR.

[0157] A cell culture vessel (e.g., tissue culture plate of flask used in a stimulation step) can be pre-coated with one or more plate-bound T cell and/or gamma delta T cell stimulating agents, for example, a gamma delta T cell receptor (gdTCR) stimulating agent, a CD3 stimulating agent, or a CD28 stimulating agent.

[0158] In some embodiments, the expansion culture medium comprises and/or the cell culture vessel is coated with a gamma delta T cell receptor (gdTCR) stimulating agent, for example, a Vdl -specific antibody, and/or a pan-gdTCR stimulating agent, such as a gdTCR stimulating antibody or a pan-gdTCR stimulating antibody. A gdTCR stimulating agent can specifically or preferentially induce signaling by a gamma delta TCR (e.g., complex), for example, compared to an alpha beta TCR. In some embodiments, the gdTCR stimulating agent is a pan-gdTCR that stimulates gdTCRs that comprise multiple gamma chain variable domains and/or delta chain variable domains. In some embodiments, the gdTCR stimulating agent stimulates gdTCRs that comprise one or more gamma chain variable domains and/or delta chain variable domains disclosed herein. In some embodiments, the gdTCR stimulating agent stimulates gdTCRs that comprise or consist of one gamma chain variable domain disclosed herein. In some embodiments, the gdTCR stimulating agent stimulates gdTCRs that comprise or consist of one delta chain variable domain disclosed herein. In some embodiments, the gdTCR stimulating agent stimulates gdTCRs that comprise or consist of one gamma chain variable domain and one delta chain variable domain disclosed herein. In some embodiments, the gdTCR stimulating agent stimulates gdTCRs that comprise two or more gamma chain variable domains disclosed herein. In some embodiments, the gdTCR stimulating agent stimulates gdTCRs that comprise two or more delta chain variable domains disclosed herein. Illustrative gdTCR stimulating agents include antibodies (e.g., clone REA591 from Miltenyi), phosphoantigens (e.g., isopentenyl pyrophosphate, IPP), and aminobisphosphonates (e.g., zoledronic acid). In some embodiments, two or more gdTCR stimulating agents are used. For example, a pan- gdTCR stimulating agent, such as a gdTCR stimulating antibody, can be combined with a stimulating agent (e.g., antibody) that is specific for a gamma and/or delta chain variable domain disclosed herein, such as a Vdl -specific agent/antibody. In some embodiments, one or more gdTCR stimulating agents used in a composition or method disclosed herein are not platebound. In some embodiments, the expansion culture medium does not contain a gdTCR stimulating agent stimulating agent, e.g., during one or more incubation steps disclosed herein. [0159] In some embodiments, the expansion culture medium comprises and/or the cell culture vessel is coated with a CD3 stimulating agent, for example, an anti-CD3 antibody. A CD3 stimulating agent can specifically or preferentially induce signaling by a CD3 complex in T cells, e.g., gamma delta T cells. In some embodiments, the CD3 stimulating agent is an OKT3 antibody. In some embodiments, the CD3 stimulating agent is plate-bound. In some embodiments, the CD3 stimulating agent is not plate-bound. In some embodiments, the expansion culture medium does not contain a CD3 stimulating agent and the culture vessel is not coated with a CD3 stimulating agent, e.g., during one or more or all incubation steps disclosed herein.

[0160] In some embodiments, the expansion culture medium comprises and/or the cell culture vessel is coated with a CD28 stimulating agent, for example, an anti-CD28 antibody. A CD28 stimulating agent can specifically or preferentially induce signaling by CD28 in T cells, e.g., gamma delta T cells. In some embodiments, the CD28 stimulating agent is plate-bound. In some embodiments, the CD28 stimulating agent is not plate-bound. In some embodiments, the expansion culture medium does not contain a CD28 stimulating agent, e.g., during one or more incubation steps disclosed herein.

[0161] An expansion culture medium can comprise a T cell or gamma delta T cell stimulating agent, (e.g., gdTCR stimulating agent, CD3 stimulating agent, or CD28 stimulating agent) coated to a surface of a culture vessel at a density of at least 0.01, at least 0.1, at least 0.5, at least 0.7, at least 1, atleast 1.3, atleast 1.5, atleast 1.75, atleast2, atleast 3, at least 5, at least 10, or at least 100 pg per square centimeter. In some embodiments, an expansion culture medium comprises a T cell or gamma delta T cell stimulating agent, (e.g., gdTCR stimulating agent, CD3 stimulating agent, or CD28 stimulating agent) coated to a surface of a culture vessel at a density of at most 0.5, at most 0.7, at most 1, at most 1.3, at most 1.5, at most 1.75, at most 2, at most 3, at most 5, at most 10, or at most 100 pg per square centimeter. In some embodiments, an expansion culture medium comprises a T cell or gamma delta T cell stimulating agent, (e.g., gdTCR stimulating agent, CD3 stimulating agent, or CD28 stimulating agent) coated to a surface of a culture vessel at a density of about 0.01, about 0.1, about 0.5, about 0.7, about 1, about 1.3, about 1.5, about 1.75, about2, about 3, about 5, about 10, or about 100 pg per square centimeter. In some embodiments, an expansion culture medium comprises a T cell or gamma delta T cell stimulating agent, (e.g., gdTCR stimulating agent, CD3 stimulating agent, or CD28 stimulating agent) coated to a surface of a culture vessel at a density of 0.1-100, 0.1-10, 0.1-5, 0.1-2, 0.1-1, 0.5-100, 0.5-10, 0.5-5, 0.5-2, 0.5-1, 1-100, 1-10, 1-5, or 1-2 pg per square centimeter. [0162] An expansion culture medium can comprise a T cell or gamma delta T cell stimulating agent, (e.g., gdTCR stimulating agent, CD3 stimulating agent, or CD28 stimulating agent) in a non-plate bound (e.g., free or soluble) form at a concentration of at least 0.01, at least 0.1, at least 0.5, at least 0.7, at least 1, at least 1.5, at least 1.75, at least 2, at least 2.5, at least 3, at least 5, at least 10, at least 50, or at least 100 pg per mL. In some embodiments, an expansion culture medium comprises a T cell or gamma delta T cell stimulating agent, (e.g., gdTCR stimulating agent, CD3 stimulating agent, or CD28 stimulating agent) in a non-plate bound (e.g, soluble) form at a concentration ofat most 0.5, at most 0.7, at most 1, at most 1.5, at most 1.75, at most 2, at most 3, at most 5, at most 10, at most 25, or at most 100 pg/mL. In some embodiments, an expansion culture medium comprises a T cell or gamma delta T cell stimulating agent, (e.g., gdTCR stimulating agent, CD3 stimulating agent, or CD28 stimulating agent) in a non-plate bound (e.g., soluble) form at a concentration of about 0.01 , about 0.1 , about 0.5, about 1, about 1.5, about 1.75, about 2, about 2.5, about 3, about 5, about 10, about 25, about 50, or about 100 pg/mL. In some embodiments, an expansion culture medium comprises a T cell or gamma delta T cell stimulating agent, (e.g., gdTCR stimulating agent, CD3 stimulating agent, or CD28 stimulating agent) in a non-plate bound (e.g., soluble) form at a concentration of 0.1-100, 0.1-10, 0.1-5, 0.1-2, 0.1-1, 0.5-100, 0.5-10, 0.5-5, 0.5-2, 0.5-1, 1-100, 1-10, 1-5, 1-3, or 1-2 pg/mL.

[0163] In some embodiments, the expansion culture medium comprises and/or the cell culture vessel is coated with an agent that combines a CD3 -stimulating, CD28-stimulating, and/or gdTCR-stimulating agent for example, a coated bead.

[0164] In some embodiments, a step of a method disclosed herein (e.g., a stimulation step) comprises incubating the population of cells in medium (e.g., an expansion culture medium) with an appropriate concentration of carbon dioxide, for example, about 5% CO2.

[0165] In some embodiments, a step of a method disclosed herein (e.g., a stimulation step) comprises incubating the population of cells in medium (e.g., an expansion culture medium) at a temperature of about 37°C. The population of cells can be incubated at a temperature of at least 35 °C, atleast 36°C, or at least 37°C. The population of cells can be incubated at a temperature of at most 38°C, at most 37°C, or at most 36°C. The population of cells can be incubated at a temperature of about 35°C, about 36°C, or about 37°C. The population of cells can be incubated at a temperature about 35-38°C, 36-38°C, or 37-38°C.

[0166] A method of generating an expanded population of therapeutic cells (e.g., gamma delta T cells or polyclonal gamma delta T cells) can comprise incubating the cells in an expansion culture medium for a suitable amount of time to improve functional attributes of the resulting cell population, e.g., to reduce or avoid exhaustion of the resulting cells, or to harvest cells prior to or at a plateau of expansion.

[0167] A method of generating an expanded population of therapeutic cells (e.g., gamma delta T cells or polyclonal gamma delta T cells) can comprise incubating the cells in an expansion culture medium for about 22-23 days, e.g., between gd T cell enrichment and harvest, between initial stimulation and harvest, or between engineering (e.g., electroporation) and harvest.

[0168] A method of generating an expanded population of therapeutic cells (e.g., gamma delta T cells or polyclonal gamma delta T cells) can comprise incubating the cells in an expansion culture medium for at least 10, at least 15, at least 18, at least 19, at least 20, at least

21, at least 22, at least 23, at least 24, or at least 25 days, e.g., between gd T cell enrichment and harvest, between initial stimulation and harvest, or between engineering (e.g., electroporation) and harvest.

[0169] A method of generating an expanded population of therapeutic cells (e.g., gamma delta T cells or polyclonal gamma delta T cells) can comprise incubating the cells in an expansion culture medium for at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most24, atmost 25, or at most 30 days, e.g., between gd T cell enrichment and harvest, between initial stimulation and harvest, or between engineering (e.g., electroporation) and harvest.

[0170] A method of generating an expanded population of therapeutic cells (e.g., gamma delta T cells or polyclonal gamma delta T cells) can comprise incubating the cells in an expansion culture medium for about 10, about 15, about 18, about 19, about 20, about 21, about

22, about 23, about 24, or about 25 days, e.g., between gd T cell enrichment and harvest, between initial stimulation and harvest, or between engineering (e.g., electroporation) and harvest.

[0171] A method of generating an expanded population of therapeutic cells (e.g., gamma delta T cells or polyclonal gamma delta T cells) can comprise incubating the cells in an expansion culture medium for 10-30, 10-25, 10-24, 10-23, 10-22, 10-21, 10-20, 10-19, 10-15, 15-30, 15-25, 15-24, 10-23, 15-22, 15-21, 15-20, 15-19, 17-30, 17-25, 17-24, 17-23, 17-22, 17- 21, 17-20, 17-19, 19-30, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-25, 21-24, 21-23, 21-22, 22-30, 22-25, 22-24, or 22-23, days, e.g., between gd T cell enrichment and harvest, between initial stimulation and harvest, or between engineering (e.g., electroporation) and harvest. [0172] A method of generating an expanded population of therapeutic cells (e.g., gamma delta T cells or polyclonal gamma delta T cells) can comprise incubating the cells in a culture vessel comprising a gas permeable membrane submerged under a column of medium (e.g., GREX culture vessel) for at least 10, at least 15, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least at least 25 days, e.g., between gd T cell enrichment and harvest, between initial stimulation and harvest, or between engineering (e.g., electroporation) and harvest.

[0173] A method of generating an expanded population of therapeutic cells (e.g., gamma delta T cells or polyclonal gamma delta T cells) can comprise incubating the cells in a culture vessel comprising a gas permeable membrane submerged under a column of medium (e.g., GREX culture vessel) for at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, or at most 30 days, e.g., between gd T cell enrichment and harvest, between initial stimulation and harvest, or between engineering (e.g., electroporation) and harvest.

[0174] A method of generating an expanded population of therapeutic cells (e.g., gamma delta T cells or polyclonal gamma delta T cells) can comprise incubating the cells in a culture vessel comprising a gas permeable membrane submerged under a column of medium (e.g., GREX culture vessel) for about 10, about 15, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 days, e.g., between gd T cell enrichment and harvest, between initial stimulation and harvest, or between engineering (e.g., electroporation) and harvest.

[0175] A method of generating an expanded population of therapeutic cells (e.g., gamma delta T cells or polyclonal gamma delta T cells) can comprise incubating the cells in a culture vessel comprising a gas permeable membrane submerged under a column of medium (e.g., GREX culture vessel) for 10-30, 10-25, 10-24, 10-23, 10-22, 10-21, 10-20, 10-19, 10-15, 15-30, 15-25, 15-24, 10-23, 15-22, 15-21, 15-20, 15-19, 17-30, 17-25, 17-24, 17-23, 17-22, 17-21, 17- 20, 17-19, 19-30, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-25, 21-24, 21-23, 21-22, 22-30, 22-25, 22-24, or 22-23 days, e.g., between gd T cell enrichment and harvest, between initial stimulation and harvest, or between engineering (e.g., electroporation) and harvest.

[0176] A method of generating an expanded population of therapeutic cells (e.g., gamma delta T cells or polyclonal gamma delta T cells) can comprise: (a) optionally, processing a population of cells to enrich for gamma delta T cells, (b) a first stimulation step, for example, culturing the gamma delta T cells in a culture vessel (e.g., plate or flask) in a first expansion culture medium comprising IL-2, IL-7, IL-15, a gdTCR-stimulating agent, a CD28 -stimulating agent, and optionally a CD3 -stimulating agent, each at concentrations/densities disclosed herein, for 36-48h or about 2 days, (c) optionally, engineering the cells (e.g., to comprise a transgene encoding a heterologous immune receptor, such as a CAR), (d) a first expansion step comprising incubating the gamma delta T cells in a second expansion culture medium comprising IL-2, IL- 7, IL-15, each at concentrations/densities disclosed herein, for about 7-9 days, (e) a second stimulation step, for example, culturing the gamma delta T cells in a culture vessel (e.g., plate or flask) in the first expansion culture medium comprising IL-2, IL-7, IL-15, a gdTCR-stimulating agent, a CD28-stimulating agent, and optionally a CD3 -stimulating agent, each at concentrations/densities disclosed herein, for 36-48h or about 2 days, (f) a second expansion step comprising incubating the gamma delta T cells in the second expansion culture medium comprising IL-2, IL-7, IL-15, each at concentrations/densities disclosed herein, for about 7-10 days, (e) harvesting the population of cells, and optionally (f) formulating the cells into a pharmaceutical composition suitable for storage administration to a subject, and/or formulating the cells in a composition suitable for storage. The cells can be split, and media and/or cytokines replenished as appropriate and as disclosed herein, e.g., about very 2-3 days.

[0177] In some embodiments, a second stimulation or restimulation is initiated on about day 12 of a method disclosed herein, for example, about 10, 11, 12, 13, or 14 days after a first stimulation, or about 8, 9, 10, 11, or 12 days after cell engineering (e.g., electroporation to introduce a nucleic acid molecule, such as a transposon system to introduce a transgene), or about 3, 4, or 5 days after splitting cells.

[0178] Methods disclosed herein can facilitate generation of a population of therapeutic cells at a large scale, with retention of desirable functional attributes of the cells.

[0179] In some embodiments, a method disclosed herein results in at least 100-fold, at least 250-fold, at least 500-fold, at least 750-fold, at least 1000-fold, at least 1250-fold, at least 1500- fold, at least 2000-fold, at least 2500-fold, at least 3000-fold, at least 3500-fold, at least 4000- fold, at least 4500-fold, at least 5000-fold, at least 6000-fold, at least 7000-fold, at least 8000- fold, at least 9000-fold, at least 10,000-fold, at least 15,000-fold, at least 20,000-fold, at least 30,000-fold, at least 40,000-fold, at least 50,000-fold, at least 75,000-fold, or at least 100,000- fold expansion of a population of cells.

[0180] In some embodiments, a method disclosed herein results in at least 100-fold, at least 250-fold, at least 500-fold, at least 750-fold, at least 1000-fold, at least 1250-fold, at least 1500- fold, at least 2000-fold, at least 2500-fold, at least 3000-fold, at least 3500-fold, at least 4000- fold, at least 4500-fold, at least 5000-fold, at least 6000-fold, at least 7000-fold, at least 8000- fold, at least 9000-fold, at least 10,000-fold, at least 15,000-fold, at least 20,000-fold, at least 30,000-fold, at least 40,000-fold, at least 50,000-fold, at least 75,000-fold, or at least 100,000- fold expansion of a population of gamma delta T cells. The fold expansion can be calculated based on the total number of (e.g., viable) gamma delta T cells at harvest compared to the number of (e.g., viable) gamma delta T cells subject to an engineering (e.g., electroporation) step. In some embodiments, the fold expansion is calculated based on the total number of (e.g., viable) gamma delta T cells at harvest compared to the number of (e.g., viable) gamma delta T cells prior to a first stimulation. In some embodiments, the fold expansion is calculated based on the total number of (e.g., viable) gamma delta T cells at harvest compared to the number of (e.g, viable) gamma delta T cells after an engineering (e.g., electroporation) step (e.g., and 1-2 hour recovery post-electroporation).

[0181] In some embodiments, a method disclosed herein results in at least 100-fold, at least 250-fold, at least 500-fold, at least 750-fold, at least 1000-fold, at least 1250-fold, at least 1500- fold, at least 2000-fold, at least 2500-fold, at least 3000-fold, at least 3500-fold, at least 4000- fold, at least 4500-fold, at least 5000-fold, at least 6000-fold, at least 7000-fold, at least 8000- fold, at least 9000-fold, at least 10,000-fold, at least 15,000-fold, at least 20,000-fold, at least 30,000-fold, at least 40,000-fold, at least 50,000-fold, at least 75,000-fold, or at least 100,000- fold expansion of a population of polyclonal gamma delta T cells. The fold expansion can be calculated based on the total number of (e.g., viable) gamma delta T cells at harvest compared to the number of (e.g., viable) gamma delta T cells subject to an engineering (e.g., electroporation) step. In some embodiments, the fold expansion is calculated based on the total number of (e.g., viable) gamma delta T cells at harvest compared to the number of (e.g., viable) gamma delta T cells prior to a first stimulation. In some embodiments, the fold expansion is calculated based on the total number of (e.g., viable) gamma delta T cells at harvest compared to the number of (e.g, viable) gamma delta T cells after an engineering (e.g., electroporation) step (e.g., and 1-2 hour recovery post-electroporation).

[0182] In some embodiments, a method disclosed herein results in at least 100-fold, at least 250-fold, at least 500-fold, at least 750-fold, at least 1000-fold, at least 1250-fold, at least 1500- fold, at least 2000-fold, at least 2500-fold, at least 3000-fold, at least 3500-fold, at least 4000- fold, at least 4500-fold, atleast 5000-fold, at least 6000-fold, at least 7000-fold, at least 8000- fold, at least 9000-fold, at least 10,000-fold, at least 15,000-fold, at least 20,000-fold, at least 30,000-fold, at least 40,000-fold, at least 50,000-fold, at least 75,000-fold, or at least 100,000- fold expansion of a population of engineered cells, for example, engineered gamma delta T cells or engineered polyclonal gamma delta T cells that express a CAR. The fold expansion can be calculated based on the total number of (e.g., viable) engineered gamma delta T cells at harvest compared to the number of (e.g., viable) gamma delta T cells subject to an engineering (e.g., electroporation) step. In some embodiments, the fold expansion is calculated based on the total number of (e.g., viable) engineered gamma delta T cells at harvest compared to the number of (e.g., viable) gamma delta T cells prior to a first stimulation. In some embodiments, the fold expansion is calculated based on the total number of (e.g., viable) engineered gamma delta T cells at harvest compared to the number of gamma delta T cells after an engineering (e.g., electroporation) step (e.g., and 1-2 hour recovery post-electroporation), e.g., all viable cells or cells that have been engineered to express the CAR.

[0183] For example, in some embodiments expansion protocols disclosed herein can generate 2,000-5,000 fold expansion of polyclonal populations of engineered (e.g., transposon- engineered CAR expressing) gamma delta T cells and greater than 10,000 fold expansion of unmodified gamma delta T cells.

[0184] In some embodiments, a method disclosed herein provides a population of cells comprising at least 1 xlO^A7, at least 5 xlO^A7, at least 1 xl0^A8, at least 5 xl0^A8, at least 1 xlO^A9, at least 3 xlO^A9, at least 5 xlO^A9, at least 7 xlO^A9, at least 1 xl0^A10, at least 3 xl0^A10, at least 5 xl0^A10, at least 7 xl0^A10, at least 1 xl0^Al 1, at least 3 xl0^Al 1 , at least 5 xl0^Al 1, at least 7 xl0^Al 1, at least 1 xlO^A12, at least 5 xlO^A12, at least or at least 1 xl0^A13 cells, for example, gamma delta T cells, polyclonal gamma delta T cells, engineered gamma delta T cells, or engineered polyclonal gamma delta T cells that express a CAR. The cells can be viable cells. In some embodiments, the population of cells comprises at most 1 xlO^A9, at most 1 xl0^A10, at most 1 xl0^Al l, atmost 1 xlO^A12, atmost 1 xl0^A13, at most 1 xlO^A14, or atmost 1 xl0^A15 cells (for example, gamma delta T cells, polyclonal gamma delta T cells, engineered gamma delta T cells, or engineered polyclonal gamma delta T cells that express a CAR).

[0185] Expanded populations of therapeutic cells disclosed herein can, in some embodiments, allow multiple patient doses of a cell therapy product to be generated, for example, from a single donor and/or single production run. For instance, in some embodiments, a method disclosed herein provides at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, atleast 100, atleast 150, at least200, atleast250, atleast 500, at least 600, atleast 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, atleast 1300, atleast 1400, at least 1500, at least 1600, at least 1700, at least 1800, atleast 1900, at least 2000, at least 2100, at least 2200, at least 2300, at least 2400, at least 2500, or at least 3000 patient doses of a cell therapy product.

[0186] A patient dose of a cell therapy product can comprise a given number of cells or at least a given number of cells. In some embodiments, a patient dose comprises at least about 5 xlO^A6, at least about 6 xlO^A6, at least about 7 xlO^A6, at least about 8 xlO^A6, at least about 9 xlO^A6, at least about 1 xlO^A7, at least about 2 xlO^A7, at least about 3 xlO^A7, at least about 4 xlO^A7, at least about 5 xlO^A7, at least about 6 xlO^A7, at least about 7 xlO^A7, at least about 8 xlO^A7, at least about 9 xlO^A7, at least about 1 xlO^A8, at least about 2 xlO^A8, at least about 3 xlO^A8, at least about 4 xlO^A8, at least about 5 xlO^A8, at least about 6 xlO^A8, at least about 7 xlO^A8, at least about 8 xlO^A8, at least about 9 xlO^A8, at least about 1 xlO^A9, at least about 2 xlO^A9, atleast about 3 xlO^A9, at least about 4 xlO^A9, at least about 5 xlO^A9, or at least about 1 xl0^A10 cells. In some embodiments, the patient dose comprises about 5 xlO^A6, about 6 xlO^A6, about 7 xlO^A6, about 8 xlO^A6, about 9 xlO^A6, about 1 xl O^A7, about 2 xlO^A7, about 3 xlO^A7, about4 xlO^A7, about 5 xlO^A7, about 6 xlO^A7, about 7 xlO^A7, about 8 xlO^A7, about 9 xlO^A7, about 1 xlO^A8, about 2 xlO^A8, about 3 xlO^A8, about 4 xl O^A8, about 5 xlO^A8, about 6 xlO^A8, about 7 xlO^A8, about 8 xlO^A8, about 9 xlO^A8, about 1 xl O^A9, about 2 xlO^A9, about 3 xlO^A9, about4 xlO^A9, about 5 xlO^A9, or about 1 xl0^A10 cells. In some embodiments, the patient dose comprises at most about 1 xlO^A7, at most about 1 xlO^A8, at most about 1 xlO^A9, or at most about 1 xl0^A10 cells. The cells can be for example, (e.g., viable) gamma delta T cells, polyclonal gamma delta T cells, engineered gamma delta T cells, or engineered polyclonal gamma delta T cells that express a CAR.

[0187] A patient dose of a cell therapy product can comprise a given number of cells or at least a given number of cells per kilogram of bodyweight of a recipient subject. In some embodiments, a patient dose comprises at least 0.1 xlO^A6, atleast 0.5 xlO^A6, at least 1 xlO^A6, at least 1.5 xlO^A6, at least 2 xlO^A6, at least 2.5 xlO^A6, at least 3 xlO^A6, at least 3.5 xlO^A6, at least 4 xlO^A6, at least 4.5 xlO^A6, at least 5 xlO^A6, at least 5.5 xlO^A6, at least 6 xlO^A6, at least 6.5 xlO^A6, at least 7 xlO^A6, at least 7.5 xlO^A6, at least 8 xlO^A6, atleast 8.5 xlO^A6, at least 9 xlO^A6, at least 9.5 xlO^A6, at least 10 xlO^A6, at least 11 xlO^A6, at least 12 xlO^A6, at least 13 xlO^A6, at least 14 xlO^A6, or atleast 15 xlO^A6 cells per kilogram of body weight of a recipient subject. In some embodiments, a patient dose comprises about 0.1 xlO^A6, about 0.5 xlO^A6, about 1 xlO^A6, about 1.5 xlO^A6, about 2 xlO^A6, about 2.5 xlO^A6, about 3 xlO^A6, about 3.5 xlO^A6, about 4 xlO^A6, about 4.5 xlO^A6, about 5 xlO^A6, about 5.5 xlO^A6, about 6 xlO^A6, about 6.5 xlO^A6, about 7 xl O^A6, about 7.5 xlO^A6, about 8 xlO^A6, about 8.5 xlO^A6, about 9 xlO^A6, about 9.5 xlO^A6, about 10 xlO^A6, about 11 xlO^A6, about 12 xlO^A6, about 13 xlO^A6, about 14 xlO^A6, or about 15 xlO^A6 cells per kilogram of bodyweight of a recipient subject. In some embodiments, a patient dose comprises at most 10 xlO^A6, at most 15 xlO^A6, or at most 100 xlO^A6 cells per kilogram of bodyweight of a recipient subject. The cells can be for example, (e.g., viable) gamma delta T cells, polyclonal gamma delta T cells, engineered gamma delta T cells, or engineered polyclonal gamma delta T cells that express a CAR. The subject can be a representative subject, for example, a representative subject assigned an average weight, such as about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 kg. Dose calculations can be made for subjects falling within a range of weights, for example, 10-150 kg, 10-125 kg, 10-100 kg, 10-90 kg, 10-80 kg, 50-150 kg, 50-125 kg, 50-100 kg, 75-150 kg, 75-125 kg, 75-100 kg, 75-90 kg, or 75-80 kg.

[0188] In some embodiments, it can be common to lose between about 30-50% of cells during activation (e.g., a first stimulation step disclosed herein), which can be accounted for when planning for the total of cells numbered present or required at an engineering step (e.g., transformation, such as electroporation).

[0189] In some embodiments, a subset of subpopulation of gamma delta T cells adhere to a surface of a culture vessel (e.g., tissue culture well or plate), and are dislodged in a method disclosed herein, e.g., by pipetting with sufficient force, washing 2-3X with media, and/or with aid of a chemical or enzymatic treatment. In some embodiments, such dislodging increases recovery of a desirable subpopulation of gamma delta T cells in a polyclonal population, for example, cells that express a TCR comprising a Vdl variable domain.

[0190] In some embodiments, methods of generating an expanded population of therapeutic cells (e.g., gamma delta T cells or polyclonal gamma delta T cells) comprise quantifying a concentration of lactate and/or a concentration of glucose in culture medium. Quantifying a concentration ofglucose and/or lactate in a culture medium can provide a useful way to assess cell metabolism and growth, and for determining the timing of replenishing media components and/or cytokines. In some embodiments, quantifying a concentration of lactate and/or a concentration of glucose in culture medium can be advantageous over, for example, methods based on color changes of medium (e.g., due to phenol red or a pH indicator). In some embodiments, gamma delta T cell growth can be sensitive to cell density and media replenishment, and e.g., cells can crash or fail to expand to generate a population of cells suitable for therapeutic applications if cells are split too soon or too late. In some embodiments, quantifying a concentration of lactate and/or a concentration of glucose in culture medium can be used when establishing the timing of replenishing media components and/or cytokines, but can be optional or unnecessary to repeat with each batch of cell expansion. In some embodiments, cells can be split and media and/or cytokines replenished cells when glucose measurements are between about 150-250 mg/dL, and lactate measurements between about 10- 12 mmol/L. In some embodiments, quantifying a concentration of lactate and/or a concentration of glucose in culture medium can be used when establishing the timing of replenishing media components and/or cytokines for a particular engineered cell, e.g., for gamma delta T cells expressing a given CAR.

[0191] In some embodiments, media is replenished about every 2, 3, or 4 days. In some embodiments, media is replenished about every 2-4 days. In some embodiments, media is replenished about every 2-3 days. In some embodiments, media is replenished about every 3-4 days. In some embodiments, replenishing media comprises adding an equal volume of media to the media previously present. In some embodiments, replenishing media comprises removing half of the spent media and replacing with fresh media. In some embodiments, when media is replenished, a proportion of the “spent” medium is retained, e.g., to retain a proportion of stimulating factors (e.g., gdTCR-stimulating agent, CD3 -stimulating agent, and/or CD28- stimulating agent) and/or to retain a proportion of paracrine factors that may support expansion of the population of gamma delta T cells. In some embodiments, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% of spent medium is retained when fresh medium is added.

[0192] In some embodiments, cells are split on about day 7 or 8 of a method disclosed herein, for example, about 7 or 8 days after a first stimulation, or about 5 or 7 days after engineering (e.g., electroporation with a transposon system to introduce a transgene encoding a CAR). In some embodiments, splitting the cells to an appropriate density (e.g., split in half with fresh media added and fresh cytokines added based on a calculation of the full final culture volume after split) can be important for expansion of the cells to a high degree.

[0193] In some embodiments, the cells are passed through an appropriate filter after stimulation, e.g., to remove dead cells and debris. In some embodiments, the cells are passed through an appropriate filter after thawing, e.g., to remove dead cells and debris. In some embodiments, the cells are passed through an appropriate filter after electroporation, e.g., to remove dead cells and debris.

[0194] At the conclusion of methods of generating an expanded population of therapeutic cells (e.g., gamma delta T cells or polyclonal gamma delta T cells), the cells can be harvested, for example, formulated in a pharmaceutical composition suitable for administration to a subject, and/or cryopreserved. In some embodiments, a harvested population of cells is suspended in a suitable cry opreservation buffer (e.g., CryoStor CS5 or CS10). The harvested population of cells can be formulated at a suitable concentration for storage, e.g., about 10-50 x!0^A6 cells/mL. III. CELL ENGINEERING

[0195] In some embodiments, compositions and methods disclosed herein comprise an engineered cell or population thereof. Methods can comprise engineering the cells, or previously engineered cells can be used. Methods disclosed herein and other suitable known methods can be used to generate engineered cells, for example, gamma delta T cells comprising a transgene that encodes a CAR. For example, cell engineering techniques disclosed herein and/or known to a skilled person can be used to modify cells to comprise a nucleic acid molecule that encodes a heterologous immune receptor (e.g., CAR) of the disclosure, thereby generating engineered cells (such as engineered gamma delta T cells).

[0196] In some embodiments, a nucleic acid molecule comprising transgene that encodes a CAR is introduced into a cell or population thereof. In some embodiments, a nucleic acid molecule comprising transgene that encodes an exogenous TCR is introduced into a cell or population thereof. In some embodiments, a nucleic acid molecule comprising transgene that encodes an exogenous gdTCR can be introduced into a cell, such as a lymphocyte, lymphoid cell, T cell (e.g., alpha-beta T cell), or myeloid cell.

[0197] Compositions and methods disclosed herein can comprise a nucleic acid molecule, for example, comprising a transgene that encodes a chimeric antigen receptor or other heterologous immune receptor (e.g., CAR). A nucleic acid molecule can comprise, for example, one or more expression regulatory regions (e.g., a promoter, enhancer, intron, and/or exon), one or more transgenes (e.g., encoding a heterologous immune receptor (e.g., CAR)), a polyadenylation signal, or a combination thereof.

[0198] A nucleic acid molecule can be a substance whose molecules comprise or consist essentially of nucleotides linked in a chain. Non-limiting examples of the nucleic acid molecule include a circular nucleic acid, a DNA, a single stranded DNA, a double stranded DNA, a genomic DNA, a plasmid, a nanoplasmid, a plasmid DNA, a viral DNA, a minicircle (e.g., lacking a bacterial origin of replication), and an RNA.

[0199] In some embodiments, a nucleic acid molecule encodes two or more polypeptides linked by one or more 2A linkers or self-cleaving peptides, which can be processed into separate polypeptides co-translationally or after translation (e.g., P2A, T2A, F2A, E2A). Inclusion of a 2A linker can increase the likelihood that an appropriate ratio of components are produced (e.g., a 1 : 1, 1 :2, 1 :3, 1 :4, or 1 :5 ratio of two components). In some cases, inclusion of a 2 A linker can increase the likelihood that equal or close to equal levels of two components of the heterologous immune receptor (e.g., TCR or split CAR) are produced. [0200] For example, a nucleic acid molecule encodes a TCR alpha chain constant region and TCR beta chain constant region, and inclusion of a 2A linker can increase the likelihood that equal or close to equal levels of a TCR alpha chain constant region and TCR beta chain are produced. In some embodiments a heterologous immune receptor (e.g., CAR) can comprise a TCR gamma chain constant region and TCR delta chain constant region, and inclusion of a 2 A linker can increase the likelihood that equal or close to equal levels of or TCR gamma chain and TCR delta chain, are produced. In some cases, use of a 2A linker can allow for fewer components in a system for transgene expression and/or genome modification, e.g., inclusion of multiple components in one vector rather than separate vectors.

[0201] An expression construct or nucleic acid molecule disclosed herein can be or can comprise DNA. An expression construct or nucleic acid molecule disclosed herein can be or can comprise double stranded DNA. For example, an expression construct or nucleic acid molecule disclosed herein can be or comprise a plasmid, such as a nanoplasmid. In some embodiments, an expression construct or nucleic acid molecule disclosed herein is or comprises a minicircle, a midge, a MIP, or a doggy bone.

[0202] In some embodiments an expression construct or nucleic acid molecule lacks an origin of replication. An expression construct or nucleic acid molecule disclosed herein can be or can comprise a circular nucleic acid molecule. An expression construct or nucleic acid molecule disclosed herein can be or can comprise a linear nucleic acid molecule. An expression construct or nucleic acid molecule disclosed herein can comprise one or more transgenes or open reading frames.

[0203] A nucleic acid molecule can be or be present in an expression construct. A nucleic acid molecule or expression construct can comprise a promoter, enhancer, or combination thereof that drive or upregulate expression of a transgene, for example, that encodes a heterologous immune receptor (e.g., CAR). The transgene can be operatively linked to and/or under regulatory control of the promoter or enhancer.

[0204] A promoter disclosed herein can be a mammalian promoter or derived from a mammalian promoter. A promoter disclosed herein can be a human promoter or derived from a human promoter. The promoter can be a promoter as found in a naturally -occurring genome. In some embodiments, a promoter is not found in a naturally -occurring genome. In some embodiments, the promoter is a synthetic or engineered promoter. The promoter can be a minimal promoter. A promoter can be a constitutive, viral, inducible, or tissue-specific promoter. [0205] A promoter can be an immune-cell selective promoter, for example, a promoter that results in preferential expression in immune cells as compared to non-immune cells. An immune cell-selective promoter can result in preferential expression in, for example, lymphocytes, T cells, CD4+ T cells, CD8+ T cells, alpha-beta T cells, gamma-delta T cells, NK cells, or NKT cells. The expression can be preferential compared to control cells, such as fibroblasts, neurons, epithelial cells, keratinocytes, or hepatocytes, etc.

[0206] A promoter can be a T-cell selective promoter, for example, a promoter that results in preferential expression in T cells as compared to non-T cells. In some embodiments, a T cell- selective promoter can limit off-target effects, e.g, limit off target effects resulting from expression of a heterologous immune receptor (e.g., CAR) in non-T cells. Non-limiting examples of T-cell selective promoters include promoters that natively drive expression of CD3 (e.g, CD3 gamma, CD3 delta, CD3 epsilon, or CD3 zeta), CD4, CD8, CD28, TCRB, TRAC, TRG, or TRD.

[0207] In some embodiments, a nucleic acid molecule disclosed herein comprises RNA, for example, mRNA.

[0208] In some embodiments, an expression construct or nucleic acid molecule disclosed herein is or comprises single stranded DNA. In some embodiments, an expression construct or nucleic acid molecule disclosed herein comprises a component of a viral genome or a viral packaging element, for example, a 5' and/or 3' inverted terminal repeat (ITR). In some embodiments, an expression construct or nucleic acid molecule disclosed herein is not single stranded DNA. In some embodiments, an expression construct or nucleic acid molecule disclosed herein lacks a component of a viral genome or lacks a viral packaging element, for example, lacks a 5' and/or 3' inverted terminal repeat (ITR).

[0209] In some embodiments, an expression construct or nucleic acid molecule disclosed herein is integrating, e.g., integrates into the genome of an engineered cell. In some embodiments, an expression construct or nucleic acid molecule disclosed herein is nonintegrating, e.g., does not integrate into the genome of an engineered cell.

[0210] A nucleic acid molecule can include one or more homology arms, for example, comprising sequences that are complementary to a genomic DNA sequence to be targeted for insertion (e.g, via homologous recombination or homology directed repair). A nucleic acid molecule can comprise one or more promoter regions, barcodes, restriction sites, cleavage sites, endonuclease recognition sites, primer binding sites, selectable markers, unique identification sequences, resistance genes, linker sequences, or any combination thereof. In some aspects, these sites may be useful for enzymatic digestion, amplification, sequencing, targeted binding, purification, providing resistance properties (e.g., antibiotic resistance for selection), or any combination thereof. A nucleic acid molecule may also include transcriptional or translational regulatory sequences, for example, one or more promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A linkers and/or polyadenylation signals.

[0211] In some embodiments, a nucleic acid molecule or expression construct disclosed herein comprises natural, synthetic, and/or artificial nucleotide analogues or bases. In some embodiments, the synthetic or artificial nucleotide analogues or bases comprise modifications at one or more of a deoxyribose moiety, ribose moiety, phosphate moiety, nucleoside moiety, or a combination thereof.

[0212] In some embodiments, a nucleotide analogue or artificial nucleotide base comprises a nucleic acid with a modification at a 2' hydroxyl group of the ribose moiety. In some instances, the modification includes an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety. Illustrative alkyl moieties include, but are not limited to, halogens, sulfurs, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary), amides, ethers, esters, alcohols and oxygen. In some instances, the alkyl moiety further comprises a modification. In some instances, the modification comprises an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso, group, a nitrile group, a heterocycle (e.g., imidazole, hydrazino or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some instances, the alkyl moiety further comprises a hetero substitution. In some instances, the carbon of the heterocyclic group is substituted by a nitrogen, oxygen or sulfur. In some instances, the heterocyclic substitution includes but is not limited to, morpholino, imidazole, and pyrrolidino.

[0213] A nucleic acid molecule, gene editing component, or other cargo can be delivered to a cell by any suitable method, for example, using any suitable vector. In some embodiments a composition or method utilizes a vector comprising any of the nucleic acid molecules described herein.

[0214] Methods to introduce nucleic acid molecules and/or gene editing components into a cell include, but are not limited to, electroporation, sonoporation, use of a gene gun, lipofection, calcium phosphate transfection, use of dendrimers, microinjection, and use of viral vectors including lentiviral, adenoviral, AAV, and retroviral vectors.

[0215] Electroporation using, for example, the Neon® Transfection System (ThermoFisher Scientific), the Xenon Electroporation System (ThermoFisher Scientific), or the AMAXA® Nucleofector (AMAXA® Biosystems) can also be used for delivery of nucleic acids into a cell. Electroporation parameters may be adjusted to optimize transfection efficiency and/or cell viability. Electroporation devices can have multiple electrical wave form pulse settings such as exponential decay, time constant and square wave. Every cell type can have a unique optimal Field Strength (E) that is dependent on the pulse parameters applied (e.g., voltage, capacitance and resistance). Application of optimal field strength causes electropermeabilization through induction of transmembrane voltage, which allows nucleic acids to pass through the cell membrane. In some cases, the electroporation pulse voltage, the electroporation pulse width, number of pulses, cell density, and tip type maybe adjusted to optimize transfection efficiency and/or cell viability, e.g., for gamma delta T cells.

[0216] In some embodiments, after electroporation, a population of cells can be recovered by incubating in a suitable recovery medium for a period of at least 10 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 1.5 hours, or at least 2 hours. In some embodiments, after electroporation, a population of cells can be recovered by incubating in a suitable recovery medium for a period of 10 minutes to 5 hours, 30 minutes to 5 hours, 1 hour to 5 hours, 2 hours to 5 hours, 10 minutes to 3 hours, 30 minutes to 3 hours, 1 hour to 3 hours, 2 hours to 3 hours, 10 minutes to 2 hours, 30 minutes to 2 hours, or 1 hour to 2 hours. The recovery medium can lack antibiotics, for example, lack penicillin and streptomycin to improve viability of the cells. The recovery medium can comprise DNase (e.g., about 1000 lU/mL DNase). In some embodiments, the cells are passed through an appropriate filter after recovery, e.g., to remove dead cells and debris.

[0217] A vector disclosed herein can be a non-viral, lipid-based vector. A non-viral, lipid- based vector can be, for example, a liposome, a lipoplex, a lipid nanoparticle, a vesicle, or a micelle. In some embodiments, a vector is or comprises a poloxamer, nanoparticle, polyplex, or dendrimer. A vector can be a nanoparticle, for example, an inorganic nanoparticle, such as a gold, silica, iron oxide, titanium, calcium phosphate, PLGA, poly(B-amino ester) (PBAE, e.g., PBAE-447), or hydrogel nanoparticle. In some embodiments a vector is not a nanoparticle, e.g., is not an inorganic nanoparticle.

[0218] A vector can be or can comprise a viral vector, a gamma-retroviral vector, a lentiviral vector, an adeno-associated viral vector, a transposon, and the like. Any vector systems can be used including, but not limited to, DNA vectors, RNA vectors, ribonucleoprotein vectors, hybrid DNA-RNA vectors, plasmid vectors, minicircle vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors, herpesvirus vectors and adeno-associated virus vectors, etc. Non-viral vector delivery systems can include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems can include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. In some cases, onevector is used. In some cases, two vectors are used. In some cases, three or more vectors are used.

[0219] In some cases, the vector is a viral vector, such as a lentiviral vector, a y-retroviral vector, or an adeno-associated virus (AAV) vector. In some embodiments, the vector is a non- viral vector, for example, a plasmid, nanoplasmid, minicircle, a midge, a MIP, or a doggybone, a lipid-based nanoparticle, a liposome, a circular nucleic acid molecule (e.g., DNA or RNA), a linear nucleic acid molecule (e.g., a DNA or RNA), or a combination thereof. In some embodiments, viral vectors are not used in a method disclosed herein.

[0220] In some cases, a nucleic acid molecule, gene editing component, or other can be delivered to cells without the use of vectors. In some cases, one or more nucleic acid molecules, gene editing components, or other cargos of the disclosure can be delivered to cells via vectors, and one or more nucleic acid molecules, gene editing components, or other cargos can be delivered without the use of vectors.

[0221] Engineering methods can comprise contacting a cell with a nucleic acid molecule, or with a vector that comprises the nucleic acid molecule, under conditions that permit uptake of the nucleic acid molecule by the cell. A nucleic acid molecule can comprise a nucleotide sequence that encodes a heterologous immune receptor (e.g., CAR) disclosed herein or a component thereof. In some cases, a nucleic acid molecule is utilized to alter a genome of a cell. An engineered cell can be generated by a method that comprises contacting a cell with a nucleic acid molecule or vector disclosed herein.

[0222] For targeted integration, a nucleic acid molecule sequence to be inserted can be flanked by homology arms comprising sequences that are complementary to a genomic DNA sequence to be targeted for insertion (e.g., via homologous recombination and/or homology- directed repair, HDR). A double stranded break can be introduced at a target site in the genome, and the homology arms can promote insertion of the nucleic acid molecule. In some cases, a nucleic acid molecule can be excised from a vector, such as a nanoplasmid (e.g., via a nuclease), and inserted into the genome of the cell.

[0223] A nucleic acid molecule can be inserted in a safe harbor locus. A safe harbor can comprise a genomic location where a nucleic acid molecule can integrate and function without substantially perturbing endogenous activity, for example, with a relatively low impact on local or global gene expression. For example, one or more nucleic acid molecules can be inserted into any one of HPRT, an AAVS site (E.G., AAVS1, AAVS2, etc.), CCR5, hROSA26, and/or any combination thereof. A nucleic acid molecule can be inserted in an intergenic region. A nucleic acid molecule can be inserted in a non-coding region. A nucleic acid molecule can be inserted within a gene. In some cases, a nucleic acid molecule can disrupt a gene it is inserted into (e.g., reduce or eliminate expression of the disrupted gene). A disrupted gene can be for example, an endogenous TCR gene (e g., TRAC, TCRB, TCRBC1, TRBC2, TRG, TRD), or an immune checkpoint gene (e.g., PD-1, CTLA-4). A nucleic acid molecule can be inserted adjacent to or near to a promoter.

[0224] In some cases, one or more nucleic acid molecules of the disclosure can be inserted randomly into the genome of a cell. For instance, a nucleic acid molecule can encode its own promoter or can be inserted into a position where it is under the control of an endogenous promoter. Alternatively or additionally, a nucleic acid molecule can be inserted into a gene, such as an intron of a gene, an exon of a gene, a promoter, or a non-coding region.

[0225] A variety of enzymes can catalyze generation of a double-stranded break in the genome and/or insertion of foreign DNA into a host genome. Non-limiting examples of gene editing tools and techniques include CRISPR systems, CRISPR-associated polypeptide (Cas), TALEN, zinc finger nuclease (ZFN), zinc finger associate gene regulation polypeptide, meganuclease, Mega-TAL, transposon-based systems, natural master transcription factors, epigenetic modifying enzymes, recombinase, flippase, transposase, RNA-binding proteins (RBP), an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof.

[0226] A transposon-based system can be utilized for insertion of a nucleic acid molecule encoding a polypeptide (e.g., CAR) of the disclosure or a component thereof into a genome. A transposon can comprise a nucleic acid molecule that can be inserted into a DNA sequence. A class I transposon can be transcribed into an RNA intermediate, then reverse transcribed and inserted into a DNA sequence. A class II transposon can comprise a DNA sequence that is excised from one DNA sequence and/or inserted into another DNA sequence. A class II transposon system can comprise (i) a transposon vector that contains a sequence (e.g., comprising a transgene) flanked by inverted terminal repeats, and (ii) a source for the transposase enzyme. A transposon system (e.g., class II transposon system) can direct the integration of a nucleic acid molecule sequence encoding a polypeptide (e.g., CAR) or a component thereof, while leaving behind the rest of the vector. A transposon and a transposase can be introduced into a cell. In some cases, a vector that encodes a transposase and comprises a nucleic acid molecule is introduced into a cell, and the transposase is expressed and mediates insertion of the transposon into the genome.

[0227] Examples of transposon-based systems that can be used include, but are not limited to, TcBuster (e.g., derived from the red flour beetle Tribolium castaneum), sleeping beauty (e.g., derived from the genome of salmonid fish); piggyback (e.g., derived from lepidopteran cells and/or the Myotis lucifugus); mariner (e.g., derived from Drosophila); frog prince (e.g., derived from Rana pipiens); Tol2 (e.g., derived from medaka fish); and spinON.

[0228] TcBuster (TcB) and hyperactive TcBuster (TcB-M) can be obtained from Bio- Techne (Minneapolis, MN). In some embodiments, a TcB transposase mRNA and transposon plasmid comprising a transgene to be genomically integrated are used, and, e.g., delivered via electroporation. Non-limiting examples of TcBuster systems that can be used are described in US Patent Application Nos. US20210277366A1, US20200323902A1, and US20180216087 Al, each of which is incorporated herein by reference in its entirety.

[0229] A CRISPR system can be utilized to facilitate insertion of a nucleic acid molecule encoding a heterologous immune receptor (e.g., CAR) or a component thereof into a cell genome. For example, a CRISPR system can introduce a double stranded break at a target site in a genome or a random site of a genome.

[0230] In some cases, a CRISPR system comprises CRISPR-associated (Cas) proteins or Cas nucleases including type I CRISPR-associated (Cas) polypeptides, type II CRISPR- associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, typeV CRISPR-associated (Cas) polypeptides, or type VI CRISPR-associated (Cas) polypeptides a derivative, variant, or functional fragment thereof.

[0231] In some embodiments, a CRISPR system comprises a Class I system or endonuclease (e.g., Type I, Type III or Type IV Cas proteins). A class I system can be of the I-A, I-B, I-C, I- U, I-D, I-E, I-F, IV-A, IV-B, III-A, III-D, III-C, or III-B subtype.

[0232] In some embodiments, a CRISPR system comprises a Class II system or endonuclease (e.g., Type II, Type V, or Type VI). A class II, Type II system can be of the II-A, II-B, II-C1, or II-C2 subtype. A class II, Type V systems can of the V-A, V-Bl, V-B2, V-C, V- D, V-E, V-Fl, V-F1(V-U3), V-F2, V-F3, V-G, V-H, V-I, V-K (V-U5), V-Ul, V-U2, or V-U4 subtype. A Class II, Type IV systems can be of the: VI- A, VI-B1, VI-B2, VI-C, or VI-D subtype.

[0233] In some embodiments, a Cas protein used in a method disclosed herein is a class II endonuclease. In some embodiments, a Cas protein used in a method disclosed herein is a class II, type V Cas endonuclease. In some embodiments, a Cas protein used in a method disclosed herein is a class II, type V-A Cas endonuclease.

[0234] Non-limiting examples of Cas proteins that can be used in the CRISPR systems include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl or Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csb l, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl S, Csfl, Csf2, CsO, Csf4, Cpfl, c2cl, c2c3, Cas9HiFi, homologues thereof, and modified versions thereof. An unmodified CRISPR enzyme can have DNA cleavage activity, such as Cas9. A CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within a target sequence and/or within a complement of a target sequence. For example, a CRISPR enzyme can direct cleavage of one or both strands within or within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. A Cas protein can be a high-fidelity Cas protein. Alternatives to S. pyogenes Cas9 may include RNA-guided endonucleases from the Cpfl family that display cleavage activity in mammalian cells.

[0235] In some embodiments, a gene editing system comprises a Cas protein, and the system further comprises a guide RNA (gRNA) which complexes with the Cas protein. In some embodiments, the gene editing moiety comprises an RBP complexed with a gRNA which is able to form a complex with a Cas protein.

[0236] In some cases, a dual nickase approach may be used to introduce a double stranded break. Cas proteins can be mutated at certain amino acids within either nuclease domains, thereby deleting activity of one nuclease domain and generating a nickase Cas protein capable of generating a single strand break. A nickase along with two distinct guide RNAs targeting opposite strands may be utilized to generate a DSB within a target site (often referred to as a “double nick” or “dual nickase” CRISPR system).

[0237] In some embodiments a polypeptide (e.g., CAR) can be expressed in an engineered cell without genomic integration of a nucleic acid molecule comprising a transgene. For example, a transgene can be expressed from an episomal vector, such as a DNA, RNA, circular DNA, circular RNA, minicircle, or the like. A polypeptide (e.g., CAR) can be transiently expressed. For example, expression of a polypeptide (e.g., CAR) can be reduced as a nucleic acid that encodes it is degraded. One method of generating engineered cells is through the use of a ribonucleic acid (RNA) system, e.g., a system that involves delivering one or more nucleic acid molecules as an RNA. In some cases, the use of RNA can minimize DNA-induced toxicity and immunogenicity sometimes observed with the use of DNA.

[0238] Cells can be genetically engineered to comprise a nucleic acid moleculethat encodes a polypeptide (e.g., CAR) ex vivo. For example, cells can be taken from a subject in one or more blood draws and/or apheresis procedures, modified ex vivo, optionally selected and/or expanded before and/or after genetic modification, and optionally re-introduced into the subject or a different subject by infusion or injection. [0239] In some cases, a selectable marker is introduced to a cell, e.g., together with or as part of a nucleic acid molecule encoding a polypeptide (e.g., CAR), so that cells that comprise the polypeptide (e.g., CAR) or modification express the selectable marker and can be selected, enriched, or expanded. In some cases, a selectable marker is an antibiotic resistance gene, and cells that do not express the antibiotic resistance gene can be killed by treatment with the antibiotic (e.g., to select or enrich for cells that comprise a polypeptide (e.g., CAR)). In some embodiments, the selectable marker is an epitope tag.

[0240] Expression of a polypeptide (e.g., CAR) can be quantified, for example, by qPCR, RNA sequencing, western blot, or flow cytometry.

[0241] In some embodiments, cells are engineered to express a cytokine or chemokine to exhibit autocrine or paracrine signaling to modulate (e.g., enhance) function of the engineered cells, for example, IL-15, IL-2, IL-7, IL-12, IFN-alpha, IFN-gamma, IL-lbeta, or a functional variant thereof.

[0242] In some embodiments, it can be useful to assess cytotoxicity of engineered cells against target cells using a small number of engineered cells harvested at an intermediate step (e.g., about day 12 of a protocol disclosed herein) to test functionality prior to completing the latter stages of an expansion protocol.

IV. CHIMERIC ANTIGEN RECEPTORS AND HETEROLOGOUS IMMUNE RECEPTORS

[0243] Populations of cells disclosed herein (e.g., polyclonal gamma delta T cells) can be engineered to express a heterologous immune receptor, such as a chimeric antigen receptor (CAR) or T cell receptor. An expression construct, cell, or nucleic acid molecule disclosed herein can comprise a transgene that encodes a heterologous immune receptor.

[0244] A heterologous immune receptor can comprise an extracellular domain (including an extracellular binding domain), a transmembrane domain, and a cytoplasmic signaling domain.

[0245] A heterologous immune receptor can be expressed by an immune cell and configured to induce activation of and/or signaling in the immune cell upon contacting a target cell that expresses a cell surface molecule. A target cell can be a cell that is associated with a disease or condition. A target cell can be a cancer cell. A target cell can be an immune cell. A target cell can be a hematologic cancer cell. A target cell can be a solid tumor cell. A target cell can be a leukemia cell. A target cell can be a lymphoma cell. A target cell can be a myeloma cell. A target cell can be a B cell. A target cell can be a CD19+ cell. A target cell can be a cell that is associated with an autoimmune or inflammatory disease. [0246] In some embodiments, a heterologous immune receptor is a chimeric antigen receptor (CAR). In some embodiments, a heterologous immune receptor is a first, second, third, fourth, or fifth generation CAR. A first generation CAR can contain a single CD3 zeta cytoplasmic signaling domain (e.g., and lack a co-stimulatory cytoplasmic signaling domain). A second generation CAR can comprise a CD3 zeta cytoplasmic signaling domain and a costimulatory cytoplasmic signaling domain, such as a CD28 or 4 IBB costimulatory domain. A third generation CAR can comprise a CD3 zeta cytoplasmic signaling domain and two costimulatory cytoplasmic signaling domains, for example, two of CD28, 41BB, and 0X40. A fourth generation CAR can comprise a CD3 zeta cytoplasmic signaling domain and a costimulatory cytoplasmic signaling domain (such as a CD28 or 4 IBB costimulatory domain), and a protein, such as interleukin 12 (IL-12), that is constitutively or inducibly expressed upon CAR activation. A fourth generation CAR can be, for example, a T cell redirected for universal cytokine-mediated killing (TRUCK). A fifth generation CAR can be based on a second generation CAR and contain a truncated cytoplasmic IL-2 receptor P-chain domain with a binding site for the transcription factor STAT3.

[0247] In some embodiments, a heterologous immune receptor is a universal CAR, for example, an extracellular binding domain can be combined with amino acid sequence(s) from one or more components of a TCR signaling complex and/or a chimeric antigen receptor (CAR) to generate a “universal” heterologous immune receptor that can be armed and disarmed based on the presence of adapter molecule(s). An adapter molecule can direct an immune cell expressing the heterologous immune receptor to a target cell (e.g., a cancer cell), and upregulate activation of the immune cell upon encountering the target cell (e.g., leading to a cytotoxic response against the target cell). A universal CAR can be capable of binding to various adapter molecules that can confer target specificity.

[0248] In some embodiments, a heterologous immune receptor is a dual CAR, a split CAR, or an inducible split CAR. A dual CAR can comprise two CARs with different extracellular binding domains, and thus signal induction based on two target antigens. A split CAR can comprise two CARs with different extracellular binding domains and separation of costimulatory domains (e.g., CD28 and 4 IBB) from CD3zeta on the distinct CAR polypeptides, thereby requiring engagement of both CARs for T cell activation.

[0249] A heterologous immune receptor can comprise a component of a TCR signaling complex, for example, an extracellular domain, transmembrane domain, and/or cytoplasmic domain of a TCR signaling complex, such as a human TCR signaling complex. [0250] In some embodiments, a heterologous immune receptor that comprises a component of a TCR signaling complex comprises two TCR chains (e.g., an alpha chain and a beta chain, or a gamma chain and a delta chain). In some embodiments, a heterologous immune receptor that comprises a component of a TCR signaling complex comprises a single chain TCR (scTCR), e.g., comprising a TCR alpha chain variable domain and a TCR beta chain variable domain joined by a suitable linker.

[0251] In some embodiments, a heterologous immune receptor that comprises a component of a TCR signaling complex is a TCR, e.g., comprises an extracellular binding domain that comprises TCR variable regions and TCR CDRs. In some embodiments, a heterologous immune receptor that comprises a component of a TCR signaling complex is not TCR, for example, comprises a non-TCR extracellular binding domain, and comprises a component of a TCR signaling complex.

[0252] A heterologous immune receptor can comprise a full length or substantially full length CD3 gamma, CD3 delta, CD3 epsilon, or CD3 zeta. A heterologous immune receptor can comprise a full length or substantially full length TCR alpha chain (e.g., constant regions, or variable plus constant regions), TCRbeta chain (e.g., constant regions, orvariable plus constant regions), TCR gamma chain (e.g., constant regions, or variable plus constant regions), or TCR delta chain (e.g., constant regions, or variable plus constant regions).

[0253] In some embodiments, a system disclosed herein allows use of two or more heterologous immune receptors (e.g., CARs) delivered alone or in combination, concurrently or sequentially.

[0254] A heterologous immune receptor disclosed herein can comprise an extracellular domain. The extracellular domain can comprise an extracellular binding domain that can specifically bind to a cell surface molecule on a target cell, thereby modulating signaling by the heterologous immune receptor.

[0255] An extracellular binding domain can utilize one or more antigen-binding domains, for example, an antigen-binding domain of or derived from an antibody. In some embodiments, an extracellular binding domain disclosed herein comprises an antigen-binding domain or fragment from an antibody, such as an scFv or a nanobody.

[0256] An extracellular binding domain of the disclosure can comprise complementarity determining regions (CDRs). For example, an antibody, antigen-binding fragment thereof, or antigen-binding domain can comprise CDRs. In some embodiments, the CDRs determine or substantially determine binding specificity and/or affinity for a surface molecule on a target cell. For example, the CDRs can be grafted onto a different suitable framework, or the framework region can be altered (e.g., via amino acid substitutions, deletions, and/or insertions), and the antigen-binding fragment or domain can retain binding for the target, and the extracellular binding domain remains functional despite the alterations outside of the CDRs.

[0257] An extracellular binding domain can comprise an antibody fragment, antigen-binding domain, or antigen-binding fragment of an antibody. Non-limiting examples of antibody fragments, antigen-binding fragments, and antigen-binding domains include Fab, Fab', F(ab')2, dimers and trimers of Fab conjugates, Fv, scFv, nanobodies, minibodies, dia-, tria-, and tetrabodies, and linear antibodies. Fab and Fab' are antigen-binding fragments that can comprise the VH and CHI domains of the heavy chain linked to the VL and CL domains of the light chain via a disulfide bond. A F(ab')2 can comprise two Fab or Fab' that are joined by disulfide bonds. A Fv can comprise the VH and VL domains held together by non-covalent interactions. A scFv (single-chain variable fragment) is a fusion protein that can comprise the VH and VL domains connected by a peptide linker. Manipulation of the orientation of the VH and VL domains and the linker length can be used to create different forms of molecules that can be monomeric, dimeric (diabody), trimeric (triabody), or tetrameric (tetrabody). Minibodies can be scFv-CH3 fusion proteins that assemble into bivalent dimers.

[0258] The extracellular binding domain can be or can comprise a single domain antibody. The single domain antibody can be or can comprise a variable region of a heavy chain only antibody. Such a single domain antibody can also be known as a nanobody or VHH. The single domain antibody can be, for example, a variable region from or derived from a heavy chain only antibody from a camelid (e.g., camels: one-humped Camelus dromedaries and two-humped Camelus bactrianus; llamas: Lama glama, Lama guanicoe, and Lama vicugna; and alpacas: Vicugna pacos), a shark (e.g., a nurse shark), a wobbegong, or a spotted ratfish. Such animals have a special type of antibody called heavy chain Abs (HCAbs), that lack the entire light chain and the first heavy chain C region (CHI) compared to regular antibodies.

[0259] An extracellular binding domain can comprise an antigen-binding domain or fragment of a chimeric, humanized, or fully human antibody. An extracellular binding domain can comprise CDRs grafted onto a humanized or fully human framework sequence.

[0260] In addition to antibodies and antigen-binding fragments or domains thereof, other compounds can also comprise antigen-binding domains that can be used in compositions, systems, and methods of the disclosure, such as in an extracellular binding domain of a heterologous immune receptor. Non-limiting examples of non-antibody antigen-binding compounds include ankyrin proteins, ankyrin repeat proteins, designed ankyrin repeat proteins (DARPins), affibodies, avimers, adnectins, anticalins, Fynomers, Kunitz domains, knottins, 0- hairpin mimetics, and receptors and derivatives thereof.

[0261] In some embodiments, an extracellular binding domain binds to a target (e.g., antigen) expressed on or associated with a hematologic cancer cell or cell type. In some embodiments, an extracellular binding domain binds to a target (e.g., antigen) expressed on or associated with a solid tumor cell or cell type.

[0262] In some embodiments, an extracellular binding domain binds to CD 19, ACE2, an Fc domain, APRIL, BAFFR, B7H6, B7H3, BCMA, CA9, CAIX, carcinoembryonic antigen, CD133, CD16, CD174, CD22, CD23, CD27, CD274, CD276, CD33, CD38, CD44, CD5, CD70, CEACAM5, CSPG4, CTLX, DNAM-1, Dsg3, E13Y IL13, E3 adnectin, EGFR, EGFRvIII, Envs, EPC AM, EPHA2, EPHB4, EPHRIN B2, ErbB, ERBB2, FAP, fibroblast activation protein, FLT3, FLT3L, FOLH1, FOLR1, FSH, FSHR, GD2, glycoprotein B, glycoprotein E2, GMCSF, GMR, gpl20, gp41, GPC3, GPNMB, HBsAg, HER2, ICAM-I, IL10, IL10R, IL11, IL1 IRa, IL13Ra2, IL1RAP, IL3RA, Insulin-B chain, Islet-specific glucose-6- phosphatase catalytic subunit-related protein, KDR, LI CAM, LFA-1, M2e, mesothelin, MET, MICA, MICB, MPL, MS4A1, MSLN, MUC1, myelin oligodendrocyte glycoprotein, NCAM1, Nectin-2, NKG2D, NKp30, PDCD1, PSCA, PSMA, PVR, ROR1, SARS-CoV2 S protein, SDC1, SLAMF7, SSTR, TIE, TACI, TEM1, TNFRSF17, TNFRSF8, TPO, transmembrane form of IgE, TriPRIL, ULBP1, ULBP1-6, ULBP2, or VEGFR2. In some embodiments, an extracellular binding domain binds to CD19. In some embodiments, the extracellular binding domain comprises an FMC63 scFv.

[0263] In some embodiments, an extracellular binding domain is or comprises a component of a receptor or a receptor ligand, for example, utilizes the naturally occurring specificity of a receptor or ligand. For example, an extracellular binding domain can comprise a receptorbinding domain or ligand-binding domain of B7H6, an Fc domain, APRIL, BAFF, BCMA, CD16, CD27, CD70, CTLX, DNAM-1, E13Y IL13, E3 adnectin, EGFR, EPHB4, EPHRIN B2, ErbBl, ErbB2, ErbB3, ErbB4, FLT3, FLT3L, FSH, FSHR, GMCSF, GMR, ICAM-I, IL10, IL10R, IL11, IL1 IRa, IL13Ra2, LFA-1, MICA, MICB, MPL, Nectin-2, or NKG2D.

[0264] In some embodiments, an extracellular binding domain is or comprises an autoantigen targeted by immune cells in an autoimmune disorder, or an epitope thereof. For example, a heterologous binding domain can comprise an autoantibody target, such as DSG3, factor VIII (FVIII), or an epitope thereof. The heterologous immune receptor can be, for example, a chimeric autoantibody receptor (CAAR). [0265] In some embodiments, an extracellular binding domain binds to an autoimmunity- associated target, for example, muscle-associated receptor tyrosine kinase (MuSK), insulin peptide-major histocompatibility complex (MHC) class II complex or insulin.

[0266] The heterologous immune receptor can comprise one or more additional extracellular domains as well as the extracellular binding domain.

[0267] In some embodiments, a heterologous immune receptor comprises an additional extracellular domain or amino acid sequence that is a linker or spacer. In some embodiments, a heterologous immune receptor comprises a hinge, such as an IgG hinge or a CD8 hinge.

[0268] A heterologous immune receptor can comprise a transmembrane domain. Any suitable transmembrane domain can be used. In some embodiments, the heterologous immune receptor comprises a transmembrane domain of CD8. In some embodiments, the heterologous immune receptor comprises a transmembrane domain of CD28. In some embodiments, the heterologous immune receptor comprises a transmembrane domain of 0X40, 4 IBB, or CD86.

[0269] The transmembrane domain can be a transmembrane domain of an immune receptor or TCR signaling complex component disclosed herein, for example, of a mammalian or a human TCR signaling complex. The transmembrane domain can comprise, for example, a transmembrane domain of TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, CD3 gamma, CD3 delta, CD3 epsilon, or CD3 zeta. In some embodiments, a transmembrane domain is not from an immune receptor or is not from a TCR signaling complex component.

[0270] A heterologous immune receptor can comprise a cytoplasmic domain or a mutant, variant, or derivative thereof. A cytoplasmic domain can comprise a cytoplasmic signaling domain or a mutant, variant, or derivative thereof. The cytoplasmic signaling domain can contribute to the ability of the heterologous immune receptor to elicit a response. For example, the cytoplasmic signaling domain can contribute to induction of signaling and/or immune cell activation upon of binding of the heterologous immune receptor (e.g., an extracellular binding domain thereof) to a surface molecule of a target cell. In some cases, the cytoplasmic signaling domain can contribute to the induction of a pro-inflammatory response, an anti-cancer immune response, an immune tolerance-promoting response, a transcriptional response, TCR signaling, T cell activation, T cell proliferation, cytokine production, a cytotoxic response against the target cell, or a combination thereof. In some cases, the cytoplasmic signaling domain can contribute to the activation of bystander immune cells that do not comprise a heterologous immune receptor of the disclosure. A cytoplasmic signaling domain can enhance the proliferation, survival, and/or function of immune cells, and/or development of effector and/or memory immune responses (e.g., memory T cells).

[0271] A cytoplasmic signaling domain can partake in an immune cell activation pathway that involves, for example, phosphorylation, dephosphorylation, calcium release, ubiquitination, de-ubiquitination, proteolytic cleavage, protein-protein interactions, a transcriptional response, or a combination thereof. An immune cell activation pathway can comprise, for example, an innate, adaptive, STING, NFkB, inflammasome, TCR, BCR, JAK/STAT, TLR, NLR, RLR, costimulatory, co-inhibitory, cytokine, or chemokine signaling pathway. A cytoplasmic signaling domain can comprise one or more immunoreceptor tyrosine-based activation motifs (IT AMs). A cytoplasmic signaling domain can comprise one or more immunoreceptor tyrosine-based inhibition motifs (ITIMs).

[0272] In some embodiments, the heterologous immune receptor contains a cytoplasmic signaling domain of CD3 zeta or a functional fragment thereof. In some embodiments, the heterologous immune receptor contains a cytoplasmic signaling domain of CD3 zeta with 1 , 2, or 3 functional or active ITAMs. In some embodiments, the heterologous immune receptor contains a cytoplasmic signaling domain of CD3 zeta with one inactivated ITAM or two inactivated ITAMs. In some embodiments, the heterologous immune receptor does not contain a cytoplasmic signaling domain of CD3 zeta or a functional fragment thereof.

[0273] A heterologous immune receptor can comprise a cytoplasmic signaling domain of a T cell signal two costimulatory signaling domain, or a functional fragment thereof. In some embodiments, a heterologous immune receptor does not contain a cytoplasmic signaling domain of a T cell signal two costimulatory signaling domain.

[0274] A cytoplasmic domain or cytoplasmic signaling domain can be derived from and/or interact with a kinase, (e.g., a protein kinase, a tyrosine kinase or a serine/threonine kinase, a receptor tyrosine kinase, a lipid kinase, a phosphoinositide kinase, a carbohydrate kinase, or a combination thereof), a phosphatase, a ubiquitin ligase, a caspase, an adapter protein, a transcription factor, an ion channel, or a combination thereof. A cytoplasmic domain or cytoplasmic signaling domain can contribute to interaction of the heterologous immune receptor with additional proteins or factors (e.g., members of a complex and/or signal transduction pathway).

[0275] A heterologous immune receptor can comprise a cytoplasmic signaling domain of a costimulatory immune receptor, or a functional fragment thereof. Non-limiting examples of costimulatory immune receptors include CD28, 2B4 (CD244, SLAMF4), 4-1BB (CD137), CD2 (LFA2, 0X34), CD21, CD226 (DNAM1), CD27 (TNFRSF7), CD30 (TNFRSF8), CD4, CD40, CD8, CD84 (SLAMF5), CRACC (CD319, BLAME), CRTAM (CD355), DcR3, DR3 (TNFRSF25), GITR (CD357), HVEM (CD270), ICOS (CD278), LIGHT, LT R (TNFRSF3), Lyl08 (NTBA, CD352, SLAMF6), Ly9 (CD229,SLAMF3), 0X40 (CD134), SLAM (CD150, SLAMF1), TIM1 (HAVCR1, KIMI), and TIM2. In some embodiments, a heterologous immune receptor does not contain a cytoplasmic signaling domain of a costimulatory immune receptor.

[0276] A heterologous immune receptor can comprise a cytoplasmic signaling domain of a co-inhibitory immune receptor, or a functional fragment thereof. A heterologous immune receptor can comprise a cytoplasmic signaling domain of an activating NK receptor or inhibitory NK receptor, or a functional fragment thereof.

[0277] A heterologous immune receptor can comprise a cytoplasmic signaling domain that is a component of a TCR signaling complex, for example, of a mammalian or a human TCR signaling complex. The cytoplasmic signaling domain can comprise, for example, a cytoplasmic signaling domain of CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, a functional fragment thereof, or a combination thereof.

[0278] In some cases, a heterologous immune receptor of the disclosure does not contain a cytoplasmic signaling domain, but can nonetheless elicit an immune cell activation signal, for example, via a cytoplasmic signaling domain in another protein that can associate with the heterologous immune receptor. In some embodiments, a heterologous immune receptor of the disclosure that comprises constant regions from one or more TCR chains can transmit an immune cell activation signal via associated CD3 proteins that comprise cytoplasmic signaling domains (e.g., CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 eta, or a combination thereof).

[0279] A heterologous immune receptor can comprise one or more cytoplasmic signaling domains or mutants, variants, or derivatives thereof. A heterologous immune receptor can comprise, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cytoplasmic signaling domains or mutants, variants, or derivatives thereof. A heterologous immune receptor can comprise at least one, at least two, at least three, at least four, or at least five cytoplasmic signaling domains. A heterologous immune receptor can comprise at most one, at most two, at most three, at most four, at most five, or at most ten cytoplasmic signaling domains.

[0280] A cytoplasmic signaling domain can be from a mammalian protein. In some cases, a cytoplasmic signaling domain is from a murine (mouse) protein. In some cases, a cytoplasmic signaling domain is from a human protein. In some cases, a cytoplasmic signaling domain can comprise modifications compared to a wild type sequence. [0281] A heterologous immune receptor of the disclosure can comprise one or more linkers for example, between different domains of the protein. A linker can be a chemical bond, for example, a covalent bond or a non-covalent bond. A linker as described herein can include a flexible or rigid linker. A linker can be a peptide. A linker can be selected to achieve a desired functionality of the heterologous immune receptor. A linker can comprise a linker sequence, for example, a linker peptide sequence. The length a linker can be adjusted to allow for proper folding or to increase or decrease biological activity of the heterologous immune receptor.

[0282] A flexible linker can have a sequence containing glycine residues. The small size of the glycine residues can provide flexibility, and allow for mobility of the connected protein domains. The incorporation of serine or threonine can maintain the stability of the linker in aqueous conditions by forming hydrogen bonds with the water molecules, thereby reducing unfavorable interactions between the linker and protein moieties. In some cases, flexible linkers can also contain additional amino acids, such as threonine and alanine, to maintain flexibility, and/or polar amino acids such as lysine and glutamine, to improve solubility.

[0283] A rigid linker can have, for example, an alpha helix-structure. An alpha-helical rigid linker can act as a spacer between protein domains. A rigid linker can have a proline-rich sequence, (XP)n, with X designating alanine, lysine, glutamine, or any amino acid, and n designating a number of repeats. The presence of proline in non-helical linkers can increase stiffness, and allow for effective separation of protein domains.

[0284] A linker can comprise a hinge region, for example an amino acid sequence derived from a hinge region of an antibody or immune receptor. In some embodiments, a linker comprises a hinge region from CD8a, IgGl, or IgG4.

[0285] In some embodiments, a heterologous immune receptor is a chimeric antigen receptor known as or within an engineered cell or construct known as Tisagenlecleucel (Kymriah®), Axicabtagene Ciloleucel (Yescarta®), Brexucabtagene Autoleucel (Tecartus™), Lisocabtagene maraleucel, Idecabtagene Vicleucel, or KTE-X19.

V. PHARMACEUTICAL COMPOSITIONS

[0286] Pharmaceutical compositions of the present disclosure can comprise a composition disclosed herein and a pharmaceutically acceptable excipient. A pharmaceutical composition can comprise, for example, a pharmaceutically acceptable excipient, vehicle, carrier, or diluent, and an expression construct or nucleic acid molecule, vector, and/or a cell (e.g., engineered cell) disclosed herein. A pharmaceutical composition can be formulated, for example, for systemic, local, parenteral, intratumoral, intravenous, intraperitoneal, subcutaneous, transdermal, or intramuscular administration. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like. Sterile phosphate-buffered saline is one example of a pharmaceutically suitable excipient.

[0287] A pharmaceutical composition can comprise a population of cells in a unit dosage form. A pharmaceutical composition can comprise a nucleic acid molecule or vector comprising the nucleic acid molecule in a unit dosage form.

[0288] In some cases, unit dosage forms, include, but are not limited to, sterile or substantially sterile parenteral solutions or suspensions, tablets, capsules, pills, powders, granules, oral solutions or suspensions, and oil water emulsions.

[0289] A formulation or composition described herein can be an aqueous solution. Compositions in some examples herein are provided as sterile or substantially sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Compositions described herein can also comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof. Sterile injectable solutions containing the cells can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.

[0290] Formulations of cells can include those for systemic, local, parenteral, intratumoral, intravenous, intraperitoneal, subcutaneous, transdermal, or intramuscular administration.

VI. THERAPEUTIC METHODS

[0291] Disclosed herein are methods that comprise administering to a subject a population of cells, for example, gamma delta T cells, such as polyclonal gamma delta T cells generated by a method disclosed herein. The cells can be administered as part of a pharmaceutical composition.

[0292] The methods can comprise treating a subject in need thereof. The subject can have a disease or condition, such as a cancer. The cancer can be a solid tumor. The cancer can be a liquid tumor. The cancer can be a hematologic tumor. The cancer can be an immune cell cancer. The cancer can be a B cell cancer.

[0293] A population of gamma delta T cells, for example, engineered polyclonal gamma delta T cells generated by a method disclosed herein, can be employed in treating a subject with cancer or an immune disorder. The cancer can be any type cancer including lymphoma, (e.g., mantle cell, diffuse large B cell, follicular, lymphoplasmacytic, marginal zone B-cell, small-cell lymphocytic, Burkitt, primary central nervous system, primary intraocular lymphoma, etc.), leukemia (e.g., chronic lymphocytic, acute lymphoblastic, hairy cell, chronic myeloid, etc), myelodysplastic syndromes, myeloproliferative disorder, or multiple myeloma. Immune disorders can encompass any dysregulation of the immune system, including autoimmune disorders (e.g., systemic lupus erythematosus, scleroderma, Sjogren’s syndrome, multiple sclerosis, rheumatoid arthritis, Hashimoto’s thyroiditis, antiphospholipid syndrome, celiac disease, Grave’s disease, myasthenic syndrome, myasthenia gravis, polyangiitis, dematomyositis, scleromyositis, pemphigus vulgaris, etc.).

[0294] In some embodiments, a population of gamma delta T cells, for example, engineered polyclonal gamma delta T cells, are used to treat a subject with a B cell related leukemia such as acute lymphoblastic leukemia, myeloma, the non-Hodgkin lymphomas (NHL) mantle cell lymphoma (MCL), or diffuse large B cell lymphoma (DLBCL).

[0295] In some embodiments, the subject has an infectious disease. In some embodiments, a method disclosed herein can be used for treating an infectious disease, for example, a viral, bacterial, or parasitic infection.

[0296] The population of cells can be administered in an amount effective to treat or prevent a disease or condition. “Treatment” (and grammatical variations thereof such as “treat” or “treating”) can refer to clinical intervention in an attempt to alter the natural course of the individual (subject) being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment can include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0297] A population of cells can be administered to a subject, for example, by parenteral administration. A population of cells can be administered to a subject, for example, by intravenous, intraperitoneal, intramuscular, subdermal, intracerebral, intracerebroventricular, intra-articular, intraarterial, intrathecal, intracapsular, subcapsular, intraorbital, intracardiac, intradermal, subcutaneous, subarachnoid, or intracranial injection or infusion. The administration can be via localized injection or infusion. The administration can be via systemic injection or infusion. The administration can be via intravenous injection or infusion. The administration can be via intratumoral injection or infusion. The administering can be local. The administering can be systemic.

[0298] Various dosing schedules can be used. In some embodiments, a population of cells is administered to a subject once. In some embodiments, the population of cells is administered to a subject two or more times. [0299] The population of cells can be autologous to the subject. The population of cells can be allogeneic to the subject, for example, from a related or unrelated donor. The population of cells can be haploidentical to the subject. The population of cells can be HLA-matched to the subject.

VII. EMBODIMENTS

[0300] Embodiment 1. A method of generating an expanded population of therapeutic cells, the method comprising: (a) seeding at least 8 xl0^A5 gamma delta T cells per square cm in a culture vessel comprising a gas permeable membrane submerged under a column of medium; and (b) incubating the gamma delta T cells in an expansion culture medium comprising a gamma delta T cell receptor (gdTCR) stimulating agent.

[0301] Embodiment 2. The method of embodiment 1, wherein the culture vessel is a Gas permeable Rapid Expansion (GREX®) cell culture vessel.

[0302] Embodiment 3. The method of any one of the preceding embodiments, wherein at least 1 xlO^A6 of the gamma delta T cells are seeded per square cm.

[0303] Embodiment 4. The method of any one of the preceding embodiments, wherein 1-1.5 xlO^A6 of the gamma delta T cells are seeded per square cm.

[0304] Embodiment 5. The method of any one of the preceding embodiments, wherein the gamma delta T cells expand at least 2,000-fold.

[0305] Embodiment 6. The method of any one of the preceding embodiments, wherein the gamma delta T cells expand at least 5,000-fold.

[0306] Embodiment?. The method of any one of the preceding embodiments, wherein the gdTCR stimulating agent comprises an anti-gdTCR antibody or antigen-binding fragment thereof.

[0307] Embodiment 8. The method of any one of the preceding embodiments, wherein the gdTCR stimulating agent comprises a pan anti-gdTCR antibody or antigen-binding fragment thereof.

[0308] Embodiment 9. The method of any one of embodiments 7-8, wherein the anti-gdTCR antibody is bound to a surface of the culture vessel.

[0309] Embodiment 10. The method of any one of the preceding embodiments, wherein the expansion culture medium further comprises an anti-CD28 antibody or antigen-binding fragment thereof.

[0310] Embodiment 11. The method of any one of the preceding embodiments, wherein the expansion culture medium and the culture vessel lack an anti-CD3 antibody or antigen-binding fragment thereof. [0311] Embodiment 12. The method of any one of the preceding embodiments, wherein after the incubating, the population of gamma delta T cells comprises at least 10% Vdl+ cells and at least 10% Vd2+ cells.

[0312] Embodiment 13. The method of any one of the preceding embodiments, wherein after the incubating, the gamma delta T cells comprise at least 1% Vdl- Vd2- gamma delta T cells.

[0313] Embodiment 14. The method of any one of the preceding embodiments, wherein after the incubating, the gamma delta T cells comprise at least 11% Vd3+ cells.

[0314] Embodiment 15. The method of any one of the preceding embodiments, wherein the method further comprises electroporating the gamma delta T cells to introduce a transgene encoding a chimeric antigen receptor into the gamma delta T cells.

[0315] Embodiment 16. The method of embodiment 15, wherein the method further comprises (i) following the electroporating, incubating the gamma delta T cells in recovery medium for about 1-2 hours, and(iii) after the incubating in the recovery medium for about 1-2 hours, adjusting the cell concentration to about 1-1.5 x 10^A6 cells per m .

[0316] Embodiment 17. The method of embodiment 15 or 16, wherein the method further comprises, prior to the electroporating, incubating the gamma delta T cells in the expansion culture medium at a volume of about 300-400 pL per square cm.

[0317] Embodiment 18. The method of any one of the preceding embodiments, further comprising assaying the population of gamma delta T cells to determine viability.

[0318] Embodiment 19. The method of any one of the preceding embodiments, further comprising assaying the population of gamma delta T cells to determine polyclonal gdTCR phenotype.

[0319] Embodiment 20. The method of any one of the preceding embodiments, further comprising assaying the population of gamma delta T cells to determine expression of exhaustion markers.

[0320] Embodiment 21. The method of any one of the preceding embodiments, wherein the incubating is at about 37°C.

[0321] Embodiment 22. The method of any one of the preceding embodiments, further comprising enriching the gamma delta T cells to reduce the frequency of non-gamma delta T cells.

[0322] Embodiment 23. The method of any one of the preceding embodiments, wherein gamma delta T cells are incubated in the culture vessel comprising a gas permeable membrane submerged under a column of medium for at least 20 days. [0323] Embodiment 24. The method of any one of the preceding embodiments, wherein the gamma delta T cells are incubated in the culture vessel comprising a gas permeable membrane submerged under a column of medium for about 2-25 days.

[0324] Embodiment 25. The method of any one of the preceding embodiments, further comprising dislodging adherent gamma delta T cells from the culture vessel.

[0325] Embodiment 26. The method of any one of embodiments 15-25, further comprising harvesting the gamma delta T cells after culturing for about 21 days in the expansion culture medium after the electroporating.

[0326] Embodiment 27. The method of any one of the preceding embodiments, further comprising quantifying glucose and lactate after a period of incubation of the population of polyclonal gamma delta T cells in the expansion culture medium.

[0327] Embodiment 28. The method of any one of the preceding embodiments, wherein the gamma delta T cells retain cytotoxic capacity after three rounds of rechallenge with target cells.

[0328] Embodiment 29. The method of any one of the preceding embodiments, wherein the method provides at least 1 x 10^A8 gamma delta T cells.

[0329] Embodiment 30. The method of any one of the preceding embodiments, wherein the method provides at least 1 x 10^A9 gamma delta T cells.

[0330] Embodiment 31. The method of any one of the preceding embodiments, wherein the method provides at least 1 x 10^Al 0 gamma delta T cells.

[0331] Embodiment 32. The method of any one of the preceding embodiments, wherein the method provides at least 100 patient doses of a cell therapy product.

[0332] Embodiment 33. The method of any one of the preceding embodiments, wherein the method provides at least 200 patient doses of a cell therapy product.

[0333] Embodiment 34. The method of any one of the preceding embodiments, wherein the method provides at least 500 patient doses of a cell therapy product.

[0334] Embodiment 35. A method of generating an expanded population of therapeutic cells, the method comprising: (a) incubating a population of engineered polyclonal gamma delta T cells in a culture vessel in an expansion culture medium comprising a gamma delta T cell receptor (gdTCR) stimulating agent; and (b) treating the population of engineered polyclonal gamma delta T cells in the expansion culture medium every 2-3 days with: (i) IL-2 freshly added to a final concentration of at least 1000 lU/mL, (ii) IL-7 freshly added to a final concentration of at least 5 ng/mL, and (iii) IL-15 freshly added to a final concentration of at least 5 ng/mL.

[0335] Embodiment 36. The method of embodiment 35, wherein the culture vessel is a Gas permeable Rapid Expansion (GREX®) cell culture vessel. [0336] Embodiment 37. The method of embodiment 36, wherein at least 1 xlO^A6 of the gamma delta T cells are seeded per square cm of the GREX cell culture vessel.

[0337] Embodiment 38. The method of embodiment 37, wherein 1-1.5 xlO^A6 of the gamma delta T cells are seeded per square cm of the GREX cell culture vessel.

[0338] Embodiment 39. The method of any one of embodiments 35-38, wherein the population of engineered polyclonal gamma delta T cells expands at least 2,000-fold.

[0339] Embodiment 40. The method of any one of embodiments 35-38, wherein the population of engineered polyclonal gamma delta T cells expands at least 5,000-fold.

[0340] Embodiment 41 . The method of any one of embodiments 35-40, wherein the gdTCR stimulating agent comprises an anti-gdTCR antibody or antigen-binding fragment thereof.

[0341] Embodiment 42. The method of any one of embodiments 35-41, wherein the gdTCR stimulating agent comprises a pan anti-gdTCR antibody or antigen-binding fragment thereof.

[0342] Embodiment 43. The method of any one of embodiments 41-42, wherein the anti- gdTCR antibody is bound to a surface of the culture vessel.

[0343] Embodiment 44. The method of any one of embodiments 35-43, wherein the expansion culture medium further comprises an anti-CD28 antibody or antigen-binding fragment thereof.

[0344] Embodiment 45. The method of any one of embodiments 35-44, wherein the expansion culture medium and the culture vessel lack an anti-CD3 antibody or antigen-binding fragment thereof.

[0345] Embodiment46. The method of any one of embodiments 35-45, wherein after the incubating, the population of polyclonal gamma delta T cells comprises at least 10% Vdl+ cells and at least 10% Vd2+ cells.

[0346] Embodiment47. The method of any one of embodiments 35-46, wherein after the incubating, the population of polyclonal gamma delta T cells comprises at least 1% Vdl- Vd2- gamma delta T cells.

[0347] Embodiment48. The method of any one of embodiments 35-47, wherein after the incubating, the population of polyclonal gamma delta T cells comprises at least 11% Vd3+ cells.

[0348] Embodiment49. The method of any one of embodiments 35-48, wherein the method further comprises electroporating to introduce a transgene encoding a chimeric antigen receptor into a population of polyclonal gamma delta T cells, thereby generating the population of engineered polyclonal gamma delta T cells.

[0349] Embodiment 50. The method of embodiment 49, wherein following the electroporating, the method comprises incubating the engineered polyclonal gamma delta T cells in recovery medium for about 1-2 hours, and (iii) after the incubating in the recovery medium for about 1-2 hours, adjusting the cell concentration to about 1-1.5 x 10^A6 cells per m .

[0350] Embodiment 51. The method of any one of embodiments 49-50, wherein the method further comprises, prior to the electroporating, incubating the polyclonal gamma delta T cells in the expansion culture medium at a volume of about 300-400 pL per square cm.

[0351] Embodiment 52. The method of any one of embodiments 35-51, wherein viral vectors are not used to generate the engineered polyclonal gamma delta T cells.

[0352] Embodiment 53. The method of any one of embodiments 35-52, wherein the method further comprises genomically integrating a transgene encoding a chimeric antigen receptor.

[0353] Embodiment 54. The method of embodiment 53, wherein the genomically integrating utilizes a transposon-based system.

[0354] Embodiment 55. The method of any one of embodiments 35-54, further comprising assaying the population of engineered polyclonal gamma delta T cells to determine viability.

[0355] Embodiment 56. The method of any one of embodiments 35-55, further comprising assaying the population of engineered polyclonal gamma delta T cells to determine polyclonal gdTCR phenotype.

[0356] Embodiment 57. The method of any one of embodiments 35-56, further comprising assaying the population of engineered polyclonal gamma delta T cells to determine expression of exhaustion markers.

[0357] Embodiment 58. The method of any one of embodiments 35-57, wherein the incubating is at about 37°C.

[0358] Embodiment 59. The method of any one of embodiments 35-58, further comprising enriching the polyclonal gamma delta T cells to reduce the frequency of non-gamma delta T cells.

[0359] Embodiment 60. The method of any one of embodiments 35-59, further comprising dislodging adherent gamma delta T cells from the culture vessel.

[0360] Embodiment 61. The method of any one of embodiments 49-60, further comprising harvesting cells after culturing for about 21 days in the expansion culture medium after the electroporating.

[0361] Embodiment 62. The method of any one of embodiments 35-61, further comprising quantifying glucose and lactate after a period of incubation of the population of engineered polyclonal gamma delta T cells in the expansion culture medium. [0362] Embodiment 63. The method of any one of embodiments 35-62, wherein the engineered polyclonal gamma delta T cells retain cytotoxic capacity after three rounds of rechallenge with target cells.

[0363] Embodiment 64. The method of any one of embodiments 35-63, wherein the engineered polyclonal gamma delta T cells exhibit at least 50% higher killing of target cells compared to a control population of cells that is not polyclonal.

[0364] Embodiment 65. The method of any one of embodiments 35-64, wherein the engineered polyclonal gamma delta T cells exhibit at least 2-fold increased proliferation in response to target cells compared to a control population of cells that is not polyclonal.

[0365] Embodiment 66. The method of any one of embodiments 35-65, wherein the engineered polyclonal gamma delta T cells exhibit at least 10% reduced exhaustion compared to a control population of cells that is not polyclonal.

[0366] Embodiment 67. The method of any one of embodiments 35-66, wherein the method provides at least 1 x 10^A8 engineered polyclonal gamma delta T cells.

[0367] Embodiment 68. The method of any one of embodiments 35-66, wherein the method provides at least 1 x 10^A9 engineered polyclonal gamma delta T cells.

[0368] Embodiment 69. The method of any one of embodiments 35-66, wherein the method provides at least 1 x 10^Al 0 engineered polyclonal gamma delta T cells.

[0369] Embodiment 70. The method of any one of embodiments 35-69, wherein the method provides at least 100 patient doses of a cell therapy product.

[0370] Embodiment 71 . The method of any one of embodiments 35-69, wherein the method provides at least 200 patient doses of a cell therapy product.

[0371] Embodiment 72. The method of any one of embodiments 35-69, wherein the method provides at least 500 patient doses of a cell therapy product.

[0372] Embodiment 73. A method of generating an expanded population of therapeutic cells, the method comprising: (a) engineering a population of polyclonal gamma delta T cells to express a chimeric antigen receptor, thereby providing a population of engineered polyclonal gamma delta T cells; and (b) incubating the population of engineered polyclonal gamma delta T cells in a culture vessel in an expansion culture medium comprising a gamma delta T cell receptor (gdTCR) stimulating agent; wherein the population of engineered polyclonal gamma delta T cells expands at least 1 ,000- fold.

[0373] Embodiment 74. The method of embodiment 73, wherein the culture vessel is a Gas permeable Rapid Expansion (GREX®) cell culture vessel. [0374] Embodiment 75. The method of embodiment 74, wherein at least 1 xlO^A6 of the engineered polyclonal gamma delta T cells are seeded per square cm of the GREX cell culture vessel.

[0375] Embodiment 76. The method of embodiment 74, wherein 1-1.5 xlO^A6 of the gamma delta T cells are seeded per square cm of the GREX cell culture vessel.

[0376] Embodiment 77. The method of any one of embodiments 73-76, wherein the population of engineered polyclonal gamma delta T cells expands at least 2,000-fold.

[0377] Embodiment 78. The method of any one of embodiments 73-77, wherein the population of engineered polyclonal gamma delta T cells expands at least 5,000-fold.

[0378] Embodiment 79. The method of any one of embodiments 73-78, wherein the gdTCR stimulating agent comprises an anti-gdTCR antibody or antigen-binding fragment thereof.

[0379] Embodiment 80. The method of any one of embodiments 73-79, wherein the gdTCR stimulating agent comprises a pan anti-gdTCR antibody or antigen-binding fragment thereof.

[0380] Embodiment 81. The method of any one of embodiments 79-80, wherein the anti- gdTCR antibody is bound to a surface of the culture vessel.

[0381] Embodiment 82. The method of any one of embodiments 73-81, wherein the expansion culture medium further comprises an anti-CD28 antibody or antigen-binding fragment thereof.

[0382] Embodiment 83. The method of any one of embodiments 73-82, wherein the expansion culture medium and the culture vessel lack an anti-CD3 antibody or antigen-binding fragment thereof.

[0383] Embodiment 84. The method of any one of embodiments 73-83, wherein after the incubating, the population of engineered polyclonal gamma delta T cells comprises at least 10% Vdl+ cells and at least 10% Vd2+ cells.

[0384] Embodiment 85. The method of any one of embodiments 73-83, wherein after the incubating, the population of engineered polyclonal gamma delta T cells comprises at least 1% Vdl- Vd2- gamma delta T cells.

[0385] Embodiment 86. The method of any one of embodiments 73-85, wherein after the incubating, the population of polyclonal gamma delta T cells comprises at least 11% Vd3+ cells.

[0386] Embodiment 87. The method of any one of embodiments 73-86, wherein the method further comprises electroporating the polyclonal gamma delta T cells to introduce a transgene encoding the chimeric antigen receptor.

[0387] Embodiment 88. The method of embodiment 87, wherein the method further comprises: (i) following the electroporating, incubating the engineered polyclonal gamma delta T cells in recovery medium for about 1-2 hours, and (iii) after the incubating in the recovery medium for about 1-2 hours, adjusting the cell concentration to about 1-1.5 x 10^A6 cells per m .

[0388] Embodiment 89. The method of any one of embodiments 87-88, wherein prior to the electroporating, the method comprises incubating the polyclonal gamma delta T cells in the expansion culture medium at a volume of about 300-400 pL per square cm.

[0389] Embodiment 90. The method of any one of embodiments 73-89, wherein viral vectors are not used to generate the engineered polyclonal gamma delta T cells.

[0390] Embodiment 91. The method of any one of embodiments 87-90, wherein the method further comprises genomically integrating the transgene encoding the chimeric antigen receptor.

[0391] Embodiment92. The method of embodiment 91, wherein the genomically integrating utilizes a transposon-based system.

[0392] Embodiment 93. The method of any one of embodiments 73-92, further comprising assaying the population of engineered polyclonal gamma delta T cells to determine viability.

[0393] Embodiment 94. The method of any one of embodiments 73-93, further comprising assaying the population of engineered polyclonal gamma delta T cells to determine polyclonal gdTCR phenotype.

[0394] Embodiment 95. The method of any one of embodiments 73-94, further comprising assaying the population of engineered polyclonal gamma delta T cells to determine expression of exhaustion markers.

[0395] Embodiment 96. The method of any one of embodiments 73-95, wherein the incubating is at about 37°C.

[0396] Embodiment 97. The method of any one of embodiments 73-96, further comprising enriching the polyclonal gamma delta T cells to reduce the frequency of non-gamma delta T cells.

[0397] Embodiment 98. The method of any one of embodiments 74-97, wherein the population of polyclonal gamma delta T cells are incubated in the GREX culture vessel for at least 20 days.

[0398] Embodiment 99. The method of any one of embodiments 74-98, wherein the population of polyclonal gamma delta T cells are incubated in the GREX culture vessel for about 2-25 days.

[0399] Embodiment 100. The method of any one of embodiments 73-99, further comprising dislodging adherent gamma delta T cells from the culture vessel. [0400] Embodiment 101. The method of any one of embodiments 73-100, further comprising harvesting cells after culturing for about 21 days in the expansion culture medium after the electroporation.

[0401] Embodiment 102. The method of any one of embodiments 73-101, further comprising quantifying glucose and lactate after a period of incubation of the population of polyclonal gamma delta T cells in the expansion culture medium.

[0402] Embodiment 103. The method of any one of embodiments 73-102, wherein the engineered polyclonal gamma delta T cells retain cytotoxic capacity after three rounds of rechallenge with target cells.

[0403] Embodiment 104. The method of any one of embodiments 73-103, wherein the engineered polyclonal gamma delta T cells exhibit at least 50% higher killing of target cells compared to a control population of cells that is not polyclonal.

[0404] Embodiment 105. The method of any one of embodiments 73-104, wherein the engineered polyclonal gamma delta T cells exhibit at least 2-fold increased proliferation in response to target cells compared to a control population of cells that is not polyclonal.

[0405] Embodiment 106. The method of any one of embodiments 73-105, wherein the engineered polyclonal gamma delta T cells exhibit at least 10% reduced exhaustion compared to a control population of cells that is not polyclonal.

[0406] Embodiment 107. The method of any one of embodiments 73-106, wherein the method provides at least 1 x 10^A8 engineered polyclonal gamma delta T cells.

[0407] Embodiment 108. The method of any one of embodiments 73-106, wherein the method provides at least 1 x 10^A9 engineered polyclonal gamma delta T cells.

[0408] Embodiment 109. The method of any one of embodiments 73-106, wherein the method provides at least 1 x 10^Al 0 engineered polyclonal gamma delta T cells.

[0409] Embodiment 110. The method of any one of embodiments 73-109, wherein the method provides at least 100 patient doses of a cell therapy product.

[0410] Embodiment 111. The method of any one of embodiments 73-109, wherein the method provides at least 200 patient doses of a cell therapy product.

[0411] Embodiment 112. The method of any one of embodiments 73-109, wherein the method provides at least 500 patient doses of a cell therapy product.

VIII. EXAMPLES

EXAMPLE 1: Thawing, isolation/enrichment of gamma delta T cells, and stimulation

[0412] Thawing [0413] If the starting cell population is cryopreserved/frozen, the cells are first thawed according to a standard protocol, washed to remove DMSO/cryoprotectant, and resuspended into pre-warmed basal medium (e.g., without cytokines and/or serum). Unfrozen, freshly isolated cells can also be used, e.g., leukopaks.

[0414] An automated cell counter is used to quantify cells. Cell count, viability, and size is documented.

[0415] Enrichment of gd T cells

[0416] Suitable methods are used to isolate or enrich gamma delta T cells and to reduce the number of non-gamma delta T cells. Illustrative products and protocols that can be used include Miltenyi manual and CliniMACs purification of gamma delta T cells.

[0417] A suitable CliniMACS protocol (e.g., for leukopaks) can utilize CliniMACs TCRaP GMP Biotin followed by negative selection with anti-biotin beads and anti-CD14 GMP beads.

[0418] A suitable gamma delta T cell isolation/enrichment protocol that can be used, e.g., for PBMCs, is MANUAL ISOLATION - Miltenyi PE or APC labeling of TCRaP, CD19 and CD14 followed by negative selection with Anti-PE or Anti-APC Microbeads, e.g., using manufacturer’s protocols for cell concentrations, magnet capacity, antibody and buffer volumes, and incubation times. The protocol can include:

[0419] 1. Label CD14, CD19 and TCRab cells with either APC or PE anti-human antibody

[0420] a. After mixing thoroughly and filtering through 70 pm (um) mesh filter, follow labeling protocol of manufacturer (e.g., l-5pL(ul) antibody/le6 cells/lOOul MACS buffer) at 4deg for 30 minutes.

[0421] 2 Label cells with either Anti-PE or Anti-APC Microbeads per manufacturer’s instructions.

[0422] a. Wash lx and resuspend cells in 80ul/le7 cells

[0423] b. add 20ul beads per le7 cells, incubate at 4deg for 15 minutes

[0424] c. Wash and resuspend cells in MACs buffer, 500ul per le7 cells per column

[0425] 3. Perform isolation, e.g., with Miltenyi LD columns for good purity (LD columns can hold le8 labeled cells and process 5e8 total cells)

[0426] a. Pre-wet columns with 2mLs of MACs buffer

[0427] b. Add 500ul of cells per column

[0428] c. wash columns 2x with ImL MACs buffer

[0429] 4. Collect flow through and keep an aliquot for counts, viability, and purity by flow cytometry.

[0430] First Stimulation [0431] Stimulate 5e5-8e5 gd T cells/square centimeter (cm2). Some illustrative culture vessels, areas, and numbers of gdT cells are:

[0432] 48 well: lcm2: 5e5-6e5 cells

[0433] 24 well: 2cm2: 1-1.5e6 cells

[0434] 12 well: 3.5cm2: 2-3e6 cells

[0435] 6 well: 9.6cm2: 5-8e6 cells

[0436] T 25: 12.5-25e6 cells

[0437] T75: 45-60e6 cells

[0438] T182: 110e6-145e6 cells

[0439] T300: 180e6-240e6 cells

[0440] Coat the plate with PBS containing 10 pg TCRy/8 Antibody per ImL PBS (lOul/mL), 130ul PBS/cm2. Mix well and add to well/plate, e.g., at 130ul per well for 48well, 260ul 24 well, 1.3mL 6well, 3.25mL T25, 10mL T75, 24mL T182, 40mL T300. An illustrative anti-human gamma delta TCR antibody is clone REA591 from Miltenyi biotec.

[0441] Allow plates/flask(s) to incubate at 37°C, 5% CO2 for 2hrs.

[0442] Allow gd T cells to rest in complete media for 2hrs during plate setup (after isolation/enrichment).

[0443] Complete media can comprise basal media as shown in TABLE 2 plus recombinant human IL-2 at 1000 lU/mL, recombinant human IL-7 at 5 ng/mL, and recombinant human IL-15 at 5 ng/mL. The cytokines can be maintained at those concentrations throughout the expansion protocol.

[0444] Run flow on pre and post isolation samples to assess purity, viability, and polyclonal phenotype.

[0445] Staining antibody panel: FVD AF780, CD3 AF700, Vdl FITC, Vd2 BV605, TCRaB BV510, CD 19 APC, CD 14 Pac Blue

[0446] Gently wash residual plate bound g/dTCR antibody 3x with PBS.

[0447] Filter enriched gdT cells to remove dead cells/debris. [0448] Pipette cells to mix well and filter rested gd T cells. Transfer gd T cells onto stim plate(s) at l-2e6 cells/mL (1.5e6 cells/mL can be ideal).

[0449] Stim media: Add soluble anti-CD28 antibody at lul/mL (2ug/mL) to complete media. Stim media is used that lacks an anti-CD3 antibody.

[0450] Incubate in stim media and allow cells to stimulate for 36-48hrs (in presence of anti- gdTCR, anti-CD28, plus IL-2, IL-7, and IL-15).

[0451] The day of thawing, isolation, and initial stimulation described in this example can be referred to as “day 0” herein.

EXAMPLE 2: Transfection via Neon or Xenon electroporation

[0452] The following protocol can be used on day 2 (e.g., 36-48 hours following stimulation as described in EXAMPLE 1) to introduce an expression construct into gamma delta T cells. A transposon/transposase system, such as TcBuster, can be used to achieve genomic integration of the expression construct (e.g., encoding one or more CARs and/or other transgenes).

1. Visually assess stimulation morphology under microscope for clumping of cells

2. Vigorously pipette cells from plate surface to disrupt clumps and remove adherent gamma delta T cells. Rinse plate 2x with media.

3. Remove and aliquot to count stimulated cells. Store remaining aliquot of cells for flow cytometry to assess viability, stimulation status, Vdl and Vd2 populations, TCRaB contamination. a. Panel: FVD AF780, CD3 AF700, Vdl FITC, Vd2 BV605, TCRab BV510, CD25 BV421, CD69 BV650

4. Keep gamma delta T cells in incubator until ready to electroporate.

5. Prepare recovery TC treated plate(s) OR GREX. Seed 3 -6e5 cells/cm2 in a TC flask or 1 - 1.5e6 cells/cm2 in GREX. Recovery plate media: ’A total volume of complete gamma delta T cell media without Pen Strep (no anti-gdTCR or anti-CD28). If cells will be electroporated with DNA reagents (i.e. Nanoplasmid transposon), use recovery media containing 1000 lU/mL DNase (StemCell Technologies use at lOul/mL). a. 48 well - 3-5e5 cells (300ul) b. 24 well - 1-1.5e6 cells (ImL) c. 12 well - 3.5-5e6 cells (2-2.5mL) d. 6 well - 9e6-10e6 cells (3-4mL) e. T25 - 12.5e6 cells (8-10mL media) f. T75 - 37e6 cells (20-25mL media) g. T182 - 91e6 cells (48m-60mL media) h. T 300 - 150e6 cells (79-85mL media) i. 24 well GREX - 2-3e6 cells/well @ le6/mL media j. 6 well GREX - 9-12e6 cells/well @ le6/mL media k. 100M GREX - 95-110e6 cells @ le6/mL media l. 500M GREX - 495-550e6 cells @ le6/mL media

6. Set up Neon/Xenon: EP parameters are 1400 volts, 10 ms, 3 pulses.

7. Spin to pellet cells, 130RPM, 10 minutes, brake on 3

8. Carefully aspirate ALL media from pelleted cells without sucking up the pellet.

9. Resuspend pellet in ImL of pre-warmed Xenon EP Buffer containing RNase Inhibitor (1 :100)

10. Depending on cell number and size of zap, calculate volume of plasmid: lug per 1 Opl tip, 10pg per 100 l tip, lOOpg per single Xenon chamber. Add Transposase (e.g., TcBuster) mRNA: Ipg per lOul tip, lOpg per 100 pl tip, lOOpg per single Xenon chamber.

11. Carefully and thoroughly mix cells in the R buffer containing RNase Inhibitor.

12. Incubate cells in R buffer + RNAse Inhibitor for 3-5 minutes then carefully and thoroughly mix with nanoplasmid and transposase mRNA.

13. Perform electroporation immediately.

14. After transfection, quickly move electroporated cells to recovery media (without antibiotic) and allow them to recover for l-2hrs at 37°C and 5% CO2.

15. Add the 2^nd half of complete media with 2X Pen Strep added such that cells are now seeded at l-1.5e6cells/mL.

EXAMPLE 3: gamma delta T cell expansion

[0453] The following protocol can be used following enrichment and stimulation as described in EXAMPLE 1 and transformation as described in EXAMPLE 2. As described in Example 2, the cells are seeded in GREX culture vessels if a sufficient number of gamma/delta T cells are available, or tissue culture plates/flasks if fewer cells are available (referred to as “plate expansion” below).

Day 3-4 visually monitor cells:

1. Visually assess cells under microscope. Unlikely they will require media or cytokines until day 5 or 6.

Day 5: (generally only applies to plate expansion) 1. Visually assess cell density under microscope.

2. (Optional) Check Lactate and Glucose

3. Disrupt cells and dislodge adherent cells for transfer ONLY when in plates NOT GREX.

4. Count cells, document viability and size

5. Transfer to appropriate flask to double media volume.

6. Add fresh media + Cytokines (complete media, no anti-gdTCR or anti-CD28; ADD FULL CYTOKINE concentration based on final concentration in final media volume, not based on the volume of the newly added media, as in all such steps)

Day 6: (generally only applies to plate expansion)

1. Visually assess cell density under microscope.

2. (Optional) Check Lactate and Glucose

3. Disrupt cells and dislodge adherent cells for transfer.

4. Count cells. If moving from a plate to GREX, keep an aliquot for flow characterization.

5. Transfer to appropriate flask to double media volume (ADD FULL CYTOKINE concentration based on final concentration in final media volume, not based on the volume of the newly added media).

6. Add fresh media + Cytokines

Day 7 : (GREX cells are generally ready to sample and split)

1. Visually assess cell density under microscope.

2. (Optional) Check Lactate and Glucose

3. Disrupt cells for split/transfer.

4. Count cells and keep aliquot for DAY 7 flow characterization. Transfer “spent” media with cells at appropriate density andvolume (e.g., keep 50%“spent” media for paracrine factors that can promote survival and expansion).

5. Add 2x fresh media + Cytokines

Day 8: If cells weren’t split and sampled on day 7

1. Visually assess cell density under microscope.

2. (Optional) Check Lactate and Glucose

3. Disrupt cells for split/transfer.

4. Count cells and keep aliquot for DAY 7 flow characterization. Transfer “spent” media with cells at appropriate density and volume.

5. Add 2x fresh media + Cytokines

Flow Panel: FVD AF780, CD3 AF700, TCRaB BV510, Vdl FITC, Vd2 BV605, optionally CAR/transgene(s) Day 10:

1 . (Optional) Check Lactate and Glucose

2. Add 2X fresh media + Cytokines

Day 12: Re-Stimulation:

1 . Harvest cells from GREX a. If wells are full, remove top half of media carefully without disturbing cells. b . Thoroughly mix cells in remaining media to create homogenous cell suspension (generally best to leave cells in GREX until cell counts are performed to determine size of re-stimulation). c. Remove an aliquot of cells to count and stain for Day 12 flow characterization.

2. Count and stain aliquot of cells while stimulation plates are incubating for 2hrs to coat with an ti-gdTCR antibody (keep engineered gd T cells in the incubator during the 2 hrs of plate prep and flow cytometry).

3. Prepare stimulation plates/flasks as indicated on Day 0 based on updated number of cells (e.g., coat tissue culture plates/flasks with anti-gdTCR antibody).

4. As on day 0, gently wash plate unbound stim antibody off with PBS 2-3x after 2hr incubation to coat the plate/flask.

5. Pipette cells to mix well and transfer gd T cells onto stim plate(s) at 1 -2e6 cells/mL stim media (1.5e6 cells/mL can be ideal) as described in EXAMPLE 1.

6. Allow cells to stimulate for 36-48hrs in stim medium (complete medium with IL-2, IL-7, and IL-15, plus anti-CD28 antibody, with plate-bound anti-gdTCR).

*Flow panel for day 12 re-stimulation: FVD AF780, CD3 AF700, TCRaB BV510, Vdl FITC, Vd2 BV605, optionally CAR/transgene(s)

Day 14: Harvest 2^nd Stimulation

1 . Visually assess stimulation under microscope for clumping of cells

2. Vigorously pipette cells from plate surface to remove adherent gamma delta T cells. Document cell count, viability and size of activated cells.

3. Transfer cells along with re-stim media into new GREX vessel at le6 cells/cm2 with cytokine concentration calculated to full volume of complete media (includes IL-2, IL-7, and IL-15, no anti-gdTCR and no additional anti-CD28 added for remainder of protocol).

Day 16:

1. (Optional) Check Lactate and Glucose 2. Add 2x Media + full Cytokines

Day 18 or 19:

1. (Optional) Check Lactate and Glucose

2. Add 2x Media + full Cytokines

Day 20 or 21:

1. (Optional) Check Lactate and Glucose

2. Add Media + full Cytokines

Day 23: Harvest Cells:

1. Slowly and carefully Remove 2/3 volume of media from GREX without disturbing cells at the bottom of the vessel

2. Mix remaining media and cells thoroughly to obtain single cell suspension

3. Remove aliquot of cells for cell counts and Day 23 flow characterization.

4. Store cells in incubator while assessing counts viability and phenotype (viability, CAR integration, TCRaB contamination, Vdl/Vdl populations, exhaustion markers).

Flow Panel A (Integration and polyclonal distribution):

FVD AF780, CD3 AF700, TCRaB BV510, Vdl FITC, Vd2 BV605, optionally CAR/transgene(s)

Flow Panel B (Exhaustion):

FVD AF780, CD3, Vdl FITC, Vd2 BV605, LAG3 BV4221, PD-1 BV510, TIM-3 BV650

• Freeze aliquots of 5-50e6 cells/vial in CryoStor CS5 or CS10. Good to have some vials with low cell number for testing cytotoxicity, pathogen testing, copy number, etc.

EXAMPLE 4: Functionality of expanded gamma delta T cells

[0454] Gamma delta cells from multiple donors were engineered with several CAR constructs and expanded via protocols disclosed herein. At the end of expansion, cells were harvested and phenotyped for viability, CAR integration, and polyclonal characterization (e.g., Vdl, Vd2, Vdl-/Vd2-). Fold expansions were calculated for harvested total gamma delta T cells relative to the number of gamma delta T cells that were subjected to electroporation or the viable number of gamma delta T cells after thaw and enrichment.

[0455] Following the expansion cells were also challenged in functional killing assays against one more cell lines expressing appropriate target antigens. [0456] For cytotoxicity assays to test functional killing of engineered and expanded gamma delta T cells: cells were seeded in 48hr or 72hr co-cultures against one or more target cell line at 3:1 effector to target (E:T) ratios. Control conditions included target cells seeded alone, and target cells with un-engineered (Pulsed) gamma delta T cells (e.g., lacking CAR). At the end of co-culture, cells were harvested, and counted for remaining target and effector cell numbers. In some cases, cytotoxicity was quantified with CellTiter-Glo 2.0 plate-based assays.

[0457] The fold expansion results, viability, percentage of cells expressing CAR, percentage of cells expressing the Vdl gdTCR subunit, and the percentage of cells expressing the Vd2 gdTCR subunit are shown in FIG. 1. The cells were engineered to express a CAR comprising a BAFF extracellular domain to target cells that express receptors of BAFF. The engineered gdT cells were co-cultured with Jeko-1 (mantle cell target cells) or IM-9 (multiple myeloma target cells). The gamma delta T cells were capable of killing the target cells, and increased killing of the multiple myeloma target cells was shown for engineered gdT cells expressing the BAFF CAR (FIG. 2A and FIG. 2B)

[0458] In another expansion experiment, cells from two donors were subjected to a 22-23 day expansion according to methods disclosed herein. The cells were not engineered to express transgenes, and were initially cultured in a plate-based protocol, and were only moved to G-Rex culture vessels following re-stimulation on day 12. Cells from donor 30 were isolated using StemCell isolation kit; donor 31 cells were isolated using CliniMACS protocol. Fold expansion was calculated from viable counts post-thaw on Day 0. Fold expansion, percentage of cells expressing the Vdl gdTCR subunit, and the percentage of cells expressing the Vd2 gdTCR subunit are shown in FIG. 3.

[0459] In a further experiment, cells were manufactured following a 23 Day protocol (FIG. 4A). Lot number 16AUG2023-TC cells were expanded in tissue-culture plates until Day 7, then moved to G-Rex. All other lots were solely expanded in G-Rex outside of stimulation. Donors 32 and 33 y8 were isolated using StemCell isolation kit. Electroporations were performed on Day 2; Pulsed conditions included transposase only. Fold expansion was calculated from viable counts post-thaw on Day 0. Only lot 16AUG2023-GR was tested for cytotoxicity. Cells were cocultured with Mantle Cell (Jeko-1) and Multiple Myeloma (IM-9) and demonstrated complete killing at 3:1 E:T over 48 hours (FIG. 4B). Absolute counts of target cells were obtained via flow cytometry.

[0460] In a further experiment, gamma delta T cells were engineered to express a “split CAR” comprising separate CAR polypeptides targeting two distinct antigens, and expanded in a 22-23 day protocol disclosed herein. Over 2,000 fold expansion was achieved, and the resulting expanded population included cells expressing Vdl and cells expressing Vd2 (FIG. 5A).

[0461] The engineered gdT cells were co-cultured with esophageal cancer (FLO-1) or cholangiocarcinoma (HuH28) target cells expressing target antigens for 72 hours. The gamma delta T cells were capable of killing esophageal cancer target cells, and increased killing of the target cells of both types was shown for engineered gdT cells expressing the split CAR (FIG. 5B and FIG. 5C).

[0462] The split CAR expressing gdT cells were also tested in 48 hour cytotoxicity assays at 3 : 1 effector to target ratios and were shown to kill target cells expressing target antigens, HuH28 (FIG. 6A), FLO-1 (FIG. 6B), and 786-0 (FIG. 6C; renal carcinoma).

[0463] Persistent functionality and resistance to exhaustion were evaluated for the split CAR-expressing gdT cells, using serial killing assays. Engineered gamma delta T cells were seeded in co-culture with target cells at a 1 :5, E:T ratio and re-seeded with fresh target cells every 2 days for 5 rounds of co-culture. Cells were harvested at each passage and both target and effector cell counts were measured. Gamma delta T cells were able to persist, proliferate (FIG. 7 A), and kill target cells for 5 rounds of killing (FIG. 7B). Engineered gamma delta T cells outperformed pulsed and engineered alpha beta CAR T cells.

[0464] In a further experiment, gamma delta T cells were engineered to express a second “split CAR” comprising separate CAR polypeptides targeting two distinct antigens on target cells, and expanded in a 22-23 day protocol disclosed herein. Over 2,300 or 3,600 fold expansion was achieved from different donors, and the resulting expanded population included cells expressing Vdl and cells expressing Vd2 (FIG. 8A). The engineered gdT cells were cocultured with glioblastoma (A-172) target cells expressing target antigens. The gamma delta T cells were capable of killing glioblastoma target cells, and increased killing of the target cells was shown for engineered gdT cells expressing the split CAR (FIG. 8B).

[0465] In a further experiment, gamma delta T cells were engineered to express a third “split CAR” comprising separate CAR polypeptides targeting two distinct antigens on target cells, and expanded in a 22-23 day protocol disclosed herein. Over 3,000 fold expansion was achieved, and the resulting expanded population included cells expressing Vdl and cells expressing Vd2 (FIG. 9)

[0466] Illustrative results from further production runs suing the 22-23 day expansion protocol are provided in TABLE 3. TABLE 3

EXAMPLE 5: Low expansion of gamma delta T cells via control protocol

[0467] Control experiments were conducted using published control protocols for expansion of gamma delta T cells. Such protocols differ from protocols disclosed herein in one or more elements. For example, compared to methods disclosed herein, the control protocols can differ by (i) not utilizing culture vessels with a gas permeable membrane submerged under a column of medium; (ii) seeding cells at a lower concentration and/or higher media volume; (iii) utilizing lower doses of cytokines (e.g., lower concentrations of IL-2, IL-7, and IL-15, such as from day 12 of the protocol); (iv) replenishing media and cytokines less frequently; (v) using a shorter recovery/rest period following isolation from PBMCs; (vi) using a shorter recovery period after electroporation; (vii) not quantifying glucose and/or lactate to inform timing for expansion or media replenishment; (viii) not collecting adherent gamma delta T cells from culture plates to retain them for subsequent steps; (ix) culturing cells at 35°C rather than 37°C; (x) shorter incubation periods before disrupting cells/aggregates; (xi) restimulating cells on day 11 rather than day 12; (xii) not retaining stimulation media when transferring cells into new culture vessels (e.g., on day 14 following the second round of stimulation); (xiii) other elements disclosed herein; or (xiv) a combination thereof.

[0468] As shown in TABLE 4, only relatively modest expansion was achieved using the control protocols, particularly for conditions that included engineering the gamma delta T cells to express a CAR. TABLE 4

EXAMPLE 6: Large scale expansion of engineered gamma delta T cells

[0469] Gamma delta T cells were engineered with a BAFF CAR construct and expanded via methods disclosed herein, e.g., as described in EXAMPLES 1-3.

[0470] Fold expansions were calculated for harvested total gamma delta T cells relative to the number of gamma delta T cells that were subjected to electroporation (with alpha beta T cells excluded).

[0471] For the Xenon to plate expansion, gamma delta T cells were seeded directly into a tissue culture plate. For the Xenon to GREX expansion, gamma delta T cells were seeded directly into a GREX culture vessel at the same concentration of cells per mL.

[0472] For the Xenon to plate expansion, on Day 12 only 360e6 cells were re-stimulated. For the smaller Xenon to GREX expansion, on Day 12 only 83e6 cells were re-stimulated.

[0473] On day 12 and at the end of expansion, cells were harvested and phenotyped for viability, CAR integration, and polyclonal characterization (e.g., Vdl, Vd2, or Vdl-/Vd2-). As shown in TABLE 5, 80-105 fold expansion was achieved by day 12. The expanded gamma delta T cells exhibited -94% viability at this time point, with -67% expressing the BAFF CAR, 42% being Vdl+, 47% being Vd2+, and 11% being Vdl/2-.

[0474] 3 ,441-4,256 fold total expansion was achieved by the end of the protocol, with -98% viability, -64.85% expressing the BAFF CAR (FIG. 10C), and polyclonality maintained with -73.4% being Vdl+, -13% being Vd2+, and -13.5% being Vdl/2- (FIG. 10D). Only -4.5% of cells present were positive for alpha beta TCR (FIG. 10B).

[0475] These results demonstrate that compositions and methods disclosed herein can facilitate generation of populations of engineered gamma delta T cells on a large and useful scale. For example, starting with a leukopak with about 80 million gamma delta T cells (general range -60-100 million), methods disclosed herein can allow for electroporation of about 40 million gamma delta T cells (e.g., on about day 2 of a protocol disclosed herein). -105 fold expansion in stage 1 can yield ~4.2e9 gamma delta T cells, with a further -40 fold expansion at stage 2 yielding ~1.68el 1 cells, which can correspond to a large number of patient doses as illustrated in TABLE 6 (calculated for a 100kg patient).

EXAMPLE 7: Functionality and persistence of Vd2+, Vdl+/Vdl-Vd2- , and polyclonal populations of CD19 gamma delta CAR T

[0476] Gamma delta T cells were engineered to express an anti-CD19 CAR, expanded to generate a polyclonal population of engineered gamma delta T cells using methods disclosed herein, and cryopreserved.

[0477] The frozen cells were thawed. Vd2+ cells were isolated from the polyclonal population using anti human Vd2 PE antibody with Miltenyi anti-PE Microbeads. The remaining Vd2- fraction contained Vdl+ cells plus a subpopulation of Vdl- and Vd2- gamma delta T cells, referred to in this example as Vdl-Vd2-. Following isolation, the cells were rested for 24 hours prior to co-culture with target cells at a 1 :6 E:T ratio of (CAR+) effector cells to REC-1 target cells (mantle cell lymphoma; MCL). The following groups were included:

[0478] (1) Vdl+ and Vdl-/Vd2- CD19 CAR T monoculture

[0479] (2) Vd2+ CD 19 CAR T monoculture

[0480] (3) Vdl+ and Vdl-/Vd2- CD19 CAR T with RECI target cells

[0481] (4) Vd2 CD 19 CAR T with RECI target cells

[0482] (5) Polyclonal (Vdl, Vd2, Vdl-/Vd2-) CD19-CAR T with RECI target cells (all 3 populations of GD T cells mixed back together).

[0483] Cells were mixed and harvested after 24 and 48 hour co-cultures, processed, and analyzed via flow cytometry. Three rechallenges were also conducted, with a portion of cells rechallenged with 5e5 new target cells every 48 hours.

[0484] Flow staining included panels of the following targets:

[0485] Exhaustion: FVD, CD19, CD3, Vdl, Vd2, GFP(CAR), TIM3, LAG3, PD1. In some embodiments the exhaustion panel can also include TIGIT.

[0486] Differentiation: FVD, CD 19, CD3, Vdl, Vd2, GFP(CAR), CD62L, CD45RO, CD27. In some embodiments the differentiation panel can also include CD45RA.

[0487] Activation: FVD, CD19, CD3, Vdl, Vd2, GFP(CAR), CD69, CD25, and CD28. In some embodiments, the activation panel can also include CD70 and/or or CD107a.

[0488] In some embodiments a cytotoxicity panel can also be used including, for example, Fixable viability dye (FVD), CD3, Vdl, Vd2, CAR, CD16, CD56, CD8, CD4, and TCRab.

[0489] The polyclonal condition contained a mix of Vdl+, Vd2+, and Vdl-Vd2- cells both at seeding and after re-challenge (FIG. 11 A). The Vd2+ condition contained predominantly Vd2+ cells both at seeding at after the second re-challenge (FIG. 11C), while the Vdl+/Vdl- Vd2- condition contained predominantly Vdl+ cells and Vdl-/Vd2- cells (FIG. 11B).

[0490] Over the initial 48 hour co-culture and after rechallenges, the percent of effector cells increased while the percent of target cells decreased, consistent with expansion of effector cells and killing of target cells. The polyclonal gamma delta CAR-T cells exhibited the quickest and most robust control of target cells (FIG. 12A), followed by the Vdl+/Vdl-Vd2- condition (FIG. 12B), then the Vd2+ single positive condition (FIG. 12C). Representative scatterplots showing the proportions of CAR+ cells for each condition are shown at 24 and 48 hours (FIG. 13A) and after rechallenges 1 and 2 (FIG. 13B). The percentage of CAR+ gamma delta T cells for the polyclonal and Vdl+/Vdl-Vd2- conditions increased with the rechallenges as CAR+ T cells were activated and proliferated (FIG. 14).

[0491] As time points progressed, higher numbers and proportions of CD3+ (T) cells were observed, while the number and proportion of CD19+ target cells decreased over time, with killing being fastest for the polyclonal population (FIG. 15A, top panels), intermediate for the Vdl+/Vdl-Vd2- population (FIG. 15A, bottom panels), and lower for the Vd2+ population (FIG. 15B). The number (FIG. 16A) and percentage (FIG. 16B) of remaining cells expressing target antigen CD19 decreased overtime forthe polyclonal population and the Vdl+/Vdl-Vd2- population.

[0492] The polyclonal and Vdl+/Vdl-Vd2- populations exhibited lower expression coexpression of TIM-3 and LAG-3, indicating reduces signs of exhaustion, compared to the Vd2+ population, including notably after three rechallenges (FIG. 17).

[0493] Staining for naive versus memory population markers was conducted for the polyclonal population of engineered gamma delta T cells after three rounds of killing. Antibodies were also included in the panel to allow a breakdown of memory phenotypes in the Vdl+, Vd2+, and Vdl-/Vd2- (shown as “Vd3” in figure) subsets of the polyclonal population. All three subsets of the polyclonal population retained central memory and effector memory phenotypes after the three rounds of killing (FIG. 18).

[0494] These results show the polyclonal product demonstrates potent anti-tumor cytotoxicity and persistence across serial killing, including with multiple target rechallenges.

[0495] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of generating an expanded population of therapeutic cells, the method comprising:

(a) seeding at least 8 xlO^A5 gamma delta T cells per square cm in a culture vessel comprising a gas permeable membrane submerged under a column of medium; and

(b) incubating the gamma delta T cells in an expansion culture medium comprising a gamma delta T cell receptor (gdTCR) stimulating agent.

2. The method of claim 1, wherein the culture vessel is a Gas permeable Rapid Expansion (GREX®) cell culture vessel.

3. The method of claim 1 , wherein at least 1 x10^A6 of the gamma delta T cells are seeded per square cm.

4. The method of claim 1, wherein 1-1.5 xlO^A6 of the gamma delta T cells are seeded per square cm.

5. The method of claim 1, wherein the gamma delta T cells expand at least 2,000-fold.

6. The method of claim 1, wherein the gamma delta T cells expand at least 5,000-fold.

7. The method of claim 1, wherein the gdTCR stimulating agent comprises an anti-gdTCR antibody or antigen-binding fragment thereof.

8. The method of claim 1 , wherein the gdTCR stimulating agent comprises a pan anti-gdTCR antibody or antigen-binding fragment thereof.

9. The method of claim 8, wherein the anti-gdTCR antibody is bound to a surface of the culture vessel.

10. The method of claim 1, wherein the expansion culture medium further comprises an anti- CD28 antibody or antigen-binding fragment thereof.

11 . The method of claim 1 , wherein the expansion culture medium and the culture vessel lack an anti-CD3 antibody or antigen-binding fragment thereof.

12. The method of claim 1, wherein afterthe incubating, the population of gamma delta T cells comprises at least 10% Vdl+ cells and at least 10% Vd2+ cells.

13. The method of claim 1 , wherein after the incubating, the gamma delta T cells comprise at least 1% Vdl- Vd2- gamma delta T cells.

14. The method of claim 1 , wherein after the incubating, the gamma delta T cells comprise at least 11% Vd3+ cells.

15. The method of claim 1 , wherein the method further comprises electroporating the gamma delta T cells to introduce a transgene encoding a chimeric antigen receptor into the gamma delta T cells.

16. The method of claim 15, wherein the method further comprises (i) following the electroporating, incubating the gamma delta T cells in recovery medium for about 1-2 hours, and (iii) after the incubating in the recovery medium for about 1-2 hours, adjusting the cell concentration to about 1-1.5 x 10^A6 cells per mL.

17. The method of claim 16, wherein the method further comprises, prior to the electroporating, incubating the gamma delta T cells in the expansion culture medium at a volume of about 300-400 pL per square cm.

18. The method of claim 1 , further comprising assaying the population of gamma delta T cells to determine viability.

19. The method of claim 1, further comprising assaying the population of gamma delta T cells to determine polyclonal gdTCR phenotype.

20. The method of claim 1, further comprising assaying the population of gamma delta T cells to determine expression of exhaustion markers.

21 . The method of claim 1, wherein the incubating is at about 37°C.

22. The method of claim 1, further comprising enriching the gamma delta T cells to reduce the frequency of non-gamma delta T cells.

23. The method of claim 1, wherein gamma delta T cells are incubated in the culture vessel comprising a gas permeable membrane submerged under a column of medium for at least 20 days.

24. The method of claim 1, wherein the gamma delta T cells are incubated in the culture vessel comprising a gas permeable membrane submerged under a column of medium for about 2- 25 days.

25. The method of claim 1, further comprising dislodging adherent gamma delta T cells from the culture vessel.

26. The method of claim 15, further comprising harvesting the gamma delta T cells after culturing for about 21 days in the expansion culture medium after the electroporating.

27. The method of claim 1 , further comprising quantifying glucose and lactate after a period of incubation of the population of polyclonal gamma delta T cells in the expansion culture medium.

28. The method of claim 5, wherein the gamma delta T cells retain cytotoxic capacity after three rounds of rechallenge with target cells.

29. The method of claim 1, wherein the method provides at least 1 x 10^A8 gamma delta T cells.

30. The method of claim 1, wherein the method provides at least 1 x 10^A9 gamma delta T cells.

31 . The method of claim 1, wherein the method provides at least 1 x 10^Al 0 gamma delta T cells.

32. The method of claim 1, wherein the method provides at least 100 patient doses of a cell therapy product.

33. The method of claim 1, wherein the method provides at least 200 patient doses of a cell therapy product.

34. The method of claim 1, wherein the method provides at least 500 patient doses of a cell therapy product.

35. A method of generating an expanded population of therapeutic cells, the method comprising:

(a) incubating a population of engineered polyclonal gamma delta T cells in a culture vessel in an expansion culture medium comprising a gamma delta T cell receptor (gdTCR) stimulating agent; and

(b) treating the population of engineered polyclonal gamma delta T cells in the expansion culture medium every 2-3 days with: (i) IL-2 freshly added to a final concentration of at least 1000 lU/mL, (ii) IL-7 freshly added to a final concentration of at least 5 ng/mL, and (iii) IL- 15 freshly added to a final concentration of at least 5 ng/mL.

36. The method of claim 35, wherein the culture vessel is a Gas permeable Rapid Expansion (GREX®) cell culture vessel.

37. The method of claim 36, wherein at least 1 xlO^A6 of the gamma delta T cells are seeded per square cm of the GREX cell culture vessel.

38. The method of claim 37, wherein 1-1.5 xlO^A6 of the gamma delta T cells are seeded per square cm of the GREX cell culture vessel.

39. The method of claim 35, wherein the population of engineered polyclonal gamma delta T cells expands at least 2,000-fold.

40. The method of claim 35, wherein the population of engineered polyclonal gamma delta T cells expands at least 5,000-fold.

41. The method of claim 35, wherein the gdTCR stimulating agent comprises an anti-gdTCR antibody or antigen-binding fragment thereof.

42. The method of claim 35, wherein the gdTCR stimulating agent comprises a pan anti- gdTCR antibody or antigen-binding fragment thereof.

43. The method of claim 42, wherein the anti-gdTCR antibody is bound to a surface of the culture vessel.

44. The method of claim 35, wherein the expansion culture medium further comprises an anti- CD28 antibody or antigen-binding fragment thereof.

45. The method of claim 35, wherein the expansion culture medium and the culture vessel lack an anti-CD3 antibody or antigen-binding fragment thereof.

46. The method of claim 35, wherein after the incubating, the population of poly clonal gamma delta T cells comprises at least 10% Vdl+ cells and at least 10% Vd2+ cells.

47. The method of claim 35, wherein after the incubating, the population of poly clonal gamma delta T cells comprises at least 1% Vdl- Vd2- gamma delta T cells.

48. The method of claim 35, wherein after the incubating, the population of polyclonal gamma delta T cells comprises at least 11% Vd3+ cells.

49. The method of claim 35, wherein the method further comprises electroporating to introduce a transgene encoding a chimeric antigen receptor into a population of polyclonal gamma delta T cells, thereby generating the population of engineered polyclonal gamma delta T cells.

50. The method of claim 49, wherein following the electroporating, the method comprises incubating the engineered polyclonal gamma delta T cells in recovery medium for about 1- 2 hours, and (iii) after the incubating in the recovery medium for about 1-2 hours, adjusting the cell concentration to about 1-1.5 x 10^A6 cells per mL.

51. The method of claim 50, wherein the method further comprises, prior to the electroporating, incubating the polyclonal gamma delta T cells in the expansion culture medium at a volume of about 300-400 p,L per square cm.

52. The method of claim 35, wherein viral vectors are not used to generate the engineered polyclonal gamma delta T cells.

53. The method of claim 35, wherein the method further comprises genomically integrating a transgene encoding a chimeric antigen receptor.

54. The method of claim 53, wherein the genomically integrating utilizes a transposon-based system.

55. The method of claim 35, further comprising assaying the population of engineered polyclonal gamma delta T cells to determine viability.

56. The method of claim 35, further comprising assaying the population of engineered polyclonal gamma delta T cells to determine polyclonal gdTCR phenotype.

57. The method of claim 35, further comprising assaying the population of engineered polyclonal gamma delta T cells to determine expression of exhaustion markers.

58. The method of claim 35, wherein the incubating is at about 37°C.

59. The method of claim 35, further comprising enriching the polyclonal gamma delta T cells to reduce the frequency of non-gamma delta T cells.

60. The method of claim 35, further comprising dislodging adherent gamma delta T cells from the culture vessel.

61. The method of claim 49, further comprising harvesting cells after culturing for about 21 days in the expansion culture medium after the electroporating.

62. The method of claim 35, further comprising quantifying glucose and lactate after a period of incubation of the population of engineered polyclonal gamma delta T cells in the expansion culture medium.

63. The method of claim 39, wherein the engineered polyclonal gamma delta T cells retain cytotoxic capacity after three rounds of rechallenge with target cells.

64. The method of claim 39, wherein the engineered polyclonal gamma delta T cells exhibit at least 50% higher killing of target cells compared to a control population of cells that is not polyclonal.

65. The method of claim 39, wherein the engineered polyclonal gamma delta T cells exhibit at least 2-fold increased proliferation in response to target cells compared to a control population of cells that is not polyclonal.

66. The method of claim 39, wherein the engineered polyclonal gamma delta T cells exhibit at least 10% reduced exhaustion compared to a control population of cells that is not polyclonal.

67. The method of claim 35, wherein the method provides at least 1 x 10^A8 engineered polyclonal gamma delta T cells.

68. The method of claim 35, wherein the method provides at least 1 x 10^A9 engineered polyclonal gamma delta T cells.

69. The method of claim 35, wherein the method provides at least 1 x 10^Al 0 engineered polyclonal gamma delta T cells.

70. The method of claim 35, wherein the method provides at least 100 patient doses of a cell therapy product.

71. The method of claim 35, wherein the method provides at least 200 patient doses of a cell therapy product.

72. The method of claim 35, wherein the method provides at least 500 patient doses of a cell therapy product.

73. A method of generating an expanded population of therapeutic cells, the method comprising:

(a) engineering a population of polyclonal gamma delta T cells to express a chimeric antigen receptor, thereby providing a population of engineered polyclonal gamma delta T cells; and

(b) incubating the population of engineered polyclonal gamma delta T cells in a culture vessel in an expansion culture medium comprising a gamma delta T cell receptor (gdTCR) stimulating agent; wherein the population of engineered polyclonal gamma delta T cells expands at least 1,000-fold.

74. The method of claim 73, wherein the culture vessel is a Gas permeable Rapid Expansion (GREX®) cell culture vessel.

75. The method of claim 74, wherein at least 1 xlO^A6 of the engineered polyclonal gamma delta T cells are seeded per square cm of the GREX cell culture vessel.

76. The method of claim 74, wherein 1-1.5 xlO^A6 of the gamma delta T cells are seeded per square cm of the GREX cell culture vessel.

77. The method of claim 73, wherein the population of engineered polyclonal gamma delta T cells expands at least 2,000-fold.

78. The method of claim 73, wherein the population of engineered polyclonal gamma delta T cells expands at least 5,000-fold.

79. The method of claim 73, wherein the gdTCR stimulating agent comprises an anti-gdTCR antibody or antigen-binding fragment thereof.

80. The method of claim 73, wherein the gdTCR stimulating agent comprises a pan anti- gdTCR antibody or antigen-binding fragment thereof.

81. The method of claim 80, wherein the anti-gdTCR antibody is bound to a surface of the culture vessel.

82. The method of claim 73, wherein the expansion culture medium further comprises an anti- CD28 antibody or antigen-binding fragment thereof.

83. The method of claim 73 , wherein the expansion culture medium and the culture vessel lack an anti-CD3 antibody or antigen-binding fragment thereof.

84. The method of claim 73, wherein after the incubating, the population of engineered polyclonal gamma delta T cells comprises at least 10% Vdl+ cells and at least 10% Vd2+ cells.

85. The method of claim 73, wherein after the incubating, the population of engineered polyclonal gamma delta T cells comprises at least 1% Vdl- Vd2- gamma delta T cells.

86. The method of claim 73, wherein after the incubating, the population of poly clonal gamma delta T cells comprises at least 11% Vd3+ cells.

87. The method of claim 73, wherein the method further comprises electroporating the polyclonal gamma delta T cells to introduce a transgene encoding the chimeric antigen receptor.

88. The method of claim 87, wherein the method further comprises: (i) following the electroporating, incubating the engineered polyclonal gamma delta T cells in recovery medium for about 1-2 hours, and (iii) after the incubating in the recovery medium for about 1-2 hours, adjusting the cell concentration to about 1-1.5 x 10^A6 cells per mL.

89. The method of claim 88, wherein prior to the electroporating, the method comprises incubating the polyclonal gamma delta T cells in the expansion culture medium at a volume of about 300-400 p,L per square cm.

90. The method of claim 73, wherein viral vectors are not used to generate the engineered polyclonal gamma delta T cells.

91. The method of claim 89, wherein the method further comprises genomically integrating the transgene encoding the chimeric antigen receptor.

92. The method of claim 91, wherein the genomically integrating utilizes a transposon-based system.

93. The method of claim 73, further comprising assaying the population of engineered polyclonal gamma delta T cells to determine viability.

94. The method of claim 73, further comprising assaying the population of engineered polyclonal gamma delta T cells to determine polyclonal gdTCR phenotype.

95. The method of claim 73, further comprising assaying the population of engineered polyclonal gamma delta T cells to determine expression of exhaustion markers.

96. The method of claim 73, wherein the incubating is at about 37°C.

97. The method of claim 73, further comprising enriching the polyclonal gamma delta T cells to reduce the frequency of non-gamma delta T cells.

98. The method of claim 74, wherein the population of polyclonal gamma delta T cells are incubated in the GREX culture vessel for at least 20 days.

99. The method of claim 74, wherein the population of polyclonal gamma delta T cells are incubated in the GREX culture vessel for about 2-25 days.

100. The method of claim 73, further comprising dislodging adherent gamma delta T cells from the culture vessel.

101. The method of claim 89, further comprising harvesting cells after culturing for about 21 days in the expansion culture medium after the electroporating.

102. The method of claim 73, further comprising quantifying glucose and lactate after a period of incubation of the population of polyclonal gamma delta T cells in the expansion culture medium.

103. The method of claim 77, wherein the engineered polyclonal gamma delta T cells retain cytotoxic capacity after three rounds of rechallenge with target cells.

104. The method of claim 77, wherein the engineered polyclonal gamma delta T cells exhibit at least 50% higher killing of target cells compared to a control population of cells that is not polyclonal.

105. The method of claim 77, wherein the engineered polyclonal gamma delta T cells exhibit at least 2-fold increased proliferation in response to target cells compared to a control population of cells that is not polyclonal.

106. The method of claim 77, wherein the engineered polyclonal gamma delta T cells exhibit at least 10% reduced exhaustion compared to a control population of cells that is not polyclonal.

107. The method of claim 73, wherein the method provides at least 1 x 10^A8 engineered polyclonal gamma delta T cells.

108. The method of claim 73, wherein the method provides at least 1 x 10^A9 engineered polyclonal gamma delta T cells.

109. The method of claim 73, wherein the method provides at least 1 x 10^Al 0 engineered polyclonal gamma delta T cells.

110. The method of claim 73, wherein the method provides at least 100 patient doses of a cell therapy product.

111. The method of claim 73, wherein the method provides at least 200 patient doses of a cell therapy product.

112. The method of claim 73, wherein the method provides at least 500 patient doses of a cell therapy product.