Methods for expanding γδ t cell populations

Abstract

The disclosure includes a method for producing an expanded population of engineered γδ T cells, comprising: (a) culturing an isolated mixed cell population comprising γδ T cells with at least one T cell mitogen to produce an activated γδ T cell population; (b) depleting αβ T cells in the activated γδ T cell population to produce an enriched γδ T cell population; (c) engineering the enriched γδ T cell population to express at least one antigen recognition moiety to produce an engineered γδ T cell population, the engineering step comprising stably integrating at least one nucleic acid construct encoding said at least one antigen recognition moiety in culture conditions comprising IL-4, IL- 13, and IL-21; and (d) culturing the engineered γδ T cell population in the presence of at least one cytokine to provide the expanded population of engineered γδ T cells.

Description

METHODS FOR EXPANDING γδ T CELL POPULATIONS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 671,691,filed July 15, 2024, and U.S. Provisional Application No.63 / 701,505, filed September 30, 2024. The entire contents of the above-identified applications are hereby fully incorporated herein by reference. FIELD OF DISCLOSURE

[0002] The present disclosure relates generally to methods for the expansion of γδ T cellpopulations and subsets thereof. BACKGROUND OF THE DISCLOSURE

[0003] Antigen recognition by T lymphocytes may be achieved by highly diverseheterodimeric receptors, the T cell receptors (TCRs). Approximately 95% of human T cells in blood and lymphoid organs express a heterodimeric ^^ TCR receptor (^^ T cell lineage). Approximately 5% of human T cells in the blood and lymphoid organs express heterodimeric ^^ TCR receptor (^^ T cell lineage). These T cell subsets may be referred to as “^^” and “^^” T cells, respectively. ^^ and ^^ T cells are different in function. Activation of ^^ T cells then occurs when an antigen presenting cell (APC) presents an antigen in the context of class I / II MHC. In contrast to ^^ T cells, ^^ T cells can recognize an antigen independent of MHC restriction. In addition, ^^ T cells combine both innate and adoptive immune recognition and responses.

[0004] In the last few years, T cell-engaging therapies employing chimeric antigen receptor(CAR) T cells have opened up a new frontier in cancer immunotherapy (June et al. CAR T cell immunotherapy for human cancer Science 2018; 359:1361–1365). Current clinical approaches rely on autologous peripheral blood T cells and primarily alpha beta (αβ) T cells targeted to tumor antigens following retroviral transduction with a CAR construct. Unfortunately, however, the effective use of αβ T cells is complicated by their high alloreactive potential in general, and by their propensity to trigger cytokine release syndrome (CRS) in particular.

[0005] CRS is associated with high circulating concentrations of several pro-inflammatorycytokines, including interleukins, interferons, tumor necrosis factors, colony-stimulating factors, Page 1 of 117 1104881063\1\AMERICASand transforming growth factors. This so-called “cytokine storm” can be compounded by numerous cellular interactions with bystander cells, such as endothelial cells, monocyte / macrophages, and dendritic cells, further increasing cytokine hypersecretion, aggravating symptoms, and inducing various grades of organ damage (Cosenza et al. Cytokine Release Syndrome Associated with T-Cell-Based Therapies for Hematological Malignancies: Pathophysiology, Clinical Presentation, and Treatment; Int J Mol Sci. 2021 Jul; 22(14): 7652). CRS symptoms can occur immediately after administration of the CAR T therapy, or can be delayed for days or even weeks after treatment, making it more difficult to predict and manage this toxic adverse event. CRS can manifest as mild, with flu-like symptoms, including fever, nausea, and chills, or can be life-threatening and severe with shock and respiratory compromise, leading to multi-system organ failure and even death. Although considerable efforts have been made to prevent and control CRS in conventional CAR T therapies, it remains an unmet clinical need. Id.

[0006] Gamma delta (γδ) T cells are thymus-derived lymphocytes that differ from αβ T cellsin their mechanism of activation and function, as well as in their anatomical distribution. In particular, while αβ T cells function exclusively in adaptive immunity, γδ T cells are innate-like immune cells that recognize malignant cells through a repertoire of activating receptors in a MHC- independent manner, similar to NK cells (Welsh et al., 1997). As such, and in contrast to αβ T cells, γδ T cells can potentially be used in an allogeneic setting without the risk of causing GvHD. Moreover, recent studies have suggested that engineered γδ T cells may produce less proinflammatory cytokines than αβ T cells, which could thereby reduce the risk of CRS in patients (Harrer et al., 2017). Given the above qualities, there is growing interest in utilizing γδ T cells instead of αβ T cells in CAR T therapies.

[0007] The ability to selectively expand γδ T cell subset populations having potent therapeuticactivity with improved purity and in clinically-relevant levels is highly desirable. Unfortunately, however, γδ T cells comprise a mere 0.5 to 5% of peripheral blood mononuclear cells (PBMCs), whereas αβ T cells make up approximately 50% (Vantourout & Hayday, 2013), thereby making the selective expansion of γδ T cells directly from PBMCs a considerable challenge. For example, ^^ T cells expansion face problems of low yield, low purity, and loss of ^^ T cell function after expansion, which may prevent large scale preparation of ^^ T cells for clinical applications. This is further compounded by the technical problems with eliminating αβ T cells from a mixed cell population grown in culture. What is needed, then, are methods for producing engineered γδ T Page 2 of 117 1104881063\1\AMERICAScells in numbers sufficient for utilization in the clinic while simultaneously minimizing the number of contaminating αβ T cells. SUMMARY OF DISCLOSURE

[0008] The present disclosure provides methods for consistently expanding γδ T cells andfor selectively expanding subsets thereof, including δ1, δ2, δ3, δ4 and / or δ5 γδ T cells and combinations thereof, comprising an optimal αβ T cell depletion step and subsequent cytokine support to maximize the growth and development of the γδ T cells and more consistently achieve the production of a requisite number γδ^ T cells from an individual donor sample. The methods herein significantly improve the yield and purity of the expanded γδ T cells while maintaining γδ^T cell function after expansion.

[0009] In one aspect, the present disclosure provides a method for producing an expandedpopulation of engineered γδ T cells, the method comprising: (a) culturing an isolated mixed cell population comprising γδ T cells with at least one T cell mitogen to produce an activated γδ T cell population; (b) depleting αβ T cells in the activated γδ T cell population from (a) to produce an enriched γδ T cell population; (c) engineering the enriched γδ T cell population from (b) to express at least one antigen recognition moiety to produce an engineered γδ T cell population, wherein the engineering step comprises stably integrating at least one nucleic acid construct encoding said at least one antigen recognition moiety in culture conditions comprising IL-4, IL-1β, and IL-21; and (d) culturing the engineered γδ T cell population from (c) in the presence of at least one cytokine to provide the expanded population of engineered γδ T cells.

[0010] In embodiments, step (b) is performed after culturing the isolated mixed cell populationwith the at least one T cell mitogen for about 4, 5, 6, or 7 days, preferably 6 days. In embodiments, step (b) further comprises depleting NK cells from the activated γδ T cell population. In embodiments, the NK cells are depleted using an agent binding to CD56.

[0011] In embodiments, step (c) is between about 16 and 96 hours, more preferably about 48hours.

[0012] In embodiments, (d) comprises sequentially culturing the engineered γδ T cellpopulation from (c) in culture conditions comprising IL-15 and IL-21 for at least about 1-7 days, preferably from about 3-5 days, more preferably about 4 days; followed by culture conditions comprising IL-15 for at least about 1-7 days, preferably between about 2-5 days, more preferably Page 3 of 117 1104881063\1\AMERICASabout 3 or 4 days to provide the expanded population of engineered γδ T cells. In embodiments, the IL-15 in the culture conditions has a concentration greater than about 10 ng / mL, preferably from about 25 ng / mL to 80 ng / mL, more preferably from about 50 ng / mL to 70 ng / mL.

[0013] In embodiments, the method further comprises: (e) depleting αβ T cells in the expandedpopulation of engineered γδ T cells from (d); (f) resting the expanded population of engineered γδ T cells after depletion for a predetermined duration of time in the presence of at least one cytokine to provide a therapeutically effective population of engineered γδ T cells; and (g) cryopreserving the therapeutically effective population of engineered γδ T cells.

[0014] In embodiments, depleting αβ T cells in step (e) comprises reducing αβ T cells to belowabout 0.5%, preferably 0.2%, of total viable cells of the expanded of engineered γδ Tcells. In embodiments, depleting αβ T cells in (e) comprises reducing αβ T cells to between about 0.05% and about 0.15% of total viable cells of the expanded population of engineered γδ T cells, preferably between about 0.05% and about 0.1% of total viable cells.

[0015] In embodiments, the predetermined duration in (f) maintains αβ T cells in thetherapeutically effective population of engineered γδ T cells below about 5% of total viable cells, preferably between about 0.2% and about 3% of total viable cells, preferably between about 0.2% and about 2% of total viable cells, more preferably between about 0.2% and about 1%, of total viable cells.

[0016] In embodiments, the predetermined duration is between about 6 hours and about 72hours, preferably between about 8 hours and about 48 hours, more preferably between about 12 hours and about 24 hours. In embodiments, the at least one cytokine in step (f) is IL-2.

[0017] In embodiments, the isolated mixed cell population in (a) is a peripheral blood sample,a cord blood sample, or a tumor.

[0018] In embodiments, the construct comprises a viral vector, optionally wherein the viralvector is a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector, preferably wherein the viral vector is a retroviral vector. In embodiments, the γδ T cells are engineered by introducing a construct by electroporation. In embodiments, the γδ T cells are engineered by gene editing, e.g., using a CRISPR-Cas system.

[0019] In embodiments, the culture conditions in step (c) do not comprise an antibody or afragment thereof. In embodiments, the culture conditions in step (c) do not comprise an antibody Page 4 of 117 1104881063\1\AMERICASor a fragment thereof that recognizes CD3. In embodiments, the culture conditions in step (c) do not comprise IFNγ.

[0020] In embodiments, the at least one T cell mitogen in a) comprises or further comprisesan antibody or a fragment thereof; preferably wherein the antibody binds to CD3 or a fragment thereof, or to a γδ TCR or a fragment thereof In embodiments, the at least one T cell mitogen comprises or further comprises an antibody or a fragment thereof that binds to a variable region of a γδ TCR, preferably a variable region of a δ1, δ2, or δ3 γδ TCR, still more preferably to a variable region of a δ1 γδ TCR.

[0021] In embodiments, the variable region of a δ1 γδ TCR comprises a Bin 3 δ1 epitopecomprising or consisting of amino acids FKKAAKSVALTISALQ or AKSGRYSVNFKKAAKSVALTISALQ of human Vδ1. In embodiments, the variable region of a δ1 γδ TCR comprises a Bin 4 δ1 epitope comprising or consisting of amino acids AQKVTQAQSSV of human Vδ1, and amino acids TDKLIFGKGTRVTVEP of human J1 or LTAQLFFGKGTQLIVEP of human J2, wherein the at least one antibody does not bind an epitope containing a K120T mutation in J1 or J2. In embodiments, the antibody is an antibody that binds to the same epitope as, or competes with, antibody δ1-08 or an antibody comprising the six complementarity determining regions (CDRs) of antibody δ1-08. In embodiments, the antibody binds to the same epitope as, or competes with, antibody δ1-35 or an antibody comprising the six CDRs of antibody δ1-35.

[0022] In embodiments, the at least one T cell mitogen is a means for selectively activatingand expanding a subset of γδ T cells, e.g., δ1, δ2, or δ3 γδ T cells, by binding to an epitope comprising a variable region of a δ1, δ2, or δ3 γδ TCR. In embodiments, the at least one T cell mitogen is a means for selectively activating and expanding δ1 γδ T cells by binding to an epitope comprising a variable region of a δ1 γδ TCR.

[0023] In embodiments, the epitope comprising a variable region of a δ1 γδ TCR is a Bin 3 δ1epitope comprising or consisting of amino acids FKKAAKSVALTISALQ or AKSGRYSVNFKKAAKSVALTISALQ of human Vδ1. In embodiments, the epitope comprising a variable region of a γδ TCR is a Bin 4 δ1 epitope comprising or consisting of amino acids AQKVTQAQSSV of human Vδ1, and amino acids TDKLIFGKGTRVTVEP of human J1 or LTAQLFFGKGTQLIVEP of human J2, wherein the at least one antibody does not bind an epitope containing a K120T mutation in J1 or J2. In embodiments, the antibody is an antibody that binds Page 5 of 117 1104881063\1\AMERICASto the same epitope as, or competes with, antibody δ1-08 or an antibody comprising the six complementarity determining regions (CDRs) of antibody δ1-08. In embodiments, the antibody binds to the same epitope as, or competes with, antibody δ1-35 or an antibody comprising the six CDRs of antibody δ1-35.

[0024] In embodiments, the at least one T cell mitogen further comprises an antibody thatbinds to CD28, CD137, CD2, CD27, and / or OX40. In embodiments, the at least one T cell mitogencomprises a combination of at least one antibody or a fragment thereof that binds to a variable region of a γδ TCR, preferably a variable region of a δ1, δ2, or δ3 γδ TCR, still more preferably toa variable region of a δ1 γδ TCR, and one or more antibodies that bind to CD28, CD137, CD2,CD27, and / or OX40. In exemplary embodiments, the at least one T cell mitogen comprises a means for selectively activating and expanding δ1 γδ T cells by binding to an epitope comprising a variable region of a δ1 γδ TCR, and an antibody that binds to CD28, CD137, CD2, CD27, and / or OX40. As demonstrated herein, the subject combinations of T cell mitogens find particularly advantageous use when immobilized on a solid support, including cell culture surfaces exhibiting reduced cellular adhesion, e.g. flasks or bags, and with beads, nanomatrices, or other solid support materials.

[0025] In exemplary embodiments, the at least one T cell mitogen comprises an antibody thatbinds to variable region of a δ1 γδ, e.g. antibody δ1-35 and / or antibody δ1-08, and an anti-CD28 antibody. In exemplary embodiments, the at least one T cell mitogen comprises an antibody that binds to variable region of a δ1 γδ, e.g. antibody δ1-35 and / or antibody δ1-08, and an anti-CD137 antibody. In exemplary embodiments, the at least one T cell mitogen comprises a means for selectively activating and expanding δ1 γδ T cells by binding to an epitope comprising a variable region of a δ1 γδ TCR, and an antibody that binds to CD28 and / or CD137.

[0026] In embodiments, the method further comprises disrupting the expression of one or moregenes in the γδ T cells, preferably performed using an RNA-guided nuclease and at least one guide RNA. In embodiments, disrupting expression the one or more genes is performed prior to step (a). In embodiments, disrupting expression of the one or more genes is performed after step (b). In embodiments, disrupting expression of the one or more genes is performed after step (b) and prior to step (c). In embodiments, the one or more genes comprises MED12. In embodiments, the one or more genes comprises MED12 and TGFβR2. Page 6 of 117 1104881063\1\AMERICAS

[0027] In embodiments, the expanded population of γδ T cells has less than 5% (e.g., 0.07%)αβ T cells. In embodiments, the expanded population of γδ T cells has less than 30% (e.g., 2.5% or 0.5%) NK cells. In embodiments, the expanded population of γδ T cells has at least 70% (e.g., at least 91%, 95%, or 98%) γδ T cells.

[0028] In another aspect, the present disclosure provides a cryopreserved therapeuticallyeffective population of engineered γδ T cells comprising at least 70% γδ T cells, less than 5% αβ T cells, and less than 30% NK cells, produced by the method herein. In embodiments, the cryopreserved therapeutically effective population of engineered γδ T cells comprises at least 91% γδ T cells, less than 0.07% αβ T cells, and less than 2.5% or 0.5% NK cells produced by the method herein.

[0029] In embodiments, the cryopreserved therapeutically effective population of engineeredγδ T cells comprises at least 1 x 109CAR+ γδ T cells with a ratio of CAR+ γδ T cells to αβ T cells of at least about 200:1. In embodiments, the cryopreserved therapeutically effective population of engineered γδ T cells comprises at least 3 x 109CAR+ δ1 T cells with a ratio of CAR+ δ1 T cells to αβ T cells of at least about 600:1. In embodiments, the cryopreserved therapeutically effective population of engineered γδ T cells comprises at least 1 x 108CAR+ γδ T cells with a ratio of CAR+ γδ T cells to αβ T cells of at least about 20:1. In embodiments, the engineered γδ T cells are CAR+. In embodiments, the engineered γδ T cells are CAR+ and MED12-. In embodiments, the engineered γδ T cells are CAR+, MED12- and TGFβR2-. In embodiments, the engineered γδ T cells are CAR+, MED12- and dnTGFβR2+.

[0030] In embodiments, the ^^^T cells are δ1, δ2, δ3, δ4, and / or δ5 γδ T cells. In embodiments,the γδ T cells are δ1 γδ T cells. In embodiments, the γδ T cells are δ2- γδ T cells. INCORPORATION BY REFERENCE

[0031] All publications, patents, and patent applications mentioned in this specification areherein 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. Page 7 of 117 1104881063\1\AMERICASBRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG.1 depicts heavy-chain framework and complementarity determining region aminoacid sequences of exemplary δ1-specific antibodies.

[0033] FIG. 2 depicts light-chain framework and complementarity determining region aminoacid sequences of the exemplary δ1-specific antibodies described in FIG.1.

[0034] FIG. 3 shows % of δ1 γδ T cells and % of αβ T cells under conditions of Study 1.

[0035] FIG. 4 shows fold expansion of total cells from day 0 under conditions of Study 1.

[0036] FIG. 5 shows %CAR expression within δ1 γδ T cells under conditions of Study 1.

[0037] FIG. 6 shows fold expansion of total cells from day 0 under conditions of Study 2.

[0038] FIG. 7 shows % of δ1 γδ T cells and % of αβ T cells under conditions of Study 2.

[0039] FIG. 8 shows %CAR expression within δ1 γδ T cells under conditions of Study 2.

[0040] FIG. 9 shows fold expansion of total cells from day 0 under conditions of Study 3.

[0041] FIG.10 shows % of δ1 γδ T cells and % of αβ T cells, and % NK cells under conditionsof Study 3.

[0042] FIG. 11 shows total viable cells, % of δ1 γδ T cells, and % of αβ T cells underconditions of Study 4.

[0043] FIG. 12 shows post-thaw Vδ1+CAR+ CD25 / CD69 activation phenotypes underconditions of Study 4.

[0044] FIGS. 13A-13C show MED12 knock-out resulted in similar or better δ1 yield tounedited control.

[0045] FIG. 14 shows MED12 knock-out improved cell health post cryopreservation.

[0046] FIG. 15 shows MED12 knock-out resulted in distinct “synthetic effector” phenotypethat was stable throughout δ1 γδ T cells expansion.

[0047] FIGS. 16A-16D show MED12 KO enhanced killing and cell survival of δ1 γδ T cells.

[0048] FIGS. 17A-17C show expansion profile and cell phenotypes by an exemplary methodof δ1 γδ T cells expansion. DETAILED DESCRIPTION I. DEFINITIONS

[0049] For purposes of interpreting this specification, the following definitions will apply, andwhenever appropriate, terms used in the singular will also include the plural and vice versa. In the Page 8 of 117 1104881063\1\AMERICASevent that any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0050] “About” as used herein when referring to a measurable value such as an amount, atemporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0051] “Ranges”: throughout this disclosure, various aspects of the disclosure can bepresented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0052] The terms “patient,” “subject,” “individual,” and the like are used interchangeablyherein, and refer to any animal amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0053] As used herein, the term “agent” refers to any protein, nucleic acid molecule (includingchemically modified nucleic acids), compound, antibody, small molecule, organic compound, inorganic compound, other molecule of interest, or cell (e.g., cell engineered to express a chimeric antigen receptor). Agents can include a therapeutic agent, a diagnostic agent, or a pharmaceutical agent. A therapeutic or pharmaceutical agent is one that alone or together with an additional agent induces the desired response (such as inducing a therapeutic or prophylactic effect when administered to a subject, including treating a subject suffering from cancer, or other disease / condition.

[0054] The term “therapeutically effective amount,” or simply “effective amount” refers to theamount of an agent or composition (e.g., composition comprising an agent) that will elicit a biological or medical response of a tissue, system, or subject that is being sought by the researcher, Page 9 of 117 1104881063\1\AMERICASveterinarian, medical doctor, or other clinician. The term “therapeutically effective amount” includes that amount of an agent, or a composition comprising an agent, that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease (e.g., cancer) being treated. The therapeutically effective amount will vary depending on the composition, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0055] As used herein, the term “T lymphocyte” or “T cell” refers to an immune cell thatexpresses or has expressed CD3 (CD3+) and a T Cell Receptor (TCR+). T cells play a central role in cell-mediated immunity. A T cell that “has expressed” CD3 and a TCR has been engineered to eliminate CD3 and / or TCR cell surface expression.

[0056] As used herein, the term “TCR” or “T cell receptor” refers to a dimeric heterologouscell surface signaling protein forming an alpha-beta or gamma-delta receptor or combinations thereof. αβ TCRs recognize an antigen presented by an MHC molecule, whereas γδ TCR can recognize an antigen independently of MHC presentation.

[0057] The term “γδ T cells (gamma delta T cells)” as used herein refers to a subset of T cellsthat express a distinct T cell receptor (TCR), namely γδ TCR, on their surface, composed of one γ-chain and one δ-chain. The term “γδ T cells” specifically includes all subsets of γδ T cells, including, without limitation, Vδ1, Vδ2, and Vδ3 γδ T cells, as well as naïve, effector memory, central memory, and terminally differentiated γδ T cells. As a further example, the term “γδ T cells” includes Vδ4, Vδ5, Vδ7, and Vδ8 γδ T cells, as well as Vγ2, Vγ3, Vγ5, Vγ8, Vγ9, Vγ10, and Vγ11 γδ T cells. In embodiments, the γδ T cells are δ1 γδ T cells. In embodiments, the γδ T cells are δ2 γδ T cells. In embodiments, the γδ T cells are δ3 γδ T cells. In embodiments, the γδ T cells are δ1-. In embodiments, the γδ T cells are δ2-. In embodiments, the γδ T cells are δ1- and δ2- . ^^ T cells for use as described herein can be obtained from an allogeneic or an autologous donor. The ^^ T cells can be, partially or entirely purified, or not purified, and expanded ex vivo.

[0058] The ^^ T cells described herein can be ^1, ^2, ^3, ^4 or δ5 ^^^T cells, and combinationsthereof. In some cases, the ^^ T cells are mostly (>50%), substantially (>90%), essentially all, orentirely ^^ ^^ T cells. In some cases, the ^^ T cells are mostly (>50%), substantially (>90%),essentially all, or entirely ^^ ^^ T cells. In some cases, the ^^ T cells are mostly (>50%),substantially (>90%), essentially all, or entirely ^^ ^^ T cells. In some cases, the ^^ T cells aremostly (>50%), substantially (>90%), essentially all, or entirely ^^^ ^^ T cells.Page 10 of 117 1104881063\1\AMERICAS

[0059] The term “δ1 γδ T cells”, “Vδ1 γδ T cells”, “Vδ1 T cells”, and “Vδ1 cells” are usedherein interchangeably to refer to a subtype of γδ T cells that expresses γδ TCR comprising one γ- chain and one δ1-chain on its surface.

[0060] As used herein, the term “αβ T cell” refers to T cells expressing α and β chains of theTCR as part of a complex with CD3 chain molecules. Each α and β chain contains one variable and one constant domain. αβ T cells primarily recognize peptide antigens presented by major histocompatibility complex (MHC) class I and class II molecules, where most of the receptor diversity is contained within the third complementarity determining region (CDR3) of the TCR α and β chains.

[0061] “Activation”, as used herein, refers to the state of a T cell that has been sufficientlystimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division.

[0062] As used herein, the term “T cell mitogen” refers to an agent that can stimulate T cellactivation. For example, a T cell mitogen may stimulate T cells through TCR signaling. Examples of T cell mitogen include cytokines, antibodies, and combinations thereof.

[0063] As used herein, the term “Natural killer (NK) cell” refers to CD56+CD3− granularlymphocytes that play important roles in immunity against viruses and in the immune surveillance of tumors, and constitute a critical cellular subset of the innate immune system (Godfrey J, et al. Leuk Lymphoma 201253:1666-1676).

[0064] As used herein, the term “peripheral blood lymphocytes” or “PBLs” is used in thebroadest sense and refers to white blood cells comprising T cells and B cells of a range of differentiation and functional stages, plasma cells, monocytes, macrophages, natural killer cells, basophils, eosinophils, etc. The term “peripheral blood mononuclear cells (PBMC)” refers to any blood cells having a round nucleus. Such cells may play a role in the immune response. Examples of PBMC include lymphocytes such as T cells, B-lymphocytes and NK cells, monocytes, and macrophages.

[0065] The term “MHC” (major histocompatibility complex) refers to a subset of genes thatencodes cell-surface antigen-presenting proteins. In humans, these genes are referred to as human leukocyte antigen (HLA) genes. Herein, the abbreviations MHC or HLA are used interchangeably. Page 11 of 117 1104881063\1\AMERICAS

[0066] As used herein, the term “cell population” refers to a number of cells obtained byisolation directly from a suitable source, usually from a mammal. The isolated cell population may be subsequently cultured in vitro. Those of ordinary skill in the art will appreciate that various methods for isolating and culturing cell populations for use with the present invention and various numbers of cells in a cell population that are suitable for use in the present invention. A cell population may be purified to homogeneity, substantial homogeneity, or to deplete one or more cell types (e.g., αβ T cells and / or NK cells) by various culture techniques and / or negative or positive selection for a specified cell type.

[0067] A mixed cell population may be, for example, a mixed heterogeneous cell populationderived from a peripheral blood sample, a cord blood sample, a tumor, a stem cell precursor, a tumor biopsy, a tissue, a lymph, skin, a sample of or containing tumor infiltrating lymphocytes, or from epithelial sites of a subject directly contacting the external milieu, or derived from stem precursor cells. Alternatively, the mixed cell population may be derived from in vitro cultures of mammalian cells, established from a peripheral blood sample, a cord blood sample, a tumor, a stem cell precursor, a tumor biopsy, a tissue, a lymph, skin, a sample of or containing tumor infiltrating lymphocytes, or from epithelial sites of a subject directly contacting the external milieu, or derived from stem precursor cells. In embodiments, a mixed cell population may be an isolated mixed cell population, e.g., isolated from a sample from a subject.

[0068] An “enriched” cell population or preparation refers to a cell population derived from astarting mixed cell population that contains a greater percentage of a specific cell type than the percentage of that cell type in the starting population. For example, a starting mixed cell population can be enriched for γδ T cells in general, and / or for a specific γδ T cell subset (e.g., δ1 γδ T cells, δ2 γδ T cells, δ3 γδ T cells, or δ2- γδ T cells). In one embodiment, the enriched γδ T cell population contains a greater percentage of γδ cells than the percentage of that cell type in the starting population. In embodiments, the enriched γδ T cell population contains a lesser percentage of αβ T cell populations and / or NK cells populations.

[0069] By “expanded” as used herein is meant that the number of the desired or target cell type(e.g., δ1 γδ T cells) in the enriched preparation is higher than the number in the initial or starting cell population. An expanded population of γδ T cells include a population of γδ T cells prepared by the method herein. In embodiments, the expanded population of γδ T cells is an expanded Page 12 of 117 1104881063\1\AMERICASpopulation of engineered γδ T cells. In embodiments, the expanded population of γδ T cells is a therapeutically effective population of engineered γδ T cells.

[0070] By “selectively expand” is meant that the target cell type (e.g., γδ T cells or a subsetthereof) is preferentially expanded over other non-target cell types, e.g., αβ T cells or NK cells, or an untargeted subset of γδ T cells.

[0071] The term “chimeric antigen receptors (CARs)” refers to artificial T cell receptors, T-bodies, single-chain immunoreceptors, chimeric T cell receptors, or chimeric immunoreceptors, for example, and encompass engineered receptors that graft an artificial specificity onto a particular immune effector cell. CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy. In embodiments, CARs direct specificity of the cell to a disease associated antigen, (e.g., a tumor associated antigen, an autoimmune associated antigen, or a pathogenic antigen). In embodiments, CARs comprise an intracellular activation domain (e.g., allowing the T cell to activate upon engagement of targeting moiety with target cell, such as a target tumor cell), a transmembrane domain, and an extracellular domain that may vary in length and comprises a disease- or disorder-associated, e.g., a tumor-antigen binding region. In embodiments, CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta a transmembrane domain and endodomain. The specificity of other CAR designs may be derived from ligands of receptors (e.g., peptides) or from pattern-recognition receptors, such as Dectins. In certain cases, the spacing of the antigen- recognition domain can be modified to reduce activation-induced cell death. In certain cases, CARs comprise domains for additional co-stimulatory signaling, such as CD3-zeta, FcR, CD27, CD28, CD137, DAP 10 / 12, and / or OX40, ICOS, TLRs, etc. In some cases, molecules can be co- expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0072] The “costimulatory domain” in the context of a chimeric receptor, also referred toherein as a chimeric antigen receptor (CAR), of the present disclosure enhances cell proliferation, cell survival and development of memory cells for cytotoxic cells that express the chimeric receptor. The chimeric receptors of the invention may include one or more costimulatory domains Page 13 of 117 1104881063\1\AMERICASselected from the costimulatory domains of proteins in the TNFR superfamily, CD28, CD137 (4- 1BB), CD134 (OX40), Dapl0, CD27, CD2, CD7, CD5, ICAM-1, LFA-1 (CD1 la / CD18), Lck, TNFR-I, PD-1, TNFR-II, Fas, CD30, CD40, ICOS LIGHT, NKG2C, B7-H3, or combinations thereof. If the chimeric receptor includes more than one costimulatory domain, these domains may be arranged in tandem, optionally separated by a linker. The term “costimulatory domain” as used herein also encompasses any modifications thereof, examples of which are described in US Patent Application No. 20200129554; US Patent Application No. 20200317777; WO2019010383; Li, W., et al., (2020) Immunity 53: 456-470; and Li, G., et al., (2017) J Immunol 198(1 Supplement): 198.4, the contents of each of which are incorporated herein in their entirety.

[0073] The “intracellular signaling domain” in the context of a chimeric receptor of the presentdisclosure transduces the effector function signal and directs the cytotoxic cell to perform its specialized function, i.e., harming and / or destroying the target cells. Examples of suitable intracellular signaling domains include, e.g., the ζ chain of the T cell receptor complex or any of its homologs, e.g., η chain, FcsRly and β chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 ζ chain, CD3 polypeptides (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. In embodiments, the intracellular signaling domain of a chimeric receptor may be human CD3 ζ chain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors and combinations thereof.

[0074] The intracellular signaling domains may include intracellular signaling domains ofseveral types of various other immune signaling receptors, including first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory, and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (Park et al., "Are all chimeric antigen receptors created equal?" J Clin Oncol., vol. 33, pp. 651-653, 2015). Additional intracellular signaling domains include signaling domains used by NK and NKT cells (Hermanson, et al., "Utilizing chimeric antigen receptors to direct natural killer cell activity," Front Immunol., vol. 6, p. 195, 2015) such as signaling domains of NKp30 (Zhang et al., "An NKp30-based chimeric antigen receptor promotes T cell effector functions and antitumor efficacy in vivo," J Immunol., vol.189, pp. 2290-2299, 2012), and DAP12 (Topfer et al., "DAP12-based activating chimeric antigen receptor for NK cell tumor immunotherapy," J Immunol., vol. 194, pp. 3201-3212, 2015), Page 14 of 117 1104881063\1\AMERICASNKG2D, NKp44, NKp46, DAP10, and CD3z. Additionally intracellular signaling domains also includes signaling domains of human Immunoglobulin receptors that contain immunoreceptor tyrosine based activation motif (ITAM) such as FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, FcRL5 (Gillis et al., "Contribution of Human Fc.gamma.Rs to Disease with Evidence from Human Polymorphisms and Transgenic Animal Studies," Front Immunol., vol.5, p.254, 2014).

[0075] In embodiments, the intracellular signaling domain includes a cytoplasmic signalingdomain of TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, or CD66d. In exemplary embodiments the intracellular signaling domain in the chimeric receptor includes a cytoplasmic signaling domain of human CD3 ζ. The term “intracellular signaling domain” as used herein also encompasses any modifications examples of which are described in US PatentApplication No. 2020 / 0317777, as well as Navarro, P., and Reyburn, HT., (2009) J Biol Chem 284(24): 16463-16472; Giurisato, E., et al., (2007) Mol Cell Biol 27(24): 8583-8599; and Wu, J., et al., (2000) J Exp Med 192(7): 1059-1068, the contents of each of which are incorporated herein in their entirety.

[0076] The term “affinity binding entity” refers to a binding moiety which binds to a specificantigen with a higher affinity than to a non-specific antigen and is endowed with an affinity of at least 10-6M, as determined by assays which are well known in the art, including surface plasmon resonance (SPR). According to a specific embodiment, the affinity is 500 nM-0.01 nM, 100 nM- 0.01 nM, 50 nM-0.01 nM, 10 nM-0.01 nM, 5 nM-0.01 nM.

[0077] According to embodiments, the affinity binding entity is an antibody. The term“antibody” is used in the broadest sense and specifically covers, for example, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions with polyepitopic specificity, polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, single chain antibodies, and fragments of antibodies (see below), including Fab, Fab’, F(ab’)2 and Fv fragments, diabodies, single domain antibodies (sdAbs), as long as they exhibit the desired biological or immunological activity. Also included among antibodies, and among fragments in particular, are portions of antibodies (and combinations of portions of antibodies, for example, scFv) that may be used as targeting arms, directed to e.g., an epitope, in chimeric antigenic receptors of the present Page 15 of 117 1104881063\1\AMERICASdisclosure. Such fragments are not necessarily proteolytic fragments but rather portions of polypeptide sequences that can confer affinity for target. The term “immunoglobulin” (Ig) is used interchangeably with antibody herein. An antibody can be, for example, human, humanized and / or affinity matured.

[0078] An “intact” antibody is one which comprises an antigen-binding site as well as a CLand at least heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

[0079] “Antibody fragments” comprise a portion of an intact antibody, preferably the antigenbinding or one or more variable regions of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng.8(10): 1057-62 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen. Also included among antibody fragments are portions of antibodies (and combinations of portions of antibodies, for example, scFv) that may be used as targeting arms, directed to e.g., an epitope, in chimeric antigenic receptors of the present disclosure. Such fragments are not necessarily proteolytic fragments but rather portions of polypeptide sequences that can confer affinity for target.

[0080] Papain digestion of antibodies produces two identical antigen-binding fragments,called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Fab’ fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were Page 16 of 117 1104881063\1\AMERICASproduced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0081] The Fc fragment comprises the carboxy-terminal portions of both H chains heldtogether by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.

[0082] “Fv” is the minimum antibody fragment which contains a complete antigen-recognitionand -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0083] “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments thatcomprise the VH and VL antibody domains connected into a single polypeptide chain. In embodiments, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form a desired structure for antigen binding. For a review of sFv, see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra. In one embodiment, an antibody derived scFv is used as the targeting arm of a CAR-modified immune cell as disclosed herein. In terms of scFv antibody fragments, where a certain order of VH and VL region in the binding domain is explicitly or implicitly described, the present disclosure also includes the alternate embodiment in which the order of VH and VL regions are reversed, e.g., in an scFv or CAR comprising an scFv binding domain. Thus, description of a VH-VL order also describes the alternate VL-VH order, e.g., in an scFv or CAR comprising an scFv binding domain. Moreover, description of a VL-VH order also describes the alternate VH-VL order, e.g., in an scFv or a CAR comprising an scFv binding domain. VH and VL regions are either joined directly or Page 17 of 117 1104881063\1\AMERICASjoined by a peptide-encoding linker, which connects the N-terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL.

[0084] An antibody that “binds” an antigen or epitope of interest is one that binds the antigenor epitope with sufficient affinity that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity.

[0085] The term “antigen” or “Ag” as used herein is defined as a molecule that provokes animmune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including proteins or peptides, can serve as an antigen.

[0086] The term “epitope” includes any protein determinant, lipid, or carbohydratedeterminant capable of specific binding to an immunoglobulin or T cell receptor. Epitopic determinants usually consist of active surface groupings of molecules such as amino acids, lipids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0087] The term “specifically binds,” as used herein refers to a receptor (which can include toan antibody or antibody fragment) which recognizes a specific molecule / ligand, but does not substantially recognize or bind other molecules in a sample. For example, a receptor that specifically binds to a molecule from one species may also bind to that molecule from one or more other species. But, such cross-species reactivity does not itself alter the classification as specific. In another example, a receptor that specifically binds to a molecule may also bind to different allelic forms of the molecule. However, such cross reactivity does not itself alter the classification as specific. In some instances, the terms "specific binding" or "specifically binding," can be used in reference to the interaction of a protein (or a peptide) with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, a receptor recognizes and binds to a specific structure rather than to proteins generally. If a receptor is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the receptor, will reduce the amount of labeled A bound to the receptor. Page 18 of 117 1104881063\1\AMERICAS

[0088] In embodiments, specific binding can be characterized by an equilibrium dissociationconstant of at least about 1x10-8M or less (e.g., a smaller KD denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

[0089] The term “anti-tumor effect” as used herein, refers to a biological effect which can bemanifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor in the first place.

[0090] The term “cancer” and “cancerous” refer to or describe the physiological condition inmammals that is typically characterized by unregulated cell growth.

[0091] The term “autoimmune disease” refers to a disorder that results from an autoimmuneresponse. An autoimmune disease is the result of an inappropriately excessive response to an antigen associated with an autoimmune disease (e.g., a self-antigen).

[0092] As used herein, the term “cell culture” refers to any in vitro culture of cells. Includedwithin this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro, including stem cells, blood cells, embryonic cord blood cells, tumor cells, transduced cells, etc.

[0093] As used herein, the term “autologous” is meant to refer to any material derived froman individual which is later to be re-introduced into the same individual.

[0094] As used herein, the term “allogeneic” refers to material derived from an individualwhich is later introduced into a different individual.

[0095] To “treat or prevent” a disease as the term is used herein, means to reduce the frequencyor severity of at least one sign or symptom of a disease or disorder experienced by a subject. In one example, a therapy (e.g., administration of a therapeutic agent of the present disclosure) treats a disease or condition by decreasing one or more signs or symptoms associated with the disease or condition, for example as compared to the response in the absence of the therapy. For example, administration of a therapeutic agent may provide an anti-tumor effect that decreases one or more signs or symptoms associated with cancer. Treating or preventing can refer to delaying the onset of symptoms, reducing the severity of symptoms, reducing the severity of an acute episode, Page 19 of 117 1104881063\1\AMERICASreducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, preventing relapse to a disease, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, increasing time to sustained progression, expediting remission, inducing remission, augmenting remission, speeding recovery, or increasing efficacy of or decreasing resistance to alternative therapeutics. In one embodiment, "treating" refers to both therapeutic treatment and prophylactic or preventive measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described herein.

[0096] As used herein, the term “administration” means to provide or give a subject one ormore agents, such as an agent that treats one or more signs or symptoms associated with a condition / disorder or disease including cancer (e.g., lymphoma), an autoimmune disease, viral infection, bacterial infection, etc., by any effective route. Exemplary routes of administration include injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes. Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0097] The term “pharmaceutically acceptable,” as used herein, refers to a material, includinga salt, carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of one or more agents, such as one or more modulatory agents. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations can include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, cryoprotectant agents, glycerol or the like as a vehicle. In addition to biologically neutral carriers, pharmaceutical agents to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or Page 20 of 117 1104881063\1\AMERICASemulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate. For example, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a γδ T population as described herein.

[0098] “Encoding” refers to the inherent property of specific sequences of nucleotides in apolynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0099] “Isolated” means altered or removed from the natural state. For example, a nucleic acidor a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0100] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence”includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0101] “Expression cassette” refers to a nucleic acid comprising expression control sequencesoperatively linked to a nucleic acid encoding a transcript or polypeptide to be expressed. An expression cassette comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression cassettes can be a component of a vector such as a cosmid, a plasmid (e.g., naked or contained in a liposome), or a virus (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus). An expression cassette can be in a host cell, such as a γδ T cell. Page 21 of 117 1104881063\1\AMERICAS

[0102] The term “dual CAR” refers to two CARs co-expressed in one cell that target differentantigens. The two CARs may be encoded by multiple nucleic acid molecules. Alternately, the two CARs may be encoded by one nucleic acid molecule. A “dual CAR construct” is a nucleic acid molecule encoding two CARs.

[0103] The term “tandem CAR” refers to a CAR with multiple affinity binding domains. Themultiple affinity binding domains may be connected by one or more linkers. Examples of dual CARs and tandem CARs designs include those described in Guedan S et al., Engineering and Design of Chimeric Antigen Receptors, Mol Ther Methods Clin Dev.2019 Mar 15; 12: 145–156, which is incorporated by reference herein in its entirety.

[0104] The term “antigen recognition moiety” refers to any molecules that can specificallybind to an antigen. An antigen recognition moiety may comprise one or more affinity binding domains that specifically bind to the antigen. For example, the antigen recognition antigen may binds to a disease-associated antigen such as a tumor associated antigen, an autoimmune diseases- associated antigen, or a pathogen infection associated antigen. Examples of antigen recognition moieties include a TCR, αβ TCR, γδ TCR, a chimeric antigen receptor (CAR), whole antibody or their antigen-binding fragment, single-chain variable fragment (scFv), a heavy chain or a light chain single domain antibody (sdAb), a Fab, a F(ab)2, or any combination thereof that binds to: (i) a cell surface tumor antigen, (ii) a peptide derived from a tumor antigen expressed on the cell surface as a complex with MHC (peptide-MHC complex), (iii) a cell surface antigen associated with an autoimmune disease or a pathogen, or (iv) a peptide derived from an antigen associated with an autoimmune disease or a pathogen expressed on the cell surface as a complex with MHC (peptide-MHC complex).

[0105] The term “RNA-guided nuclease” as used herein refers to a polypeptide that binds to aparticular target nucleotide sequence in a sequence-specific manner and is directed to the target nucleotide sequence by a guide RNA molecule that is complexed with the polypeptide and hybridizes with the target sequence. In embodiments, an RNA-guided nuclease is capable of cleaving the target sequence upon binding. The term “RNA-guided nuclease” also encompasses nuclease-dead RNA-guided nucleases that are capable of binding to, but not cleaving a double stranded target sequence. A dead RNA-guided nuclease may have nickase activity (i.e., capable of cleave only one strand of a double-stranded nucleic acid), or may not have the capability of cleaving any strand of a nucleic acid at all. Examples of suitable RNA-guided nucleases include Page 22 of 117 1104881063\1\AMERICASCas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Ca12a, Cas12b, Cas12e, Cas12d, Cas13a, Cas13, Cas13c, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2C1, C2C3, C2C4, C2C5, C2C6, C2C7, C2C8, c2C9, C2C10, CasX, and CasY. Further examples of RNA-guided nucleases include those described in Makarova et al. “Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants”, Nature Reviews Microbiology, 18:67-81 (Feb 2020), which is incorporated by reference herein in its entirety.

[0106] The term “guide RNA” as used herein refers to a nucleotide sequence having sufficientcomplementarity with a target nucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of an associated RNA-guided nuclease to the target nucleotide sequence. In general, a guide RNA comprises at least a spacer sequence (crRNA) that hybridizes to a target nucleic acid sequence of interest and a CRISPR repeat sequence. In embodiments, the gRNA also has a second RNA sequence, tracrRNA sequence. In embodiments, a guide RNA is a single RNA, e.g., a single molecule comprising both the spacer and tracrRNA sequences. γδ T CELL ACTIVATION

[0107] A γδ T cell population or a desired subset thereof in a mixed cell population (e.g., anisolated mixed cell population) may be activated by culturing the mixed cell population with at least one T cell mitogen. In embodiments, the at least one T cell mitogen comprises an antibody or a fragment thereof that binds to CD3, or to a γδ TCR. In embodiments, the at least one T cell mitogen comprises an antibody or a fragment thereof that binds to a variable region of a γδ TCR, preferably a variable region of a δ1, δ2, or δ3 γδ TCR, still more preferably to a variable region of a δ1 γδ TCR.

[0108] In embodiments, the at least one T cell mitogen comprises, or is, a means forselectively activating and expanding a subset of γδ T cells, e.g., δ1, δ2, or δ3 γδ T cells, by binding to an epitope comprising a variable region of a δ1, δ2, or δ3 γδ TCR. In embodiments, the at least one T cell mitogen comprises or is, a means for selectively activating and expanding δ1 γδ T cells by binding to an epitope comprising a variable region of a δ1 γδ TCR. In embodiments, the at least one T cell mitogen comprises at least one antibody. Page 23 of 117 1104881063\1\AMERICAS

[0109] In embodiments, an antibody that can be used for selectively activating and expandingδ1 γδ T cells binds to an epitope comprising a variable region of a δ1 γδ TCR. In some examples, the antibody is an antibody that binds to the same epitope as, or competes with, an antibody that binds to an epitope specific to a δ1 γδ TCR. Examples of such epitopes include those described below (e.g., Bin 1 δ1 epitope, Bin 1b δ1 epitope, Bin 2 δ1 epitope, Bin 2b δ1 epitope, Bin 2c δ1 epitope, Bin 5 δ1 epitope, Bin 6 δ1 epitope, Bin 7 δ1 epitope, Bin 8 δ1 epitope, and Bin 9 δ1 epitope) and in WO2017197347, which is incorporated by reference herein in its entirety.

[0110] In embodiments, the epitope, that is (e.g., specifically) recognized by the antibody isan epitope comprising or consisting of amino acids 47-70 of human Vδ1 (SKEMIFLIRQGSDEQNA) and J1 (TDKLIFGKGTRVTVEP) or J2 (LTAQLFFGKGTQLIVEP), wherein the antibody does not bind an epitope containing a K120T mutation in J1 or J2. This epitope is referred to herein as a Bin 1 δ1 epitope. A human Vδ1 J region can vary due to (D)(J) recombination, an exemplary Vδ1 J region of a δ1 chain of a γδ TCR having the Bin 1 δ1 epitope is SKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGL QDTDKLIFGKGTRVTVEP; and another exemplary Vδ1 J region Bin 1 δ1 epitope of a δ1 chain of a γδ TCR is SKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGEAPSAW GKHLTAQLFFGKGTQLIVEP.

[0111] In some cases, antibodies that bind a Bin 1 δ1 epitope bind a δ1 chain of a γδ TCRhaving the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVE P; but do not bind a δ1 chain of a γδ TCR having the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDSWDTRQMFFGTGIK LFVEP.

[0112] The antibodies that bind a Bin 1 δ1 epitope can also bind a δ1 chain of a γδ TCRhaving the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA Page 24 of 117 1104881063\1\AMERICASKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGEAPSAWGKHLTAQLFFGKGTQLI VEP.

[0113] In embodiments, the epitope, that is (e.g., specifically) recognized by an antibody is anepitope comprising or consisting of amino acids 47-70 of human Vδ1 (SKEMIFLIRQGSDEQNA) and J1 (TDKLIFGKGTRVTVEP), wherein the antibody does not bind an epitope containing a K120T mutation in J1. This epitope is referred to herein as a Bin 1b δ1 epitope. Exemplary antibodies that bind a Bin 1b δ1 epitope include δ1-37. An exemplary Vδ1 J region of a δ1 chain of a γδ TCR having the Bin 1b δ1 epitope is SKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSV ALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP.

[0114] In some cases, antibodies that bind a Bin 1b δ1 epitope bind a δ1 chain of a γδ TCRhaving the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVE P; but do not bind a δ1 chain of a γδ TCR having the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDSWDTRQMFFGTGIK LFVEP.

[0115] The antibodies that bind a Bin 1b δ1 epitope also do not bind a δ1 chain of a γδ TCRhaving the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGEAPSAWGKHLTAQLFFGKGTQLI VEP.

[0116] In embodiments, the epitope that is (e.g., specifically) recognized by an antibody is anepitope comprising or consisting of amino acids 11-21 of human Vδ1 (VSMPVRKAVTL). This epitope is referred to herein as a Bin 2 δ1 epitope. Exemplary antibodies that bind a Bin 2 δ1 epitope include δ1-285.

[0117] In some cases, antibodies that bind a Bin 2 δ1 epitope bind a δ1 chain of a γδ TCRhaving the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVE P; but do not bind a δ1 chain of a γδ TCR having the sequence of: Page 25 of 117 1104881063\1\AMERICASAQKVTQVQRAMSSQLGEAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVE PRSQPHTKPSVFVMKNGTNVACLVKEF.

[0118] In embodiments, the epitope that is (e.g., specifically) recognized by an antibody is anepitope comprising or consisting of amino acids 11-21 of human Vδ1 (VSMPVRKAVTL), wherein the antibody that binds this epitope does not bind an epitope containing a mutation of R16 in Vδ1, such as an R16N mutation. This epitope is referred to herein as a Bin 2b δ1 epitope. Exemplary antibodies that bind a Bin 2b δ1 epitope include R9.12.

[0119] In some cases, antibodies that bind a Bin 2b δ1 epitope bind a δ1 chain of a γδ TCRhaving the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVE P; but do not bind a δ1 chain of a γδ TCR having the sequence of: AQKVTQAQSSVSMPVNKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVE P; and / or do not bind a δ1 chain of a γδ TCR having the sequence of: AQKVTQVQRAMSSQLGEAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVE PRSQPHTKPSVFVMKNGTNVACLVKEF.

[0120] In embodiments, the epitope that is (e.g., specifically) recognized by an antibody is anepitope comprising or consisting of amino acids 11-21 of human Vδ1 (VSMPVRKAVTL), wherein the antibody that binds this epitope also binds (cross-reacts) with δ3, δ4, and δ5 γδ TCRs. This epitope is referred to herein as a Bin 2c δ1 epitope. Exemplary antibodies that bind a Bin 2c δ1 epitope include δ1-39.

[0121] In some cases, antibodies that bind a Bin 2c δ1 epitope bind a δ1 chain of a γδ TCRhaving the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVE P; but do not bind a δ1 chain of a γδ TCR having the sequence of: AQKVTQVQRAMSSQLGEAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA Page 26 of 117 1104881063\1\AMERICASKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVE PRSQPHTKPSVFVMKNGTNVACLVKEF.

[0122] In embodiments, the epitope that is (e.g., specifically) recognized by an antibody is anepitope comprising or consisting of amino acids 80-95 of human Vδ1 (FKKAAKSVALTISALQ) or 70 to 95 of human Vδ1 (AKSGRYSVNFKKAAKSVALTISALQ). This epitope is referred to herein as a Bin 3 δ1 epitope. Exemplary antibodies that bind a Bin 3 δ1 epitope include δ1-08; and δ1-23.

[0123] In some cases, antibodies that bind a Bin 3 δ1 epitope bind a δ1 chain of a γδ TCRhaving the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVE P, but do not bind a δ1 chain of a γδ TCR having the sequence of: AQKVTQVQRAMSSQLGEAVTLSCQYETSLSWYDIFWYKQLPSGEMTFLIHQISSDQNAK NGRYSVNFQERHKFISLTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEPR SQPHTKPSVFVMKNGTNVACLVKEFYPKD.

[0124] In embodiments, the epitope that is (e.g., specifically) recognized by an antibody is anepitope comprising or consisting of amino acids 1-11 of human Vδ1 (AQKVTQAQSSV) and J1 or J2. This epitope is referred to herein as a Bin 4 δ1 epitope. In some cases, the Bin 4 δ1 epitope binding antibody does not bind an epitope containing a K120T mutation in J1 or J2. Exemplary antibodies that bind a Bin 4 δ1 epitope include δ1-35; and δ1-203.

[0125] In some cases, antibodies that bind a Bin 4 δ1 epitope bind a δ1 chain of a γδ TCRhaving the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVE P; but do not bind a δ1 chain of a γδ TCR having the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDSWDTRQMFFGTGIK LFVEP.

[0126] In some cases, antibodies that bind a Bin 4 δ1 epitope bind a δ1 chain of a γδ TCRhaving the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA Page 27 of 117 1104881063\1\AMERICASKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGEAPSAWGKHLTAQLFFGKGTQLI VEP.

[0127] In embodiments, the epitope that is (e.g., specifically) recognized by an antibody ofinterest is an epitope comprising or consisting of amino acids 28-47 of human Vδ1 (SWWSYYIFWYKQLPS) and J1. This epitope is referred to herein as a Bin 5 δ1 epitope. Exemplary antibodies that bind a Bin 5 δ1 epitope include δΙ-113; δ1- 155; δ1-183; δ1-191; δ1- 278; and δ1-282. An exemplary Vδ1 J1 region of a δ1 chain of a γδ TCR having the Bin 5 δ1 epitope is SWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDS AKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP.

[0128] In some cases, antibodies that bind a Bin 5 δ1 epitope bind a δ1 chain of a γδ TCRhaving the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVE P; but do not bind a δ1 chain of a γδ TCR having the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGEAPSAWGKHLTAQLFFGKGTQLI VEP.

[0129] In embodiments, the epitope that is (e.g., specifically) recognized by an antibody is anepitope comprising or consisting of amino acids 21-28 of human Vδ1 (LNCLYETS) and J1. This epitope is referred to herein as a Bin 6 δ1 epitope. Exemplary antibodies that bind a Bin 6 δ1 epitope include TS 8.2 and δ1-143. An exemplary Vδ1 J1 region of a δ1 chain of a γδ TCR having the Bin 6 δ1 epitope is LNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTIS ALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP.

[0130] In some cases, antibodies that bind a Bin 6 δ1 epitope bind a δ1 chain of a γδ TCRhaving the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVE P; but do not bind a δ1 chain of a γδ TCR having the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA Page 28 of 117 1104881063\1\AMERICASKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDSWDTRQMFFGTGIK LFVEP.

[0131] In embodiments, the epitope that is (e.g., specifically) recognized by an antibody is anepitope comprising or consisting of amino acids 47-70 of human Vδ1 (SKEMIFLIRQGSDEQNA) and J1 or J2. This epitope is referred to herein as a Bin 7 δ1 epitope. Exemplary antibodies that bind a Bin 7 δ1 epitope include δ1-149; δ1- 253, and δ1-257. An exemplary Vδ1 J region of a δ1 chain of a γδ TCR having the Bin 7 δ1epitope is SKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGL QDTDKLIFGKGTRVTVEP; and another exemplary νδ1 J region Bin 7 δ1 epitope of a δ1 chain of a γδ TCR is SKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGEAPSAW GKHLTAQLFFGKGTQLIVEP.

[0132] In some cases, antibodies that bind a Bin 7 δ1 epitope bind a δ1 chain of a γδ TCRhaving the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVE P; but do not bind a δ1 chain of a γδ TCR having the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDSWDTRQMFFGTGIK LFVEP.

[0133] In embodiments, the epitope that is (e.g., specifically) recognized by an antibody is anepitope comprising or consisting of amino acids 70-80 of human Vδ1 (AKSGRYSVNF) and J1 or J2. This epitope is referred to herein as a Bin 8 δ1 epitope. Exemplary antibodies that bind a Bin 8 δ1 epitope include δ1-192. An exemplary Vδ1 J region of a δ1 chain of a γδ TCR having the Bin 8 δ1 epitope is AKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTV EP; and another exemplary Vδ1 J region Bin 8 δ1 epitope of a δ1 chain of a γδ TCR is AKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGEAPSAWGKHLTAQLFFGKGTQ LIVEP.

[0134] In some cases, antibodies that bind a Bin 8 δ1 epitope bind a δ1 chain of a γδ TCRhaving the sequence of: Page 29 of 117 1104881063\1\AMERICASAQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVE P; but do not bind a δ1 chain of a γδ TCR having the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDSWDTRQMFFGTGIK LFVEP.

[0135] In embodiments, the epitope that is (e.g., specifically) recognized by an antibody ofinterest is an epitope comprising or consisting of amino acids 80-95 of human νδ1 (FKKAAKSVALTISALQ). This epitope is referred to herein as a Bin 9 δ1 epitope. Exemplary antibodies that bind a Bin 9 δ1 epitope include δ1-201.

[0136] In some cases, antibodies that bind a Bin 9 δ1 epitope bind a δ1 chain of a γδ TCRhaving the sequence of: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNA KSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVE P; but do not bind a δ1 chain of a γδ TCR having the sequence of AQKVTQVQRAMSSQLGEAVTLSCQYETSLSWYDIFWYKQLPSGEMTFLIHQISSDQNAK NGRYSVNFQERHKFISLTISALQLEDSAKYFCALGTGVRGLQDTDKLIFGKGTRVTVEP.

[0137] The δ1-specific antibodies described herein selectively bind δ1-containing γδ-TCRsover δ2-containing γδ-TCRs. As such, the foregoing δ1-specific antibodies do not bind, e.g., the sequence of and / or a γδ-TCR comprising the sequence of: AIELVPEHQTVPVSIGVPATLRCSMKGEAIGNYYINWYRKTQGNTMTFIYREKDIYGPGF KDNFQGDIDIAKNLAVLKILAPSERDEGSYYCACDPLGGPPDKLIFGKGTRVTVEP.

[0138] In one example, the epitope is Bin 3 δ1 epitope comprising or consisting of amino acidsFKKAAKSVALTISALQ or AKSGRYSVNFKKAAKSVALTISALQ of human Vδ1. In another example, the epitope is Bin 4 δ1 epitope comprising or consisting of amino acids AQKVTQAQSSV of human Vδ1, and amino acids TDKLIFGKGTRVTVEP of human J1 or LTAQLFFGKGTQLIVEP of human J2, wherein the at least one antibody does not bind an epitope containing a K120T mutation in J1 or J2.

[0139] Examples of antibodies that can be used as a T cell mitogen herein include thosecomprising the framework regions and CDRs in FIGs.1 and 2. In one example, the antibody binds to the same epitope as, or competes with, antibody δ1-08. In another example, the antibody Page 30 of 117 1104881063\1\AMERICAScomprises the six complementarity determining regions (CDRs) of antibody δ1-08. In another example, the antibody binds to the same epitope as, or competes with, antibody δ1-35. In another example, the antibody comprises the six CDRs of antibody δ1-35. Further examples of antibodies that can be used as T mitogens in the subject invention include those disclosed in WO2017197347, WO2021113558, WO2023156604, WO2022034562, WO2022175414, WO2022175413, WO2021032963, and WO2021032960, which are incorporated by reference herein in their entireties.

[0140] In embodiments, an antibody that can be used for selectively activating and expandingδ2 γδ T cells binds to an epitope comprising a variable region of a δ2 γδ TCR. In some examples, the antibody is an antibody that binds to the same epitope as, or competes with, an antibody that binds to an epitope specific to a δ2 γδ TCR, including those described in WO / 2016 / 081518 and WO 2017 / 197347, each of which is incorporated by reference herein in its entirety. In one example, the antibody is δ2-37 described in WO 2017 / 197347. In another example, the antibody is δ2-22 described in WO 2017 / 197347. In another example, the antibody is δ2-14 described in WO 2017 / 197347.

[0141] In embodiments, an antibody that can be used for selectively activating and expandingδ3 γδ T cells binds to an epitope comprising a variable region of a δ3 γδ TCR. In some examples, the antibody is an antibody that binds to the same epitope as, or competes with, an antibody that binds to an epitope specific to a δ3 γδ TCR, including those described in WO 2019 / 099744, which is incorporated by reference herein in its entirety. In one example, the antibody is δ3-08 described in WO 2019 / 099744. In another example, the antibody is δ3-23 described in WO 2019 / 099744.

[0142] Further examples of T mitogens finding advantageous use in the subject inventioninclude anti-CD3 (e.g. OKT3), anti-CD2, anti-CD27, anti-CD28, anti-CD30, anti-CD70, anti- CD137, anti-OX40 antibodies, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), CD70 (CD27 ligand), concavalin A (ConA), pokeweed (PWM), protein peanut agglutinin (PNA), soybean agglutinin (SBA), Les Culinaris Agglutinin (LCA), Pisum Sativum Agglutinin (PSA), Helix pomatia agglutinin (HP A), Vicia graminea Lectin (VGA), Phaseolus Vulgaris Erythroagglutinin (PHA-E), Phaseolus Vulgaris Leucoagglutinin (PHA-L), Sambucus Nigra Lectin (SNA, EBL), Maackia Amurensis, Lectin Π (MAL Π), Sophora Japonica Agglutinin (SJA), Dolichos Biflorus Agglutinin (DBA), Lens Page 31 of 117 1104881063\1\AMERICASCulinaris Agglutinin (LCA), Wisteria Floribunda Lectin (WFA, WFL) or another suitable mitogen capable of stimulating T cell proliferation.

[0143] In embodiments, a T cell mitogen is an antibody that binds to CD28. Examples ofantibodies that bind to CD28 include 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248.23.2, and EX5.3D10, lulizumab pegol, and TGN1412. Additional examples of antibodies that bind to CD28 include those described in Poirier N. et al., CD28-specific immunomodulating antibodies: what can be learned from experimental models? Am J Transplant, 2012 Jul;12(7):1682-90, which is incorporated by reference in its entirety. Further examples of antibodies that bind to CD28 include those described in US8785604, US10364287, US8679841, WO2023114701, and US11919958, each of which is incorporated by reference herein in its entirety.

[0144] In embodiments, a T cell mitogen is an anti-CD137 antibody. Examples of antibodiesthat bind to CD137 (also known as 4-1BB) include BMS-663031, BMS-469492, XmAb-5592 (Xencor), PF-05082566 (Pfizer), Urelumab (BMS-663513), Utomilumab (PF-05082566), GEN 1042 (BioNTech SE (Genmab)), LOAd703 (Lokon Pharma), ADG-106 (Adagene), PRS-343 (Pieris Pharmaceuticals), CTX-471 (Compass Therapeutics), INBRX-105 (Elpiscience; Inhibrx), LVGN6051 (Lyvgen Biopharma), MCLA-145 (Merus NV), MP-0310 (Molecular Partners), ATOR-1017 (Alligator Bioscience AB), RG-6076 (Roche), RG-7827 (Roche), and AGEN-2373 (Agenus). Additional examples of antibodies that bind to CD137 include those described in Chu D et al., An Update on Anti-CD137 Antibodies in Immunotherapies for Cancer, Int J Mol Sci. 2019 Apr; 20(8): 1822; Bartkowiak T et al., 4-1BB Agonists: Multi-Potent Potentiators of Tumor Immunity, Front Oncol. 2015 Jun 8:5:117.; Vinay DS et al., Immunotherapy of cancer with 4- 1BB, Mol Cancer Ther. 2012 May;11(5):1062-70, each of which is incorporated by reference herein in its entirety. Further examples of antibodies that bind to CD137 include those described in WO2014144666, WO2006088447, WO2006088464, US20080166336, US20050095244, US7288638, US6362325, US7214493, US6887673, US8337850, US8821867, US7288638, US6303121, US6569997, US6905685, US6355476, US6362325, US6974863, US6210669, and US5928893, each of which is incorporated by reference herein in its entirety.

[0145] In embodiments, for activation, the mixed cell population is cultured in a mediumcontaining the one or more T cell mitogens for about 1 day, 2 days, 3 days, 4 days 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or any range therein. For example, the mixed cell population may be cultured in a medium containing at least one T cell mitogens for about 1 to 3 days, 1 to 4 days, Page 32 of 117 1104881063\1\AMERICAS1 to 5 days, 2 to 3 days, 2 to 4 days, 2 to 5 days, 3 to 4 days, 3 to 5 days, 4 to 6 days, 5 to 7 days, 6 to 8 days, 7 to 9 days, or 8 to 10 days. In one example, the mixed cell population may be cultured in a medium containing at least one T cell mitogen for about 5 to 7 days. In another example, the mixed cell population may be cultured in a medium containing at least one T cell mitogen for about 6 days.

[0146] In embodiments, the at least one T cell mitogen (e.g., antibody) is immobilized on asurface such as a cell culture surface (e.g., the surface of a cell culture plate, wells, containers (e.g. flasks or bags), beads, or other solid supports in or covered by cell culture media such as, e.g., nanomatrices). Depending on the cell adhesion properties of the substrate, a combination of T cell mitogens can advantageously be used.

[0147] In embodiments, the one or more T cell mitogens is immobilized on a bead. A beadmay be a solid or semi-solid support structure can have any suitable shape, e.g., spherical, ovoid, cylindrical or any other recognized particle shape with regular or irregular dimensions.

[0148] In embodiments, a bead is conjugated with one or more T cell mitogens, e.g., theantibodies described herein. In some examples, a bead is conjugated with an antibody that binds to CD3. In some examples, a bead is conjugated with an antibody that binds to CD28. In some examples, a bead is conjugated with an antibody that binds to a γδ TCR or a fragment thereof (e.g., a δ1 γδ TCR). In embodiments, a bead is conjugated with multiple antibodies that bind to different targets. In some examples, a bead is conjugated with an antibody that binds to CD3 and an antibody that binds to CD28. In some examples, a bead is conjugated with an antibody that binds to a γδ TCR (e.g., a δ1 γδ TCR) and an antibody that binds to CD28.

[0149] In embodiments, a bead is conjugated with a combination of at least one antibody or afragment thereof that binds to a variable region of a γδ TCR, preferably a variable region of a δ1, δ2, or δ3 γδ TCR, still more preferably to a variable region of a δ1 γδ TCR, and one or moreantibodies that bind to CD28, CD137, CD2, CD27, and / or OX40. In embodiments, a bead isconjugated with a combination of a means for selectively activating and expanding δ1 γδ T cells by binding to an epitope comprising a variable region of a δ1 γδ TCR, and an antibody that binds to CD28, CD137, CD2, CD27, and / or OX40. In embodiments, the T cell mitogen combination comprises an antibody that binds to variable region of a δ1 γδ TCR, e.g. antibody δ1-35 and / or antibody δ1-08, and an anti-CD28 antibody and / or anti-CD137 antibody. Page 33 of 117 1104881063\1\AMERICAS

[0150] Examples of beads include those described in Trickett A et al., T cell stimulation andexpansion using anti-CD3 / CD28 beads, J Immunol Methods.2003 Apr 1;275(1-2):251-5; Li Y et al., Comparison of anti-CD3 and anti-CD28-coated beads with soluble anti-CD3 for expanding human T cells: differing impact on CD8 T cell phenotype and responsiveness to restimulation, J Transl Med. 2010 Oct 26:8:104; Gansuvd B et al., Expansion of CD4+CD25+ suppressive regulatory T cells from rhesus macaque peripheral blood by FN18 / antihuman CD28-coated Dynal beads, Hum Immunol.2007 Jun;68(6):478-90; Walter S et al., Cutting edge: predetermined avidity of human CD8 T cells expanded on calibrated MHC / anti-CD28-coated microspheres, J Immunol. 2003 Nov 15;171(10):4974-8; Diehn M et al., Genomic expression programs and the integration of the CD28 costimulatory signal in T cell activation, Proc Natl Acad Sci U S A. 2002 Sep 3;99(18):11796-801, each of which is incorporated by reference herein in its entirety. Further examples of beads include those described in US9309368, US10017586, US7160707, US8722429, US20030119185, and US7572631, each of which is incorporated by reference in its entirety.

[0151] In embodiments, the bead may be added to the culture medium at a suitable bead to cellratio (the ratio of the number of beads to the number of cells). In some examples, the bead to cell ratio may be 100:1 to 1:100, e.g., from 1:100 to 1:80, from 1:90 to 1:70, from 1:80 to 1:60, from 1:70 to 1:50, from 1:60 to 1:40, from 1:50 to 1:30, from 1:40 to 1:20, from 1:30 to 1:10, from 1:20 to 1:1, from 1:10 to 10:1, from 1:1 to 20:1, from 10:1 to 30:1, from 20:1 to 40:1, from 30:1 to 50:1, from 40:1 to 60:1, from 50:1 to 70:1, from 60:1 to 80:1, from 70:1 to 90:1, or from 80:1 to 100:1.

[0152] In embodiments, a bead is conjugated with an antibody with a suitable antibody load(the average number of antibody molecules per bead). In some examples, a bead may be conjugated with an antibody with a 101to 108antibodies per bead, e.g., from 101to 102, from 102to 103, from 103to 104, from 104to 105, from 105to 106, from 106to 107, or from 107to 108antibodies per bead.

[0153] In embodiments, a bead is conjugated with a first antibody (e.g., an antibody that bindsto a γδ TCR such as a δ1 γδ TCR) and a second antibody (e.g., an antibody that binds to CD28 or CD137) with a suitable molar ratio between the two antibodies. In some examples, the molar ratio between the first antibody and the second antibody is 1:100 to 100:1, e.g., from 1:100 to 1:80, from 1:90 to 1:70, from 1:80 to 1:60, from 1:70 to 1:50, from 1:60 to 1:40, from 1:50 to 1:30, from 1:40 to 1:20, from 1:30 to 1:10, from 1:20 to 1:1, from 1:10 to 10:1, from 1:1 to 20:1, from 10:1 Page 34 of 117 1104881063\1\AMERICASto 30:1, from 20:1 to 40:1, from 30:1 to 50:1, from 40:1 to 60:1, from 50:1 to 70:1, from 60:1 to 80:1, from 70:1 to 90:1, or from 80:1 to 100:1.

[0154] In embodiments, the one or more T cell mitogens is immobilized on a cell culture bag.Suitable examples of such cell culture bags include those described in Garland RJ et al., The use of Teflon cell culture bags to expand functionally active CD8+ cytotoxic T lymphocytes, J Immunol Methods, 1999 Jul 30;227(1-2):53-63; Vomittag P et al., A guide to manufacturing CAR T cell therapies, Curr Opin Biotechnol, 2018 Oct:53:164-181, each of which is incorporated by reference in its entirety. Further examples of such bags include those described in US10655097, US11959056, US20210198606, and US20160178490, each of which is incorporated by reference in its entirety. In some examples, the cell culture bag comprises a treated form of fluorinated ethylene propylene film and have a high surface energy. In one example, the bag is a VueLife® AC bag (Saint Gobain).

[0155] In embodiments, the at least one T cell mitogen for use with cell culture bags comprisesa combination of at least one antibody or a fragment thereof that binds to a variable region of a γδ TCR, preferably a variable region of a δ1, δ2, or δ3 γδ TCR, still more preferably to a variableregion of a δ1 γδ TCR, and one or more antibodies that bind to CD28, CD137, CD2, CD27, and / orOX40. In embodiments, the at least one T cell mitogen for use with cell culture bags comprises a combination of a means for selectively activating and expanding δ1 γδ T cells by binding to an epitope comprising a variable region of a δ1 γδ TCR, and an antibody that binds to CD28, CD137, CD2, CD27, and / or OX40. In exemplary embodiments, the T cell mitogen combination comprises an antibody that binds to variable region of a δ1 γδ TCR, e.g. antibody δ1-35 and / or antibody δ1- 08, and an anti-CD28 antibody and / or anti-CD137 antibody.

[0156] In embodiments, the one or more T cell mitogens is immobilized on a polymericnanomatrix. In embodiments, the polymeric nanomatrix more easily contacts the cell surface membrane of target cells, e.g., T cells to be activated and / or proliferated. The polymeric nanomatrix may comprise one or more T cell mitogens and provide optimal access of the T cell mitogen(s) to their cell surface receptors or antigens. In embodiments, the polymeric nanomatrix can provide enough cross-linking to activate T cells regardless of the small size of the structure.

[0157] A polymeric nanomatrix comprises polymers such as collagen, purified proteins,purified peptides, polysaccharides, glycosaminoglycans, or extracellular matrix compositions, or any combination thereof. A polysaccharide may include for example, cellulose, agarose, dextran, Page 35 of 117 1104881063\1\AMERICASchitosan, hyaluronic acid, or alginate. Other polymers may include polyesters, polyethers, polyanhydrides, polyalkylcyanoacrylates, polyacrylamides, polyorthoesters, polyphosphazenes, polyvinylacetates, block copolymers, polypropylene, polytetrafluorethylene (PTFE), or polyurethanes. The polymer may be lactic acid or a copolymer. A copolymer may comprise lactic acid and glycolic acid (PLGA). In embodiments, the polymeric nanomatrix comprise of dextran. In some examples, the polymeric nanomatrix is biodegradable to cells.

[0158] In embodiments, the polymeric nanomatrix is conjugated with one or more T cellmitogens, e.g., the antibodies described herein. In some examples, the polymeric nanomatrix is conjugated with an antibody that binds to CD3. In some examples, the polymeric nanomatrix is conjugated with an antibody that binds to CD28. In some examples, the polymeric nanomatrix is conjugated with an antibody that binds to a γδ TCR or a fragment thereof (e.g., a δ1 γδ TCR). In embodiments, the polymeric nanomatrix is conjugated with multiple antibodies that bind to different targets. In some examples, the polymeric nanomatrix is conjugated with an antibody that binds to CD3 and an antibody that binds to CD28. In some examples, the polymeric nanomatrix is conjugated with an antibody that binds to a γδ TCR (e.g., a δ1 γδ TCR) and an antibody that binds to CD28.

[0159] In embodiments, the polymeric nanomatrix is conjugated with a combination of i) atleast one antibody or a fragment thereof that binds to a variable region of a γδ TCR, preferably a variable region of a δ1, δ2, or δ3 γδ TCR, still more preferably to a variable region of a δ1 γδ TCR,and ii) one or more antibodies that bind to CD28, CD137, CD2, CD27, and / or OX40. Inembodiments, the polymeric nanomatrix is conjugated with a combination of a means for selectively activating and expanding δ1 γδ T cells by binding to an epitope comprising a variable region of a δ1 γδ TCR, and an antibody that binds to CD28, CD137, CD2, CD27, and / or OX40. In exemplary embodiments, the T cell mitogen combination comprises an antibody that binds to variable region of a δ1 γδ TCR, e.g. antibody δ1-35 and / or antibody δ1-08, and an anti-CD28 antibody and / or anti-CD137 antibody.

[0160] Examples of polymeric nanomatrices include those described in US10513687 ; WangX et al., Evaluation of Miltenyi ExpAct and TransAct CD3 / 28 Beads for CAR-T Cell Manufacturing, Vector and Cell Engineering / Manufacturing I, Volume 24, Supplement 1, S182, May 2016; Mauer D et al., Potent Polyclonal T Cell Activation and Expansion Through GMP- Grade Transact Nano-Matrices, Clinical Translation of Vector Production and Protocol Page 36 of 117 1104881063\1\AMERICASPreparation II, Volume 22, Supplement 1, S286, May 2014; Casati Anna et al., Clinical-scale selection and viral transduction of human naïve and central memory CD8+ T cells for adoptive cell therapy of cancer patients, Cancer Immunol Immunother. 2013 Aug 1;62(10):1563–1573, each of which is incorporated by reference herein in its entirety. In one example, the polymeric nanomatrix is T cell TransAct (Miltenyi Biotec).

[0161] In embodiments, the polymeric nanomatrix is added to the culture medium at a suitablenanomatrix to cell ratio (the ratio of the number of nanomatrix particles to the number of cells). In some examples, the nanomatrix to cell ratio may be at least 100:1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1, at least 800:1, at least 900:1, or at least 1000:1. In some examples, the nanomatrix to cell ratio may be from 100:1 to 1000:1, e.g., from 100:1 to 200:1, from 200:1 to 300:1, from 300:1 to 400:1, from 400:1 to 500:1, from 500:1 to 600:1, from 600:1 to 700:1, from 700:1 to 800:1, from 800:1 to 900:1, or from 900:1 to 1000:1.

[0162] In embodiments, the polymeric nanomatrix is from 1 to 1000 nm, e.g., 1 nm to 800 nm,1 nm to 600 nm, 1 nm to 500 nm, 1 nm to 300 nm, 1 nm to 200 nm, 10 nm to 200v, 50 nm to 200 nm, 100 nm to 200 nm, 1 nm to 100 nm, 10 nm to 100 nm, or 50 nm to 100 nm, in size.

[0163] In embodiments, the polymeric nanomatrix is conjugated with a first antibody and asecond antibody with a suitable molar ratio between the two antibodies. In some examples, the molar ratio between the first antibody and the second antibody is 1:100 to 100:1, e.g., from 1:100 to 1:80, from 1:90 to 1:70, from 1:80 to 1:60, from 1:70 to 1:50, from 1:60 to 1:40, from 1:50 to 1:30, from 1:40 to 1:20, from 1:30 to 1:10, from 1:20 to 1:1, from 1:10 to 10:1, from 1:1 to 20:1, from 10:1 to 30:1, from 20:1 to 40:1, from 30:1 to 50:1, from 40:1 to 60:1, from 50:1 to 70:1, from 60:1 to 80:1, from 70:1 to 90:1, or from 80:1 to 100:1.

[0164] In embodiments, the γδ T cells activation may be performed using self-assemblingprotein nanoparticles with functional antibodies (e.g., anti-CD3 and anti-CD-28 antibodies) bound to the nanoparticle surface through high-affinity non-covalent interactions. Examples of such nanoparticles include those described in US20220196655 (“Self-Assembling Protein Nanocage Decorated with Antibodies (Sapna) and Parts Thereof”) and Imam Z et al., Expansion of stem-like t cells by novel nanosparktm stem-t activator, Cytotherapy, Volume 26, Issue 6, Supplement, June 2024, Pages S171-S172, each of which is incorporated by reference in its entirety. In some examples, such nanoparticles are NanoSpark STEM-T Soluble T Cell Activator or NanoSpark EVEN-T Soluble T Cell Activator (Nanotein). Page 37 of 117 1104881063\1\AMERICAS

[0165] In embodiments, the γδ T cells activation may be performed using soluble antibodycomplexes that bind cell surface ligands (e.g., CD3 and CD28). Binding of such antibody complexes may result in the cross-linking of the cell surface ligands, thereby providing the required primary and co-stimulatory signals for T cell activation. Examples of such soluble antibody complexes include those described in WO2016033690, “Soluble antibody complexes for t cell or NK cell activation and expansion” and Siatskas C et al., Activation, expansion, and culture of human t cells incorporating immunoculttm cgmp-compliant ancillary reagents, Cytotherapy, Volume 26, Issue 6, Supplement, June 2024, Pages S195-S196, each of which is incorporated by reference in its entirety. In some examples, such soluble antibody complexes are ImmunoCult Human CD3 / CD28 T Cell Activators (Stemcell Technologies, Catalog # 10971).

[0166] In embodiments, the mixed cell population is a peripheral blood sample (e.g., PBLs orPBMCs), a leukapheresis sample, a cord blood sample, a tumor, a stem cell precursor, a tumor biopsy, a tissue, a lymph, or from epithelial sites of a subject directly contacting the external milieu, or derived from stem precursor cells. In some examples, the mixed cell population is a peripheral blood sample, a cord blood sample, or a tumor. Peripheral blood mononuclear cells can be collected from a subject, for example, with an apheresis machine, and enriched using density gradient separation media including the Ficoll-Paque™ PLUS (GE Healthcare) or red blood cell lysis method, or another suitable device / system. γδ T cells can be purified from the collected sample with, for example, flow cytometry techniques. Cord blood cells can also be obtained from cord blood during the birth of a subject. See WO 2016 / 081518, incorporated by reference herein in its entirety.

[0167] In embodiments, the mixed cell population is derived from a single donor. In otherembodiments, the mixed cell population is derived from multiple donors (e.g., 2, 3, 4, 5, or from 2-5, 2-10, or 5-10 donors, or more). γδ T CELL ENRICHMENT

[0168] The activated γδ T cell population in the mixed cell population may be enriched bydepleting undesired type of cells, e.g., monocytes, macrophages, B cells, NK cells, αβ T cells, or a combination thereof. In embodiments, the activated γδ T cell population is enriched by depleting αβ T cells. In embodiments, the activated γδ T cell population is enriched by depleting NK cells. Page 38 of 117 1104881063\1\AMERICASIn embodiments, the activated γδ T cell population is enriched by depleting αβ T cells and NK T cells.

[0169] The depletion of certain types of cells from the mixed cell population may be performedusing techniques known in the art. In embodiments, the enrichment of γδ T cells is performed with positive and / or negative selection of cell surface markers expressed on the collected cells can be used to directly isolate a γδ T cell population. For instance, a γδ T cell population can be enriched based on positive or negative expression of markers such as CD2, CD3, CD4, CD8, CD24, CD25, CD44, Kit, TCR ^, TCR ^, TCR ^ (including one or more TCR ^ sub-types), TCR ^ (including one or more TCR ^ sub-types), NKG2D, CD70, CD27, CD28, CD30, CD16, OX40, CD46, CD161, CCR7, CCR4, NKp30, NKp44, NKp46, DNAM-1, CD242, JAML, and other suitable cell surface markers.

[0170] In embodiments, the depletion may be performed by culturing the mixed cellpopulation with one or more antibodies that bind to specific molecules on the cells to be depleted. In embodiments, the one or more antibodies may be coupled to magnetic beads that can be used to magnetically deplete or enrich target cells when these cells are passed through a magnetic column. Alternatively, or additionally, the one or more antibodies may be used to label cells and depletion may be performed using anti-immunoglobulin coupled microbeads. Additional example methods for depletion of αβ T cells include those described in Nishimoto KP et al., Allogeneic CD20-targeted γδ T cells exhibit innate and adaptive antitumor activities in preclinical B-cell lymphoma models, Clin Transl Immunology.2022 Feb 2;11(2):e1373; Almeida AR et al., Delta One T Cells for Immunotherapy of Chronic Lymphocytic Leukemia: Clinical-Grade Expansion / Differentiation and Preclinical Proof of Concept, Clin Cancer Res. 2016 Dec 1;22(23):5795-5804; Ferry GM et al., A Simple and Robust Single-Step Method for CAR-Vδ1 γδ T Cell Expansion and Transduction for Cancer Immunotherapy, Front Immunol. 2022 May 31:13:863155, each of which is incorporated by reference herein in its entirety.

[0171] In embodiments, NK cells may be depleted using an agent (e.g., an antibody)specifically binds to CD56. In some examples, the NK cells may be depleted using microbeads comprising anti-CD56 antibody, e.g., Miltenyi Biotec Part No.130-050-401. In some examples, the NK cells may be depleted using methods and reagents described in Ferry GM et al., A Simple and Robust Single-Step Method for CAR-Vδ1 γδ T Cell Expansion and Transduction for Cancer Immunotherapy, Front Immunol.2022 May 31:13:863155). Page 39 of 117 1104881063\1\AMERICAS

[0172] In embodiments, the depletion is performed after culturing the mixed cell populationwith at least one T cell mitogen for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days and before transduction. In one example, the depletion may be performed after culturing the mixed cell population with at least one T cell mitogen for about 5 days. In some examples, the depletion may be performed after culturing the mixed cell population with at least one T cell mitogen for about 1 to 3 days, 2 to 4 days, 3 to 5 days, 4 to 6 days, 5 to 7 days, 6 to 8 days, 7 to 9 days, or 8 to 10 days. In one example, the depletion may be performed after culturing the mixed cell population with at least one T cell mitogen for about 4 to 6 days. γδ T CELL ENGINEERING

[0173] The enriched γδ T cells may be engineered to express at least one antigen recognitionmoiety. The engineering may comprise stably integrating one or more nucleic acid constructs encoding the at least one antigen recognition moiety in a culture medium comprising at least one antibody and / or at least one cytokine.

[0174] In embodiments, the culture medium for engineering comprises one or more of IL-4,IL-21, and IL-1β. In one example, the culture medium for engineering may comprise IL-4, IL-21, and IL-1β.

[0175] In embodiments, the culture medium for engineering comprises one or more cytokinesbut does not comprise an antibody that stimulates T cell proliferation. In one example, the culture medium for engineering comprises one or more cytokine but does not comprise an anti-CD3 antibody. For example, the culture medium for engineering may comprise IL-4, IL-21, and IL-1β, and does not comprise anti-CD3 antibody.

[0176] In embodiments, the medium does not comprise IFNγ. In another example, the culturemedium for engineering may comprises IL-4, IL-21, and IL-1β, and does not comprise IFNγ. In another example, the culture medium for engineering may comprises IL-4, IL-21, and IL-1β, and does not comprise IFNγ or anti-CD3 antibody.

[0177] In embodiments, the culture medium for engineering may further comprise an anti-CD3 antibody (e.g., the OKT3 antibody as described Almeida AR et al., Delta One T Cells for Immunotherapy of Chronic Lymphocytic Leukemia: Clinical-Grade Expansion / Differentiation and Preclinical Proof of Concept, Clin Cancer Res. 2016 Dec 1;22(23):5795-5804; Ferry GM et al., A Simple and Robust Single-Step Method for CAR-Vδ1 γδ T Cell Expansion and Transduction Page 40 of 117 1104881063\1\AMERICASfor Cancer Immunotherapy, Front Immunol.2022 May 31:13:863155), and IL-4, IL-21, and IL- 1β. In embodiments, the medium may further comprise IFNγ.

[0178] For the engineering step, the γδ T cells may be cultured in the medium for about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more days, e.g., about 1 to 3 days, 2 to 4 days, 3 to 5 days, 4 to 6 days, 5 to 7 days, 6 to 8 days, 7 to 9 days, or 8 to 10 days. In one example, for the engineering step, the γδ T cells may be cultured in the medium for about 2 to 4 days, e.g., 2, 3, or 4 days. In some examples, for the engineering, step, the γδ T cells may be cultured in the medium for between about 16 hours and 120 hours, between about 48 hours and 96 hours, between about 60 hours and 84 hours, e.g., about 60 hours, about 66 hours, about 72 hours, about 78 hours, or about 84 hours. In one example, for the engineering step, the γδ T cells may be cultured in the medium for about 48 hours.

[0179] The engineering of the γδ T cells may be performed using various methods known inthe art. A polynucleotide encoding an expression cassette that comprises at least one antigen recognition moiety may be stably introduced into the γδ T cell by a transposon / transposase system or a viral-based gene transfer system, such as retroviruses, lentiviruses, adenoviruses, adeno- associated viruses, or another suitable method, such as transfection, electroporation, transduction, lipofection, calcium phosphate (CaPO4), nanoengineered substances, such as Ormosil. Viral methods that can be used for engineering includes those described in WO 1993020221, which is incorporated herein in its entirety. In some examples, the nucleic acid constructs may be introduced to the γδ T cells by viral transduction. For example, the nucleic acid constructs may comprise a viral vector, e.g., a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno- associated viral vector. In one example, the viral vector may be a retroviral vector.

[0180] In some cases, the polynucleotide is derived from a human or from another species. Anantibody fragment or antigen binding fragment polynucleotide that is derived from a non-human species can be modified to increase their similarity to antibody variants produced naturally in humans, and an antibody fragment or antigen binding fragment can be partially or fully humanized. An antibody fragment or antigen binding fragment polynucleotide can also be chimeric, for example a mouse-human antibody chimera. An engineered γδ T cell that expresses a CAR can also be engineered to express a ligand to the antigen recognized by the antigen recognition moiety.

[0181] Various techniques known in the art can be used to introduce a cloned, or syntheticallyengineered, nucleic acid comprising the genetic code for an antigen recognition moiety into a specific location within the genome of an engineered γδ T cell. The RNA-guided Cas9 nuclease Page 41 of 117 1104881063\1\AMERICASfrom the microbial clustered regularly interspaced short palindromic repeats (CRISPR) system, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganuclease technologies, as described, respectively by WO201409370, WO2003087341, WO2014134412, and WO2011090804, each of which is incorporated by reference herein in its entirety, can be used to provide efficient genome engineering in γδ T cells. The technologies described herein can also be used to insert the expression cassette into a genomic location that simultaneously provides a knock-out of one gene and a knock-in of another gene. For example, a polynucleotide comprising an expression cassette of the disclosure can be inserted into a genomic region that encodes for an MHC gene. Such engineering can simultaneously provide the knock-in of one or more genes, e.g. the genes comprised in the expression cassette, and a knock-out of another gene, e.g. an MHC locus. In one case, a Sleeping Beauty transposon that includes a nucleic acid coding for the antigen recognition moiety is introduced into the cell γδ T cell that is being engineered. A mutant Sleeping Beauty transposase that provides for enhanced integration as compared to the wild-type Sleeping Beauty, such as the transposase described in US 7,985,739, which is incorporated by reference herein in its entirety, may be used to introduce a polynucleotide in the engineered γδ T cell.

[0182] In embodiments, an antigen recognition moiety (e.g., a CAR) is a receptor to a ligandthat is expressed on a cancer cell, e.g., a tumor associated antigen. Examples of suitable tumor associated antigens include CD19, CD20, CD30, CD22, CD37, CD38, CD56, CD33, CD138, CD123, CD79b, CD70, CD75, CA6, GD2, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), RON, CEACAM5, CA-125, MUC-16, 5T4, NaPi2b, ROR1, ROR2, PLIF, Her2 / Neu, EGFRvIII, GPMNB, LIV-1, glycolipidF77, fibroblast activation protein (FAP), PSMA, STEAP- 1, STEAP-2, mesothelin, c-Met, CSPG4, PVRL-4, VEGFR2, PSCA, CLEC12a, LI CAM, GPC2, GPC3, folate binding protein / receptor, SLC44A4, Cripto, CTAG1B, AXL, IL-13R, IL-3Ra2, SLTRK6, gp100, MARTI, Tyrosinase, SSX2, SSX4, NYESO-1, WT-1, PRAME, epithelial tumor antigen (ETA), MAGEA family genes (such as MAGEA3. MAGEA4), KKLC1, mutated ras, VRaf, p53, MHC class I chain- related molecule A (MICA), or MHC class I chain-related molecule B (MICB), or one or more antigens of HPV, CMV, or EBV, BCMA, GPC3, TyrD, B7H6, CD70, or PSMA.

[0183] In some cases, the antigen recognition moiety targets an MHC class I molecule (HLA-A, HLA-B, or HLA-C) in complex with a tumor-associated peptide. Methods and compositions Page 42 of 117 1104881063\1\AMERICASfor generating and using antigen recognition moieties that target a tumor-associated peptide in complex with a MHC class I molecule include those described in Weidanz et al., Int. Rev. Immunol. 30:328-40, 2011; Scheinberg et al, Oncotarget. 4(5):647-8, 2013; Cheever et al, Clin. Cancer Res.15(17):5323-37, 2009; Dohan & Reiter Expert Rev Mol Med.14:e6, 2012; Dao et al., Sci Transl Med. 2013 Mar 13;5(176):176ra33; U.S. 9,540,448; and WO 2017 / 011804. In some embodiments, the targeted tumor-associated peptide of the peptide MHC complex is a peptide of Wilms’ tumor protein 1 (WT1), human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 (CYP1B), KRAS, or BRAF.

[0184] In embodiments, an antigen recognition moiety (e.g., a CAR) recognizes an antigenassociated with an autoimmune disease.

[0185] Such antigens include endogenous antigens that stimulate the production of anautoimmune response, such as production of autoantibodies. Antigens associated with autoimmune diseases also include antigens from a normal tissue that is the target of a cell mediated or an antibody-mediated immune response that may result in the development of an autoimmune disease. Examples of antigens associated with autoimmune diseases include aggrecan, alanyl- tRNA syntetase (PL-12), alpha beta crystallin, alpha fodrin (Sptan 1), alpha-actinin, α1 antichymotrypsin, α1 antitripsin, α1 microglobulin, aldolase, aminoacyl-tRNA synthetase, an amyloid, an annexin, an apolipoprotein, aquaporin, bactericidal / permeability-increasing protein (BPI), β-globin precursor BP1, β-actin, β-lactoglobulin A, β-2-glycoprotein I, β2-microglobulin, a blood group antigen, C reactive protein (CRP), calmodulin, calreticulin, cardiolipin, catalase, cathepsin B, a centromere protein, chondroitin sulfate, chromatin, collagen, a complement component, cytochrome C, cytochrome P4502D6, cytokeratins, decorin, dermatan sulfate, DNA topoisomerase I, elastin, Epstein-Barr nuclear antigen 1 (EBNA1), elastin, entaktin, an extractable nuclear antigen, Factor I, Factor P, Factor B, Factor D, Factor H, Factor X, fibrinogen, fibronectin, formiminotransferase cyclodeaminase (LC-1), gp210 nuclear envelope protein, GP2 (major zymogen granule membrane glycoprotein), a glutenin, glycoprotein gpIIb / IIIa, glial fibrillary acidic protein (GFAP), glycated albumin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), haptoglobin A2, heat shock proteins, hemocyanin, heparin, a histone, histidyl-tRNA synthetase (Jo-1), a hordein, hyaluronidase, immunoglobulins, an integrin, interstitial retinol-binding protein 3, intrinsic factor, Ku (p70 / p80), lactate dehydrogenase, laminin, liver cytosol antigen type 1 (LC1), liver / kidney microsomal antigen 1 (LKM1), lysozyme, melanoma differentiation- Page 43 of 117 1104881063\1\AMERICASassociated protein 5 (MDAS), Mi-2 (chromodomain helicase DNA binding protein 4), a mitochondrial protein, muscarinic receptors, myelin-associated glycoprotein, myosin, myelin basic protein, myelin proteolipid protein, myelin oligodendrocyte glycoprotein, myeloperoxidase (MPO), rheumatoid factor (IgM anti-IgG), neuron-specific enolase, nicotinic acetylcholine receptor A chain, nucleolin, a nucleoporin, nucleosome antigen, PM / Sc1100, PM / Scl 75, pancreatic β-cell antigen, pepsinogen, peroxiredoxin 1, phosphoglucose isomerase, phospholipids, phosphatidyl inositol, platelet derived growth factors, polymerase beta (POLB), potassium channel KIR4.1, proliferating cell nuclear antigen (PCNA), proteinase-3, proteolipid protein, proteoglycan, prothrombin, recoverin, rhodopsin, ribonuclease, a ribonucleoprotein, ribosomes, a ribosomal phosphoprotein, RNA, an Sm protein, Sp100 nuclear protein, SRP54 (signal recognition particle 54 kDa), a selectin, smooth muscle proteins, sphingomyelin, streptococcal antigens, superoxide dismutase, synovial joint proteins, T1F1 gamma collagen, threonyl-tRNA synthetase (PL-7), tissue transglutaminase, thyroid peroxidase, thyroglobulin, thyroid stimulating hormone receptor, transferrin, triosephosphate isomerase, tubulin, tumor necrosis factor-alpha, topoisomerase, U1- dnRNP 68 / 70 kDa, U1-snRNP A, U1-snRNP C, U-snRNP B / B′, ubiquitin, vascular endothelial growth factor, vimentin, and vitronectin.

[0186] In embodiments, an antigen recognition moiety (e.g., a CAR) recognizes a pathogenicantigen. A pathogenic antigen may be a bacterial, viral, or fungal molecule, such as a bacterial,viral, or fungal protein. In embodiments, an antigen presenting cell may internalize pathogenicmolecules (e.g., pathogenic proteins, nucleic acids, lipids, or fragments produced by a pathogenic organism such as a bacterium or a virus), for instance with phagocytosis or by receptor-mediated endocytosis, and display a fragment of the antigen bound to an appropriate MHC molecule. For instance, various 9 mer fragments of a pathogenic protein may be displayed by an APC. Engineered, enriched modified T cells populations of the disclosure may be designed to recognize various antigens and antigen fragments of a pathogenic bacterium or a virus.

[0187] Examples of pathogenic bacteria can be found in the: a) Bordetella genus, such asBordetella pertussis species; b) Borrelia genus, such Borrelia burgdorferi species; c) Brucelia genus, such as Brucella abortus, Brucella canis, Brucela meliterisis, and / or Brucella suis species; d) Campylobacter genus, such as Campylobacter jejuni species; e) Chlamydia and Chlamydophila genuses, such as Chlamydia pneumonia, Chlamydia trachomatis, and / or Chlamydophila psittaci species; f) Clostridium genus, such as Clostridium botulinum, Clostridium difficile, Clostridium Page 44 of 117 1104881063\1\AMERICASperfringens, Clostridium tetani species; g) Corynebacterium genus, such as Corynebacterium diphtheria species; h) Enterococcus genus, such as Enterococcus faecalis, and / or Enterococcus faecium species; i) Escherichia genus, such as Escherichia coli species; j) Francisella genus, such as Francisella tularensis species; k) Haemophilus genus, such as Haemophilus influenza species; 1) Helicobacter genus, such as Helicobacter pylori species; m) Legionella genus, such as Legionella pneumophila species; n) Leptospira genus, such as Leptospira interrogans species; o) Listeria genus, such as Listeria monocytogenes species; p) Mycobacterium genus, such as Mycobacterium leprae, mycobacterium tuberculosis, and / or mycobacterium ulcerans species; q) Mycoplasma genus, such as Mycoplasma pneumonia species; r) Neisseria genus, such as Neisseria gonorrhoeae and / or Neisseria meningitidia species; s) Pseudomonas genus, such as Pseudomonas aeruginosa species; t) Rickettsia genus, such as Rickettsia rickettsii species; u) Salmonella genus, such as Salmonella typhi and / or Salmonella typhimurium species; v) Shigella genus, such as Shigella sonnei species; w) Staphylococcus genus, such as Staphylococcus aureus, Staphylococcus epidermidis, and / or Staphylococcus saprophyticus spedes; x) Streptpcoccus genus, such as Streptococcus agalactiae, Streptococcus pneumonia, and / or Streptococcus pyogenes species; y) Treponema genus, such as Treponema pallidum species; z) Vibrio genus, such as Vibrio cholera; and / or aa) Yersinia genus, such as Yersinia pestis spedes.

[0188] Examples of pathogenic viruses can be found in the following families of viruses andare illustrated with exemplary spedes: a) Adenoviridae family, such as Adenovirus spedes; b) Herpesviridae family, such as Herpes simplex type 1, Herpes simplex type 2, Varicella-zoster virus, Epstein-barr virus, Human cytomegalovirus, Human herpesvirus type 8 species; c) Papillomaviridae family, such as Human papillomavirus species; d) Polyomaviridae family, such as BK virus, JC virus species; e) Poxviridae family, such as Smallpox species; f) Hepadnaviridae family, such as Hepatitis B virus species; g) Parvoviridae family, such as Human bocavirus, Parvovirus B19 species; h) Astroviridae family, such as Human astrovirus spedes; i) Caliciviridae family, such as Norwalk virus species; j) Flaviviridae family, such as Hepatitis C virus (HCV), yellow fever virus, dengue virus, West Nile virus species; k) Togaviridae family, such as Rubella virus species; 1) Hepeviridae family, such as Hepatitis E virus species; m) Retroviiidae family, such as Human immunodeficiency virus (HIV) species; n) Orthomyxoviridaw family, such as Influenza virus species; o) Arenaviridae family, such as Guanarito virus, Junin virus, Lassa virus, Machupo virus, and / or Sabia virus species; p) Bunyaviridae family, such as Crimean-Congo Page 45 of 117 1104881063\1\AMERICAShemorrhagic fever virus species; q) Filoviridae family, such as Ebola virus and / or Marburg virus spedes; Paramyxoviridae family, such as Measles virus, Mumps virus, Parainfluenza virus, Respiratory syncytial virus, Human metapneumovirus, Hendra virus and / or Nipah virus spedes; r) Rhabdoviridae genus, such as Rabies virus species; s) Reoviridae family, such as Rotavirus, Orbivirus, Coltivirus and / or Banna virus species. In some examples, a virus is unassigned to a viral family, such as Hepatitis D.

[0189] Two or more antigen recognition moieties may be expressed in the γδ T cell fromgenetically different, substantially different, or substantially identical, ^^ TCR polynucleotides stably expressed from the engineered γδ T cell or from genetically distinct ^^ TCR polynucleotides stably incorporated in the engineered γδ T cell. In the case of genetically distinct ^^ TCR(s), ^^ TCR(s) recognizing different antigens associated with the same condition may be utilized. In embodiments, a γδ T cell is engineered to express different TCRs, from human or mouse origin, from one or more expression cassettes that recognize the same antigen in the context of different MHC haplotypes. In embodiments, a γδ T cell is engineered to express one TCR and two or more antibodies directed to the same or different peptides from a given antigen complexed with different MHC haplotypes. In some cases, expression of a single TCR by an engineered γδ T cell facilitates proper TCR pairing. An engineered γδ T cell that expresses different TCRs can provide a universal allogeneic engineered γδ T cell. In a second preferred embodiment, a γδ T cell is engineered to express one or more different antibodies directed to peptide-MHC complexes, each directed to the same or different peptide complexed with the same or different MHC haplotypes. In some cases, an antigen recognition moiety can be an antibody that binds to peptide- MHC complexes.

[0190] A γδ T cell may be engineered to express TCRs from one or more expression cassettesthat recognize the same antigen in the context of different MHC haplotypes. In some cases, an engineered γδ T cell is designed to express a single TCR, or a TCR in combination with a CAR to minimize the likelihood of TCR mispairing within the engineered cell. The antigen recognition moieties expressed from two or more expression cassettes preferably have different polynucleotide sequences, and encode antigen recognition moieties that recognize different epitopes of the same target, e.g., in the context of different HLA haplotypes. An engineered γδ T cell that expresses such different TCRs or CARs can provide a universal allogeneic engineered γδ T cell. Page 46 of 117 1104881063\1\AMERICAS

[0191] In embodiments, two or more antigen recognition moieties may be expressed fromgenetically identical, or substantially identical, antigen-specific chimeric (CAR) polynucleotides engineered in the γδ T cell. Two or more antigen recognition moieties may be expressed from genetically distinct CAR polynucleotides engineered in the γδ T cell. The genetically distinct CAR(s) may be designed to recognize different antigens associated with the same condition.

[0192] A γδ T cell may alternatively be bi-specific. A bi-specific engineered γδ T cell canexpress two or more antigen recognition moieties. A bi-specific engineered γδ T cell can express both TCR and CAR antigen recognition moieties. A bi-specific engineered γδ T cell can be designed to recognize different antigens associated with the same condition. An engineered γδ T cell can express two or more CAR / TCR(s) bi-specific polynucleotides that recognize an identical or substantially identical antigen. An engineered γδ T cell can express two or more CAR / TCR(s) bi-specific constructs that recognize distinct antigens. In some cases, a bi-specific construct of the disclosure binds to an activating and an inactivating domain of a target cell, thereby providing increased target specificity. The γδ T cell may be engineered to express at least 1 antigen recognition moiety, at least 2 antigen recognition moieties, at least 3 antigen recognition moieties, at least 4 antigen recognition moieties, at least 5 antigen recognition moieties, at least 6 antigen recognition moieties, at least 7 antigen recognition moieties, at least 8 antigen recognition moieties, at least 9 antigen recognition moieties, at least 10 antigen recognition moieties, at least 11 antigen recognition moieties, at least 12 antigen recognition moieties, or another suitable number of antigen recognition moieties.

[0193] A polynucleotide containing the genetic code for an antigen recognition moiety maycomprise mutations or other transgenes that affect the growth, proliferation, activation status of the engineered γδ T cell or an antigen specific to disease-associated cells. A γδ T cell of the disclosure may be engineered to express a polynucleotide comprising an activation domain that is linked to the antigen recognition moiety, such as a molecule in TCR-CD3 complex or a co- stimulatory factor. An engineered ^^ T cell can express an intracellular signaling domain that is a T-lymphocyte activation domain. The ^^ T cell may be engineered to express an intracellular activation domain gene or an intracellular signaling domain. The intracellular signaling domain gene, may be, for example CD3ζ, CD28, CD2, ICOS, JAML, CD27, CD30, OX40, NKG2D, CD4, OX40 / CD134, 4-1BB / CD137, Fc^RI^, IL-2RB / CD 122, IL- 2RG / CD132, DAP molecules, CD70, cytokine receptor, CD40, or any combination thereof. In some cases, the engineered γδ T cell is Page 47 of 117 1104881063\1\AMERICASalso engineered to express a cytokine, an antigen, a cellular receptor, or other immunomodulatory molecule.

[0194] The appropriate antigen recognition moiety to be expressed by the engineered γδ T cellcan be selected based on the disease to be treated. For example, in some cases an antigen recognition moiety is a TCR. A polynucleotide encoding a CAR may be introduced into the engineered γδ T cell by stably inserting the polynucleotide into the genome of the γδ T cell. In some cases, the engineered antigen recognition moiety is an engineered T cell receptor, and the expression cassette incorporated into the genome of an engineered γδ T cell comprises a polynucleotide encoding an engineered TCR ^ (TCR alpha) gene, an engineered TCR ^ (TCR beta) gene, an TCR δ (TCR delta) gene, or an engineered TCR γ (TCR gamma) gene. In some cases, the expression cassette incorporated into the genome of the engineered γδ T cell comprises a polynucleotide encoding an antibody fragment or an antigen binding portion thereof, e.g., an antibody fragment, a single-chain variable fragment (scFv), a single domain antibody (sdAb), a Fab, F(ab)2, an Fc, the light or heavy chains on an antibody, the variable or the constant region of an antibody, or any combination thereof that binds to a cell surface tumor antigen as part of the Chimeric Antigen Receptor (CAR) construct, or a bi-specific construct, comprising a CAR and a T cell receptor (TCR), or CARs with antibodies directed to different antigens.

[0195] In embodiments, the antigen recognition moiety is a CAR. The CAR may be expressedon the surface of the engineered γδ T cell and comprise an affinity binding domain specific for a disease associated antigen (e.g., tumor antigen, autoimmune antigen, or pathogenic antigen), or a means for specifically binding a disease associated antigen.

[0196] The CAR may comprise a transmembrane domain that couples the affinity bindingdomain of the CAR to one or more intracellular domains of the CAR. The transmembrane domain of a CAR of the present disclosure is a region that is capable of spanning the plasma membrane of a cell (e.g., a γδ T cell). In embodiments, the transmembrane domain is interposed between the affinity binding domain / means for specifically binding and the one or more intracellular domains of a CAR.

[0197] In embodiments, the transmembrane domain is naturally associated with one or moreof the domains in the CAR. In embodiments, the transmembrane domain can be selected or modified by one or more amino acid substitutions to avoid binding of such domains to the Page 48 of 117 1104881063\1\AMERICAStransmembrane domains of the same or different surface membrane proteins, to minimize interactions with other members of the receptor complex.

[0198] For example, a transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derived from any membrane- bound or transmembrane protein. Transmembrane regions of particular use in this invention may be derived from (i.e. comprise at least the transmembrane region(s) of) 4-1BB / CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8alpha, CD8beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, a ligand that specifically binds with CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD11a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or a combination thereof. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

[0199] In embodiments, the transmembrane domain further comprises a hinge region. Asubject CAR of the present invention may also include a hinge region. The hinge region of the CAR is a hydrophilic region which is located between the affinity binding domain / means for specifically binding and the transmembrane domain. In embodiments, this domain facilitates proper protein folding for the CAR. The hinge region is an optional component for the CAR. The Page 49 of 117 1104881063\1\AMERICAShinge region may include a domain selected from Fc fragments of antibodies, hinge regions of antibodies, CH2 regions of antibodies, CH3 regions of antibodies, artificial hinge sequences or combinations thereof. Examples of hinge regions include, without limitation, a CD8α hinge, CD8β hinge, CD28 hinge, 4-1BB hinge, CD7 hinge, artificial hinges made of polypeptides which may be as small as, three glycines (Gly), as well as CHI and CH3 domains of IgGs (such as human IgG4). Naturally-occurring hinge domains may be used as wild-type hinge regions or the molecules may be altered.

[0200] In embodiments, a CAR includes a hinge region that couples the affinity bindingdomain / means for specifically binding with the transmembrane domain, which, in turn, couples to one or more intracellular domain(s). The hinge region is preferably capable of supporting the affinity binding domain / means for specifically binding to recognize and bind to the target antigen on the target cells (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In embodiments, the hinge region is a flexible domain, thus allowing the affinity binding domain / means for specifically binding to have a structure to optimally recognize the specific structure and density of the target antigens on a cell such as tumor cell (Hudecek et al., supra). The flexibility of the hinge region permits the hinge region to adopt many different conformations. In embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In embodiments, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8- derived hinge region).

[0201] In one example, the hinge domain comprises, or consists of, the sequence set forth inSEQ ID NO: 17. In another example, the transmembrane domain comprises, or consists of, the sequence set forth in SEQ ID NO: 18. In another example, the hinge domain / transmembrane domain comprises, or consists of, the sequence set forth in SEQ ID NO: 19.

[0202] In embodiments, a CAR comprises at least one costimulatory domain, wherein thecostimulatory domain comprises functional costimulatory signaling domain derived from e.g., a MHC class I molecule, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, and the like. For example, it is within the scope of this disclosure that the CAR can include 2, 3, 4 or more costimulatory domains. It is also within the scope of this disclosure that when more than one costimulatory domain is included, the costimulatory domains may be the same, or they may be different. In embodiments, the Page 50 of 117 1104881063\1\AMERICAScostimulatory domains are derived from one or more of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, B7-H3, CEACAM1, CRTAM, CD2, CD3C, CD4, CD7, CD8α, CD8β, CD11a, CD11b, CD11c, CD11d, IL2Rβ, IL2γ, IL7Rα, IL4R, IL7R, IL15R, IL21R, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CDS, CD49a, CD49D, CD49f, CD54 (ICAM), CD69, CD70, CD80, CD83, CD84, CD86, CD96 (Tactile), CD100 (SEMA4D), CD103, CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), CD160 (BY55), CD162 (SELPLG), CD244 (2B4), CD270 (HVEM), CD226 (DNAM1), CD229 (Ly9), CD278 (ICOS), ICAM-1, LFA-1 (CD11a / CD18), FcR, FcγRI, FcγRII, FcγRIII, LAT, NKG2C, SLP76, TRIM, ZAP70, GITR, BAFFR, LTBR, LAT, GADS, LIGHT, HVEM (LIGHTR), KIRDS2, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, NKG2C, NKG2D, IA4, VLA-1, VLA-6, SLAM (SLAMF1, CD150, IPO-3), SLAMF4, SLAMF6 (NTB-A, Ly108), SLAMF7, SLAMF8 (BLAME), SLP-76, PAG / Cbp, NKp80 (KLRF1), NKp44, NKp30, NKp46, BTLA, JAML, CD150, PSGL1, TSLP, TNFR2, and TRANCE / RANKL, or a portion thereof, and combinations thereof.

[0203] In one example, the co-stimulatory domain comprises, or consist of, the sequence setforth in SEQ ID NO: 16.

[0204] In embodiments, a CAR comprises at least one intracellular signaling domain. Inembodiments, the at least one intracellular signaling domain is additional to one or more costimulatory domains. In embodiments, the one or more intracellular signaling domains are included to increase proliferation, persistence, and / or cytotoxic activity of the γδ cell, harboring the CAR as herein disclosed. For example, in some embodiments, the intracellular signaling domain(s) comprise CD3ζ, repeat (e.g., 2-5) DAP10 YINM motifs, signaling domains derived from LFA-1, DAP12, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD79a, CD79b, CD5, CD22, FcεRI, CD66d, and the like. It is within the scope of this disclosure that the endodomain of a disclosed CAR can include a plurality (e.g., 2, 3, 4, or more) of intracellular signaling domains. In a case where more than one intracellular signaling domain is included, the intracellular signaling domains may be the same, or they may be different.

[0205] In one example, the CD3 ζ signaling domain comprises, or consists of, the sequence setforth in SEQ ID NO: 20.

[0206] In embodiments, an isolated nucleic acid encoding a CAR can also encode for one ormore multi-cistronic linker region(s) configured to facilitate translation of the CAR polypeptide Page 51 of 117 1104881063\1\AMERICASand one or more additional polypeptides. In embodiments, nucleic acids encoding the one or more additional polypeptides and associated linker region can be positioned at the 3’ end of the isolated nucleic acid, or at the 5’ end of the isolated nucleic acid, or in some examples at both the 5’ end and the 3’ end of the isolated nucleic acid. In some examples, the linker region(s) can encode a self-cleavage and / or a cleavage polypeptide sequence. In some examples, the self-cleavage sequence is a 2A self-cleaving sequence (e.g., T2A, P2A, E2A, F2A) which can induce ribosomal skipping during translation of the CAR. In embodiments, the cleavage sequence is a furin sequence. In some examples, the cleavage sequence (e.g., furin cleavage sequence as set forth in SEQ ID NO: 190) is amino terminal to a self-cleavage sequence, for example furin-P2A (FP2A). In embodiments, the multi-cistronic linker region encodes an internal ribosome entry site. In embodiments, the addition of an optional linker “GSG” or “SGSG” and the like can improve cleavage efficiency. In this way, the one or more additional polypeptides can be release from the CAR and directed to the secretory pathway.

[0207] In embodiments, a γδ T cell may be engineered to express two or more CAR constructs.In some examples, the γδ T cell may be engineered to express two CAR constructs (dual CAR). In some examples, the γδ T cell may be engineered to express a first CAR comprising a first antigen recognition moiety that specifically binds to CD70 (e.g., a full-length CD27 domain as described in PCT / US2024 / 030119, which is incorporated by reference herein in its entirety). The γδ T cell may further express a second CAR comprising a second antigen recognition moiety.

[0208] In embodiments, the second CAR in a multispecific CAR T cell comprises one or moreaffinity binding domains that bind to one or more of CD19, CD20, CD22, CD37, CD38, CD40, CD40L, CD52, CD79b, CD123, CD138, BAFF-R, BCMA, FcRL5, GPRC50, TAC1, FcgRIIB, IL-36, IL-36R, IL-6, IL-6R, alpha4 integrin, CXCR6, DSG3, PD1, VISTA, BTLA, LAG3, ICOS, and ICOS-L. In embodiments, the antigen is a B cell antigen selected from the group comprising or consisting of CD19, CD20, CD22, CD37, CD38, CD40, CD52, CD79b, CD123, CD138, BAFF- R, BCMA, FcRL5, GPRC50, TAC1, and FcgRIIB. In embodiments, the antigen is a plasma cell antigen selected from the group comprising or consisting of CD38, CD138, BCMA, FcRL5, GPRC5D, TACI, and FcgRIIB. In embodiments, the antigen is a T cell antigen selected from the group comprising or consisting of CD52, CD40L, alpha4 integrin, CXCR6, PD1, VISTA, BTLA, LAG3, ICOS, and ICOS-L. Page 52 of 117 1104881063\1\AMERICAS

[0209] In embodiments, the second CAR in a multispecific T cell comprises an affinity bindingdomain that binds to CD19. Examples of the affinity binding domains that bind to CD19 include those described in PCT / US2024 / 030119, EP3214091, US10221245, US8906682, US10421810, US10639329, WO2010 / 052014, WO2015 / 109131, WO2017 / 134140, and WO 2020 / 018922, each of which is incorporated by reference herein in its entirety. Further examples of affinity binding domains that bind to CD19 include the CD19-binding domains in tisagenlecleucel, blinatumomab, tafasitamab, XmAb5574 (Xencor), AFM-11, inebilizumab, loncastuximab, MEDI-551 (Cellective Therapeutics), MDX-1342 (Medarex), A3B1, and FMC63-28Z.

[0210] In embodiments, the second CAR comprises an affinity binding domain that binds toFcRL5. Examples of the affinity binding domains that bind to FcRL5 include those described in WO2016205520, WO2014210064, WO2010114940, and WO2016090337, each of which is incorporated by reference in its entirety.

[0211] In embodiments, the second CAR in a multispecific CAR T cell comprises one affinitybinding domain. Alternatively, the second CAR can be a tandem CAR, e.g., a CAR comprising multiple affinity binding domains, that bind to different disease-associated antigens. In some examples, the affinity binding domain of the second CAR in a multispecific CAR T cell comprises a tandem sdAb, diabody, DART, sc-diabody, (scFv)2 / BITE, or a multivalent antibody or fragment described in Nuñez-Prado, et al., The coming of age of engineered multivalent antibodies, Drug Discov Today.2015 May;20(5):588-94.

[0212] In embodiments, the second CAR in a multispecific CAR T cell comprises affinitybinding domains targeting CD19 and CD20, e.g., those described in Tong C et al., Optimized tandem CD19 / CD20 CAR-engineered T cells in refractory / relapsed B-cell lymphoma., Blood. 2020 Oct 1;136(14):1632-1644. In embodiments, the second CAR comprises affinity binding domains targeting FcRL5 and BCMA, e.g., those described in Jiang D et al., Chimeric antigen receptor T cells targeting FcRH5 provide robust tumor-specific responses in murine xenograft models of multiple myeloma., Nature Communications volume 14, Article number: 3642 (2023). In embodiments, the second CAR comprises affinity binding domains targeting CD19 and BCMA, e.g., those described in Shi M., Bispecific CAR T cell therapy targeting BCMA and CD19 in relapsed / refractory multiple myeloma: a phase I / II trial, Nat Commun.2024 Apr 20;15(1):3371. In embodiments, the second CAR comprises affinity binding domains targeting CD19 and BCMA, e.g., those described in Kang L et al., Characterization of novel dual tandem CD19 / BCMA Page 53 of 117 1104881063\1\AMERICASchimeric antigen receptor T cells to potentially treat multiple myeloma, Biomark Res.2020 May 13:8:14. In embodiments, the second CAR comprises affinity binding domains targeting CD38 and BCMA, e.g., those described in Feng Y. et al., Novel BCMA-OR-CD38 tandem-dual chimeric antigen receptor T cells robustly control multiple myeloma, Oncoimmunology, 2021 Aug 17;10(1):1959102. Further examples of tandem CARs include those CD19 / CD20 CARs, CD19 / CD22 CARs, CD19 / CD123 CARs, CD19 / CD79b CARs, CD19 / CD38 CARs, CD19 / CD37 CARs, CD19 / CD20 / CD22 CARs, e.g., those described in Furqan F. et al., Multispecific CAR T Cells Deprive Lymphomas of Escape via Antigen Loss, Annu Rev Med.2023 Jan 27:74:279-291.

[0213] Additional examples of CAR constructs, domains and sequences in CARs (e.g., theaffinity binding domains, hinges, transmembrane domains, signaling domains, etc.), and related methods include those describe in PCT / US2023 / 024073, PCT / US2023 / 034227, PCT / US2023 / 030115, PCT / US2024 / 030119, 63 / 627746, and 63 / 607808, each of which is incorporated by reference in its entirety.

[0214] Proper TCR function may be enhanced by two functioning ^ (zeta) proteins comprisingITAM motifs. Proper TCR function may also be enhanced by expression of ^^ or ^^ activation domains, such as CD3ζ, CD28, CD2, CTLA4, ICOS, JAML, PD-1, CD27, CD30, 41-BB, OX40, NKG2D, HVEM, CD46, CD4, Fc^RI^, IL-2RB / CD122, IL-2RG / CD132, DAP molecules, and CD70. The expressed polynucleotide may include the genetic code for an antigen recognition moiety, a linker moiety, and an activation domain. Translation of the polynucleotide by the engineered γδ T cell may provide an antigen recognition moiety and an activation domain linked by a protein linker. Often, the linker comprises amino acids that do not obstruct the folding of the antigen recognition moiety and the activation domain. A linker molecule can be at least about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, about 11 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, or about 20 amino acids in length. In some cases, at least 50%, at least 70% or at least 90% of the amino acids in the linker are serine or glycine.

[0215] In some cases, an activation domain can comprise one or more mutations. Suitablemutations may be, for example, mutations that render an activation domain constitutively active. Altering the identity of one or more nucleic acids changes the amino acid sequence of the translated amino acid. A nucleic acid mutation can be made such that the encoded amino acid is modified to Page 54 of 117 1104881063\1\AMERICASa polar, non-polar, basic or acidic amino acid. A nucleic acid mutation can be made such that the antigen recognition moiety is optimized to recognize an epitope from a tumor. The engineered antigen recognition moiety, an engineered activation domain, or another engineered component of a γδ T cell may include more than 1 amino acid mutation, 2 amino acid mutations, 3 amino acid mutations, 4 amino acid mutations, 5 amino acid mutations, 6 amino acid mutations, 7 amino acid mutations, 8 amino acid mutations, 9 amino acid mutations, 10 amino acid mutations, 11 amino acid mutations, 12 amino acid mutations, 13 amino acid mutations, 14 amino acid mutations, 15 amino acid mutations, 16 amino acid mutations, 17 amino acid mutations, 18 amino acid mutations, 19 amino acid mutations, 20 amino acid mutations, 21 amino acid mutations, 22 amino acid mutations, 23 amino acid mutations, 24 amino acid mutations, 25 amino acid mutations, 26 amino acid mutations, 27 amino acid mutations, 28 amino acid mutations, 29 amino acid mutations, 30 amino acid mutations, 31 amino acid mutations, 32 amino acid mutations, 33 amino acid mutations, 34 amino acid mutations, 35 amino acid mutations, 36 amino acid mutations, 37 amino acid mutations, 38 amino acid mutations, 39 amino acid mutations, 40 amino acid mutations, 41 amino acid mutations, 42 amino acid mutations, 43 amino acid mutations, 44 amino acid mutations, 45 amino acid mutations, 46 amino acid mutations, 47 amino acid mutations, 48 amino acid mutations, 49 amino acid mutations, or 50 amino acid mutations.

[0216] In some cases, a γδ T cell of the disclosure does not express one or more MHCmolecules. Deletion of one or more MHC loci in an engineered γδ T cell can decrease the likelihood that the engineered γδ T cell will be recognized by the host immune system. The human Major Histocompatibility Complex (MHC) loci, known as the human leukocyte antigen (HLA) system, comprises a large gene family that is expressed in antigen presenting cells, including γδ T cells. The HLA-A, HLA-B, and HLA-C molecules function to present intracellular peptides as antigens to antigen presenting cells. The HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR molecules function to present extracellular peptides as antigens to antigen presenting cells. Some alleles of the HLA genes have been associated with GVHD, autoimmune disorders, and cancer. An engineered γδ T cell described herein can be further engineered to lack, or to disrupt gene expression of one or more HLA genes. An engineered γδ T cell described herein can be further engineered to lack, or to disrupt gene expression of one or more components of the MHC complex, such as complete deletion of one or more of the MHC genes, deletion of specific exons, or deletion of the ^2microglobulin (B2m). Genetic excision or genetic disruption of at least one Page 55 of 117 1104881063\1\AMERICASHLA gene can provide a therapeutic γδ T cell that can be administered to a subject with any HLA haplotype without causing host-versus-graft disease. An engineered γδ T cell as described herein can be a universal donor for a human subject with any HLA haplotype.

[0217] A γδ T cell can be engineered to lack one or various HLA locus (loci). An engineeredγδ T cell can be engineered to lack an HLA-A allele, an HLA-B allele, an HLA-C allele, an HLA- DR allele, an HLA-DQ allele, or an HLA-DP allele. In some cases, an HLA allele is associated with a human condition, such as an auto-immune condition. For instance, the HLA-B27 allele has been associated with arthritis and uveitis, the HLA-DR2 allele has been associated with systemic lupus erythematosus, and multiple sclerosis, the HLA-DR3 allele has been associated with 21- hydroxylase deficiency, the HLA-DR4 has been associated with rheumatoid arthritis and type 1 diabetes. An engineered γδ T cell that lacks, for example, the HLA-B27 allele can be administered to a subject afflicted with arthritis without being readily recognized the immune system of the subject. In some cases, deletion of one or more HLA loci provides an engineered γδ T cell that is a universal donor for any subject with any HLA haplotype.

[0218] In some cases, engineering a γδ T cell includes the deletion of a portion of the γδ T cellgenome. In some cases, the deleted portion of the genome comprises a portion of the MHC locus (loci). In some instances, the engineered γδ T cell is derived from a wild-type human γδ T cell, and the MHC locus is an HLA locus. In some cases, the deleted a portion of the genome comprises a portion of a gene corresponding to a protein in the MHC complex. In some cases, the deleted portion of the genome comprises the ^2 microglobulin gene. In some instances, the deleted portion of the genome comprises an immune checkpoint gene, such as PD-1, CTLA-4, LAG3, ICOS, BTLA, KIR, TIM3, A2aR, B7-H3, B7-H4, and CECAM-1. In some cases, an engineered γδ T cell can be designed to express an activation domain that enhances T cell activation and cytotoxicity. Non-limiting examples of activation domains that can be expressed by an engineered γδ T cell include: CD2, ICOS, 4-1 BB (CD137), OX40 (CD134), CD27, CD70, CD80, CD86, DAP molecules, CD122, GITR, and FcεRIγ.

[0219] In embodiments, the engineered γδ T cells comprise one or more disrupted genes. Forexample, one or more genes whose expression is disrupted can comprise adenosine A2a receptor (ADORA), CD276, V-set domain containing T cell activation inhibitor 1 (VTCN1), B and T lymphocyte associated (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), killer cell immunoglobulin-like receptor, three domains, Page 56 of 117 1104881063\1\AMERICASlong cytoplasmic tail, 1 (KIR3DL1), lymphocyte-activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cellular receptor 2 (HAVCR2), V-domain immunoglobulin suppressor of T-cell activation (VISTA), natural killer cell receptor 2B4 (CD244), cytokine inducible SH2-containing protein (CISH), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site (AAVS SITE (E.G. AAVS1, AAVS2, ETC.)), or chemokine (C—C motif) receptor 5 (gene / pseudogene) (CCR5), CD160 molecule (CD160), T- cell immunoreceptor with Ig and ITIM domains (TIGIT), CD96 molecule (CD96), cytotoxic and regulatory T-cell molecule (CRTAM), leukocyte associated immunoglobulin like receptor 1(LAIR1), sialic acid binding Ig like lectin 7 (SIGLEC7), sialic acid binding Ig like lectin 9 (SIGLEC9), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), tumor necrosis factor receptor superfamily member 10a (TNFRSF10A), caspase 8 (CASP8), caspase 10 (CASP10), caspase 3 (CASP3), caspase 6 (CASP6), caspase 7 (CASP7), Fas associated via death domain (FADD), Fas cell surface death receptor (FAS), transforming growth factor beta receptor II (TGFBRII), transforming growth factor beta receptor I (TGFBR1), SMAD family member 2 (SMAD2), SMAD family member 3 (SMAD3), SMAD family member 4 (SMAD4), SKI proto- oncogene (SKI), SKI-like proto-oncogene (SKIL), TGFB induced factor homeobox 1 (TGIF1), interleukin 10 receptor subunit alpha (IL10RA), interleukin 10 receptor subunit beta (IL10RB), heme oxygenase 2 (HMOX2), interleukin 6 receptor (IL6R), interleukin 6 signal transducer (IL6ST), c-src tyrosine kinase (CSK), phosphoprotein membrane anchor with glycosphingolipid microdomains 1(PAG1), signaling threshold regulating transmembrane adaptor 1 (SIT1), forkhead box P3 (FOXP3), PR domain 1 (PRDM1), basic leucine zipper transcription factor, ATF-like (BATF), guanylate cyclase 1, soluble, alpha 2 (GUCY1A2), guanylate cyclase 1, soluble, alpha 3 (GUCY1A3), guanylate cyclase 1, soluble, beta 2 (GUCY1B2), guanylate cyclase 1, soluble, beta 3 (GUCY1B3), cytokine inducible SH2-containing protein (CISH), prolyl hydroxylase domain (PHD1, PHD2, PHD3) family of proteins, Cbl proto-oncogene B (CBL-B), Zinc Finger Protein 91(ZFP91), Roquin, CD58, ICAM-1, Regnase-1, RASA2, MED12, Fas, Arid1a, or any combinationthereof.

[0220] In embodiments, a gene whose expression is disrupted is CISH, a negative regulator ofTCR signaling. Disruption of the CISH gene may provide a functional advantage over control cells that have an intact CISH gene in improving the sensitivity to certain cytokines (e.g., IL-2 / IL- 15), increasing T cell proliferation, and / or limiting T cell exhaustion. In some examples, the CISH Page 57 of 117 1104881063\1\AMERICASgene may be disrupted by methods described in Daher M. et al, Targeting a cytokine checkpoint enhances the fitness of armored cord blood CAR-NK cells, Blood. 2021 Feb 4;137(5):624-636, which is incorporated by reference herein in its entirety. In embodiments, the CISH gene is disrupted by gene editing using a CRISPR-Cas system comprising one or more guide RNAs, e.g., guide RNAs described in PCT / US2024 / 030119, which is incorporated by reference herein in its entirety.

[0221] In embodiments, a gene whose expression is disrupted is CBL-B, a negative regulatorof T cell activation. Disruption of the CBL-B gene may provide a functional advantage over control cells that have an intact CBL-B gene in enhancing T cell activation. In some examples, the CBL- B gene may be disrupted by methods described in Augustin R. et al., Targeting Cbl-b in cancer immunotherapy, J Immunother Cancer. 2023 Feb;11(2):e006007; Hooper K. et al., Knockout of CBLB Greatly Enhances Anti-Tumor Activity of CAR T Cells, Blood (2018) 132 (Supplement 1): 338; and Guo X. et al., CBLB ablation with CRISPR / Cas9 enhances cytotoxicity of human placental stem cell-derived NK cells for cancer immunotherapy, J Immunother Cancer. 2021 Mar;9(3):e001975, each of which is incorporated by reference herein in its entirety. In embodiments, the CBL-B gene is disrupted by gene editing using a CRISPR-Cas system comprising one or more guide RNAs, e.g., guide RNAs described in PCT / US2024 / 030119.

[0222] In embodiments, a gene whose expression is disrupted is Roquin (e.g., Roquin-1).Disruption of the Roquin gene may provide a functional advantage over control cells that have an intact Roquin gene in increasing T cell proliferation and enhancing antitumor activity. In some examples, the Roquin gene may be disrupted by methods described in Mai D et al., Combined disruption of T cell inflammatory regulators Regnase-1 and Roquin-1 enhances antitumor activity of engineered human T cells, Proc Natl Acad Sci U S A. 2023 Mar 21;120(12):e2218632120, which is incorporated by reference herein in its entirety.

[0223] In embodiments, a gene whose expression is disrupted is ZFP91. Disruption of theZFP91 gene may provide a functional advantage over control cells that have an intact ZFP91 gene in improving T cell glycolytic fitness and effector function. In some examples, the ZFP91 gene may be disrupted by method described in Wang F. et al., J Clin Invest. 2021 Oct 1;131(19):e144318, which is incorporated by reference herein in its entirety.

[0224] In embodiments, a gene whose expression is disrupted is CD58. In embodiments, agene whose expression is disrupted is ICAM-1. In embodiments, both CD58 and ICAM-1 are Page 58 of 117 1104881063\1\AMERICASdisrupted. Disruption of the CD58 and / or ICAM-1 genes may provide a functional advantage over control cells that have an intact CD58 and / or ICAM-1 gene in disrupting T cell adhesion and co- stimulatory interactions to reduce Host vs Graft allocytotoxicity. In one example, the ICAM-1 gene may be disrupted as described in Teo HY et al. IL12 / 18 / 21 Preactivation Enhances the Antitumor Efficacy of Expanded γδ T Cells and Overcomes Resistance to Anti-PD-L1 Treatment, Cancer Immunol Res.2023 Jul 5;11(7):978-999, which is incorporated by reference in its entirety. In embodiments, the ICAM-1 gene is disrupted by gene editing using a CRISPR-Cas system (e.g., CRISPR-Cas or CRISPR-Mad7 system) comprising one or more guide RNAs, e.g., guide RNAs described in PCT / US2024 / 030119. In embodiments, the CD58gene is disrupted by gene editing using a CRISPR-Cas system (e.g., CRISPR-Cas or CRISPR-Mad7 system) comprising one or more guide RNAs, e.g., guide RNAs described in PCT / US2024 / 030119.

[0225] In embodiments, a gene whose expression is disrupted is Regnase-1 gene. Regnase-1(Regulatory RNase 1), also known as ZC3H12A or MCPIP-1, is a ribonuclease that promotes decay of target mRNA through recognition of 3’ UTR stem loop motifs. Disruption of the Regnase- 1 gene may provide a functional advantage over control cells that have an intact Regnase-1 gene in enhancing T cell expansion and persistence. In one example, the Regnase-1 gene may be disrupted as described in Jun Wei et al., Targeting Regnase-1 programs long-lived effector T cells for cancer therapy. Nature.2019 Dec; 576(7787): 471–476, which is incorporated by reference in its entirety.

[0226] In embodiments, a gene whose expression is disrupted is RAS p21 protein activator 2(RASA2) gene. RASA2 is a RAS GTPase-activating protein (RasGAP) that is a signaling checkpoint in T cells, which can be downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. Disruption of RASA2 may enhance MAPK signaling and CAR T cell cytolytic activity in response to target antigen. RASA2-deficient T cells may exhibit increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent disease-associated cell killing. In some examples, the RASA2 gene may be disrupted by the method described in Julia Carnevale et al., RASA2 ablation in T cells boosts antigen sensitivity and long-term function. Nature. 2022 Sep;609(7925):174-182, which is incorporated by reference herein in its entirety.

[0227] In embodiments, a gene whose expression is disrupted is MED12 gene. Mediatorcomplex subunit 12 (MED12) is a Mediator subunit, and can regulate metabolic activity and Page 59 of 117 1104881063\1\AMERICASfitness, e.g., increased glycolysis, oxidative phosphorylation, and spare respiratory capacity. Disruption of the MED12 gene may provide a functional advantage over control cells that have an intact MED12 gene in enhancing T cell activation and effector function. In some examples, the disruption of the MED12 gene is performed using an RNA-guided nuclease and at least one guide RNA. In some examples, the RNA-guided nuclease and at least one guide RNA may be delivered with a nanoparticle-based vehicle. In some examples, nucleic acid(s) encoding the RNA-guided nuclease and at least one guide RNA may be delivered with a nanoparticle-based vehicle. In one example, the MED12 gene may be disrupted as described in Katherine A Freitas et al., Enhanced T cell effector activity by targeting the Mediator kinase module, Science. 2022 Nov 11;378(6620):eabn5647, which is incorporated by reference in its entirety. Further examples of methods and reagents for MED12 gene disruption include those described in WO2021076744, WO2021188828, WO2022098864, WO2024030970, WO2024044672, WO2024059641, and WO2024064642, each of which is incorporated by reference in its entirety. In some examples, the engineering comprises disrupting both TGFβR2 and MED12 genes.

[0228] In embodiments, a gene whose expression is disrupted is Fas gene. Disruption of theFas gene may provide a functional advantage over control cells that have an intact Fas gene in reducing T cell allocytotoxicity. In one example, the ICAM-1 gene may be disrupted as described in Jiangtao Ren et al., A versatile system for rapid multiplex genome-edited CAR T cell generation, Oncotarget. 2017 Mar 7; 8(10): 17002–17011, which is incorporated by reference herein in its entirety.

[0229] In embodiments, a gene whose expression is disrupted is Arid1a gene. Arid1a regulatesthe acquisition of the epigenetic state of terminal exhaustion. Disruption of the Arid1a gene may provide a functional advantage over control cells that have an intact Arid1a gene in improving tumor control and enhancing persistence of T cells. In one example, the Arid1a gene may be disrupted as described in Julia A Belk et al., Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence, Cancer Cell. 2022 Jul 11;40(7):768-786.e7.

[0230] In embodiments, a gene whose expression is disrupted is TGFβR2. Disruption of theTGFβR2 may provide a functional advantage over control cells that have an intact TGFβR2 gene in improving in the presence of a TGF-beta-secreting tumor, including enhanced anti-tumor activity. In embodiments, TGFβR2 gene is disrupted by a CRISPR / Cas system. In some examples, Page 60 of 117 1104881063\1\AMERICASthe TGFβR2 gene may be disrupted as described in Tang N et al., TGF-β inhibition via CRISPR promotes the long-term efficacy of CAR T cells against solid tumors, JCI Insight. 2020 Feb 27;5(4):e133977; or Alishah K. et al., CRISPR / Cas9-mediated TGFβRII disruption enhances anti- tumor efficacy of human chimeric antigen receptor T cells in vitro, J Transl Med.2021; 19: 482, each of which is incorporated by reference in its entirety.

[0231] Any portion of the genome of an engineered γδ T cell can be deleted to disrupt theexpression of an endogenous γδ T cell gene. Non-limiting examples of genomic regions that can be deleted or disrupted in the genome of a γδ T cell include a promoter, an activator, an enhancer, an exon, an intron, a non-coding RNA, a micro-RNA, a small-nuclear RNA, variable number tandem repeats (VNTRs), short tandem repeat (STRs), SNP patterns, hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, or simple sequence repeats. In some cases, the deleted a portion of the genome ranges between 1 nucleic acid to about 10 nucleic acids, 1 nucleic acid to about 100 nucleic acids, 1 nucleic acid to about 1,000 nucleic acids, 1 nucleic acid to about 10,000 nucleic acids, 1 nucleic acid to about 100,000 nucleic acids, 1 nucleic acid to about 1,000,000 nucleic acids, or other suitable range.

[0232] The disruption of one or more genes (e.g., MED12 and / or TGFβR2) may be performedat a time point different from introducing the nucleic acid construct(s) encoding the at least one antigen recognition moiety. In embodiments, the disruption of one or more genes (e.g., MED12 and / or TGFβR2) may be performed prior to activation of γδ T cells. In embodiments, the disruption of one or more genes (e.g., MED12 and / or TGFβR2) may be performed after depletion of αβ T cells. In some examples, the disruption of one or more genes (e.g., MED12 and / or TGFβR2) may be performed 1 day after depletion of αβ T cells. In some examples, the disruption of one or more genes (e.g., MED12 and / or TGFβR2) may be performed after depletion of αβ T cells and prior to introducing the nucleic acid construct(s) encoding the at least one antigen recognition moiety. In some examples, the disruption of one or more genes (e.g., MED12 and / or TGFβR2) may be performed after depletion of αβ T cells and 1 day prior to introducing the nucleic acid construct(s) encoding the at least one antigen recognition moiety. In some examples, the disruption of one or more genes (e.g., MED12 and / or TGFβR2) may be performed after introducing the nucleic acid construct(s) encoding the at least one antigen recognition moiety. In embodiments, the disruption of one or more genes (e.g., MED12 and / or TGFβR2) may be performed at the same time as introducing the nucleic acid construct(s) encoding the at least one antigen recognition moiety. Page 61 of 117 1104881063\1\AMERICAS

[0233] A γδ T cell may be engineered from an isolated non-engineered γδ T cell that alreadyexpresses an antigen recognition moiety. The engineered γδ T cell can retain a tumor cell recognition moiety that is endogenously expressed by the isolated wild-type γδ T cell, e.g., isolated from tumor infiltrating lymphocytes of a tumor sample. In some cases, the engineered γδ T cell tumor cell recognition moiety replaces the wild-type γδ TCR.

[0234] A γδ T cell can be engineered to express one or more homing molecules, such as alymphocyte homing molecule. Homing molecules can be, for instance, lymphocyte homing receptors or cell adhesion molecules. A homing molecule can help an engineered γδ T cell to migrate and infiltrate a solid tumor, including a targeted solid tumor upon administration of the engineered γδ T cell to the subject. Non-limiting examples of homing receptors include members of the CCR family, e.g.: CCR2, CCR4, CCR7, CCR8, CCR9, CCR10, CLA, CD44, CD103, CD62L, E-selectin, P-selectin, L-selectin, integrins, such as VLA-4 and LFA-1. Non-limiting examples of cell adhesion molecules include ICAM, N-CAM, VCAM, PE-CAM, L1-CAM, Nectins (PVRL1, PVRL2, PVRL3), LFA-1, integrin alphaXbeta2, alphavbeta7, macrophage-1 antigen, CLA-4, glycoprotein IIb / IIIa. Additional examples of cell adhesion molecules include calcium dependent molecules, such as T-cadherin, and antibodies to matrix metaloproteinases (MMPs) such as MMP9 or MMP2.

[0235] The steps involved in T cell maturation, activation, proliferation, and function may beregulated through co-stimulatory and inhibitory signals through immune checkpoint proteins. Immune checkpoints are co-stimulatory and inhibitory elements intrinsic to the immune system. Immune checkpoints aid in maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses to prevent injury to tissues when the immune system responds to disease conditions, such as cell transformation or infection. The equilibrium between the co- stimulatory and inhibitory signals used to control the immune response from either ^^ and ^^ T cells can be modulated by immune checkpoint proteins. Immune checkpoint proteins, such as PD1 and CTLA4 are present on the surface of T cells and can be used to turn an immune response “on” or “off.” Tumors can dysregulate checkpoint protein function as an immune-resistance mechanism, particularly against T cells that are specific for tumor antigens. An engineered ^^ T cell of the disclosure can be further engineered to lack one or more immune checkpoint locus (loci), such as PD-1, CTLA-4, LAG3, ICOS, BTLA, KIR, TIM3, A2aR, CEACAM1, B7-H3, and B7-H4. Page 62 of 117 1104881063\1\AMERICASAlternatively, the expression of an endogenous immune check point gene in an engineered ^^ T cell of the disclosure can be disrupted with gene editing technologies.

[0236] Immunological checkpoints can be molecules that regulate inhibitory signalingpathways (exemplified by CTLA4, PD1, and LAG3) or molecules that regulate stimulatory signaling pathways (exemplified by ICOS) in an engineered ^^ T cell of the disclosure. Several proteins in the extended immunoglobulin superfamily can be ligands for immunological checkpoints. Non-limiting examples of immune checkpoint ligand proteins include B7-H4, ICOSL, PD-L1, PD-L2, MegaCD40L, MegaOX40L, and CD137L. In some cases, immune checkpoint ligand proteins are antigens expressed by a tumor. In some cases, the immune checkpoint gene is a CTLA-4 gene. In some cases, the immune checkpoint gene is a PD-1 gene.

[0237] In embodiments, the engineered γδ T cells comprise a polynucleotide (e.g., differentor the same as the polynucleotide encoding the at least one antigen recognition moiety) encoding one or more additional polypeptides. In embodiments, the one or more additional polypeptides is selected from the group comprising or consisting of lymphotoxin beta receptor (LTBR), low-affinity nerve growth factor receptor (LNGFR), a dominant negative (dn) receptor for TGF-beta or Fas, a truncated form of the human epidermal growth factor receptor (EGFRt), and membrane-bound IL-12 (mbIL-12), or any combination thereof. In embodiments, the one or more additional polypeptides is selected from a fluorescent protein, a gamma chain cytokine, CD19, CD20, LNGFR, EGFRt, LTBR, dnTGFβR2, dominant negative Fas, membrane-bound IL- 12, CAR that binds to CD70, stem cell factor (SCF) or any combination thereof.

[0238] In embodiments, the one or more additional polypeptides include a protein thatfunctions to increase resistance to exhaustion and activation-induced apoptosis and / or upregulate one or more proinflammatory cytokines, costimulatory molecules and / or antigen presentation machinery. A representative example includes but is not limited to lymphotoxin beta receptor (LTBR). LTBR is typically expressed in a subset of myeloid cells but is absent in lymphocytes. When expressed in T cells, LTBR may induce transcriptional remodeling that imparts the T cell with one or more of the above-mentioned advantageous functions (Legut et al., Blood. (2021); 138(1): 1726).

[0239] In embodiments, the one or more additional polypeptides includes a polypeptide thatimparts host cells with the capability to resist tumor antigen-specific cellular immunity, for example that mediated by transforming growth factor beta (TGF-β). For example, an isolated Page 63 of 117 1104881063\1\AMERICASnucleic acid may encode a dominant negative receptor for TGF-beta (dnTGFβR2), e.g., as described in Foster et al., J Immunother. (2008); 31: 500-505, WO2019 / 173324A1, WO2020 / 183131A1, and WO2020042647A1. Incorporation of such a dominant negative receptor for TGF-beta may provide a functional advantage over control cells that lack such a dominant negative receptor for TGF-beta in the presence of a TGF-beta-secreting tumor, including enhanced anti-tumor activity. In embodiments, an isolated nucleic acid encodes a signal peptide operably linked to facilitate directing of the one or more additional polypeptides to the secretory pathway. Such one or more additional polypeptides can be those that reside inside certain organelles, are secreted from the host cell, or are inserted into cellular membranes. In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 1. In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 2. In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 4. In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 6. In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 8. In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 10.

[0240] In embodiments, the one or more additional polypeptides includes the EGFRt aminoacid sequence as set forth in SEQ ID NO: 3. In embodiments, the signal peptide comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 2 is operably linked to SEQ ID NO: 3. In embodiments, the one or more additional polypeptides includes the dominant-negative TGFβ receptor II (dnTGFβR2) amino acid sequence as set forth in SEQ ID NO: 5. In embodiments, the signal peptide comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 4 is operably linked to SEQ ID NO: 5. In embodiments, the one or more additional polypeptides includes the full-length LTBR amino acid sequence as set forth in SEQ ID NO: 7 In embodiments, the signal peptide comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 6 is operably linked to SEQ ID NO: 7. In embodiments, the one or more additional polypeptides includes the LNGFR amino acid sequence as set forth in SEQ ID NO: 9. In embodiments, the signal peptide comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 8 is operably linked to SEQ ID NO: 9. In embodiments, the one or more additional polypeptides includes the sIL-15 amino acid sequence as set forth in SEQ ID NO: 11. In Page 64 of 117 1104881063\1\AMERICASembodiments, the signal peptide comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 10 is operably linked to SEQ ID NO: 11.

[0241] In embodiments, the one or more additional polypeptides includes a chimeric switchreceptor comprising an extracellular domain of a TGFβ receptor for binding to TGFβ (e.g., TGFβRI and / or TGFβRII), and an intracellular domain of a cytokine receptor. The chimeric switch receptors can convert a TGFβ signal into a cytokine signal that promotes cytotoxicity. Examples of such chimeric switch receptors include those descried in WO2012138858, WO2016122738, WO2018094244, WO2014172584, WO2019109980, and WO2022037562, each of which is incorporated by reference in its entirety.

[0242] In embodiments, the one or more additional polypeptides includes a dominant negativeFas (dnFas). Incorporation of such a dominant negative Fas in a T cell may provide a functional advantage over control cells that lack such a dominant negative Fas in the prevention of Fas ligand- induced apoptosis and allowing for T cell persistence and antitumor efficacy. Examples of the dnFas include that described in Yamamoto TN et al., T cells genetically engineered to overcome death signaling enhance adoptive cancer immunotherapy, J Clin Invest.2019 Feb 25;129(4):1551- 1565, which is incorporated by reference herein in its entirety.

[0243] In embodiments, the one or more additional polypeptides includes a membrane-boundIL-12 (mbIL-12). Incorporation of such a mbIL-12 in a T cell may provide a functional advantage over control cells that lack such a mbIL-12 in enhancing effector functions of the T cells and / or limiting the systemic toxicity associated with IL-12. Examples of the mbIL-12 include those described in Hu J. et al., Cell membrane-anchored and tumor-targeted IL-12 (attIL12)-T cell therapy for eliminating large and heterogeneous solid tumors, J Immunother Cancer. 2022 Jan;10(1):e003633; Hombach A. et al., IL12 integrated into the CAR exodomain converts CD8+ T cells to poly-functional NK-like cells with superior killing of antigen-loss tumors, Mol Ther. 2022 Feb 2;30(2):593-605; and Lee EH et al., Antigen-dependent IL-12 signaling in CAR T cells promotes regional to systemic disease targeting, bioRxiv.2023 Jan 7;2023.01.06.522784, each of which is incorporated by reference herein in its entirety.

[0244] In embodiments, the one or more additional polypeptides an exogenous stem cell factor(SCF). The term “stem cell factor (SCF)” refers to a cytokine that that exerts its biological functions by binding to and activating the receptor tyrosine kinase c-Kit. Endogenous SCF is a stromal cell-derived cytokine synthesized by fibroblasts and other cell types. It is a glycoprotein Page 65 of 117 1104881063\1\AMERICASthat plays a key role in hematopoiesis acting both as a positive and negative regulator, often in synergy with other cytokines. It also plays a role in mast cell development, gametogenesis, and melanogenesis. SCF through c-Kit interaction regulates cell viability, proliferation, and differentiation both in physiological and pathological conditions (see e.g., Mazzoldi, E.L., et al. A juxtacrine / paracrine loop between C-Kit and stem cell factor promotes cancer stem cell survival in epithelial ovarian cancer. Cell Death Dis 10, 412 (2019)). In embodiments, SCF includes a soluble form SCF (e.g., comprising or consisting of SEQ ID NO: 12 (human SCF based on NP_000890, first 165 aa of the mature peptide without signal peptide)). In embodiments, SCF includes a transmembrane form SCF (e.g., comprising or consisting of SEQ ID NO:13 (human SCF based on NP_000890, mature peptide without signal peptide), see also Aderson DM et al. Cell Growth Differ.1991 Aug;2(8):373-8), or comprising or consists of SEQ ID NO: 14 (human SCF based on NP_ NP_003985, mature peptide without signal peptide), see also Johan Lennartsson et al. Physiol Rev.2012 Oct;92(4):1619-49). Examples of soluble and transmembrane forms of SCF also include those described in J G Flanagan et al., Transmembrane form of the kit ligand growth factor is determined by alternative splicing and is missing in the Sld mutant Cell. 1991 Mar 8;64(5):1025-35; DM Anderson et al., Alternate splicing of mRNAs encoding human mast cell growth factor and localization of the gene to chromosome 12q22-q24, Cell Growth Differ.1991 Aug;2(8):373-8; and Johan Lennartsson et al., Stem cell factor receptor / c-Kit: from basic science to clinical implications, Physiol Rev. 2012 Oct;92(4):1619-49, each of which is incorporated by reference in its entirety.

[0245] In embodiments, the exogenous SCF may be a recombinant or synthetic stem cellfactor, such as ancestim, a 166-amino-acid protein produced by E. coli bacteria into which a gene has been inserted for soluble human stem cell factor (SEQ ID NO: 15) as described in McNiece IK and Briddel RA, The Cytokine Handbook (Fourth Edition), 2003); and MG da Silva et al. Ancestim (recombinant human stem cell factor, SCF) in association with filgrastim does not enhance chemotherapy and / or growth factor-induced peripheral blood progenitor cell (PBPC) mobilization in patients with a prior insufficient PBPC collection, Bone Marrow Transplant.2004 Oct;34(8):683-91, each of which is incorporated by reference herein in its entirety.

[0246] Additional examples of recombinant and synthetic stem cell factor molecules includethose described in WO1991005795 (titled “Stem cell factor”); US6020469 (titled “Stem cell factor formulations and methods”); US6759215 (titled “Method of preparing human stem cell factor Page 66 of 117 1104881063\1\AMERICASpolypeptide”); and US6218148 (titled “DNS encoding stem cell factor”), each of which is incorporated by reference herein in its entirety. In embodiments, the exogenous SCF is a SCF analog peptide, e.g., those described in US5885962 (titled “Stem cell factor analog compositions and method”); and Tilayov T. et al. Engineering Stem Cell Factor Ligands with Different c-Kit Agonistic Potencies, Molecules. 2020 Oct 21;25(20):4850, each of which incorporated herein in its entirety.

[0247] In embodiments, the one or more additional polypeptides include one or more solublegamma chain cytokines expressed as separate polypeptides from the CAR. The one or more soluble common gamma chain cytokines can include but are not limited to IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IL-23. In embodiments, the common gamma chain cytokine is selected from IL-2, IL-7, and IL-15. In embodiments, the common gamma chain cytokine is IL-15. IL-15 sequences, including codon optimized nucleic acid sequences encoding soluble IL-15 (sIL-15) are disclosed herein and in WO 2007 / 037780.

[0248] In embodiments, the engineered γδ T cells may further include a polynucleotideconstruct that encodes a protein which imparts a desired functionality to the host cells. For example, such a nucleic acid may encode for a chimeric DAP10 adaptor polypeptide, described in U.S. Provisional Application No. 63 / 272,613 and U.S. Provisional Application No. 63 / 347,194, the contents of each of which is hereby expressly incorporated herein by reference in their entirety. In embodiments, a chimeric DAP10 adaptor polypeptide is capable of associating with a chimeric antigen receptor of the present disclosure, for example a CAR of the present disclosure may comprise a DAP10-interacting domain. In such an example, the chimeric DAP10 adaptor polypeptide may also associate with one or more additional endogenous or exogenous polypeptides, for example endogenous or exogenous NKG2D. In embodiments, a chimeric DAP10 adaptor polypeptide may not associate with a CAR of the present disclosure, but instead may interact with an endogenous or exogenous polypeptide (e.g., NKG2D) that includes a DAP10- interacting domain.

[0249] Examples of such exogenous proteins and disrupted genes, as well as reagents andmethods of making γδ T cells with the exogenous proteins or disrupted genes, include those described in PCT / US2023 / 034227 and US63 / 607808, which are incorporated herein in their entireties. Page 67 of 117 1104881063\1\AMERICASγδ T CELL EXPANSION

[0250] The engineered γδ T cells may be expanded by simultaneously or sequentially culturingthe cells with at least one cytokine. Examples of cytokines that can be used to expand the γδ T cells include IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-18, IL-19, IL-21, IL 23, IL-33, IFNγ, granulocyte-macrophage colony stimulating factor (GM-CSF), or granulocyte colony stimulating factor (G-CSF). In embodiments, the at least one cytokine comprises or consists of a common gamma chain cytokine, e.g., IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, or a combination thereof. In embodiments, the at least one cytokine comprises or consists of IL-2, IL-15, IL-7, IL-21, or any combination thereof. In embodiments, the at least one cytokine comprises or consists of IL-2. In embodiments, the at least one cytokine comprises or consists of IL-15. In embodiments, the at least one cytokine comprises or consists of IL-7. In embodiments, the at least one cytokine comprises or consists of IL-21. In embodiments, the at least one cytokine comprises or consists of IL-15 and IL-21. In embodiments, the at least one cytokine comprises or consists of IL-7 and IL-21. In embodiments, the at least one cytokine comprises or consists of IL-7 and IL-15. In embodiments, the at least one cytokine comprises or consists of IL-15, IL-7, and IL-21.

[0251] In embodiments, the expansion is performed by sequentially culturing the engineeredγδ T cells in multiple culture conditions (e.g., culture media). For example, the expansion may be performed by sequentially culturing the engineered γδ T cells in a first culture condition comprising IL-15 and IL-21, and a second culture condition comprising IL-15. In some examples, the expansion may be performed by sequentially culturing the engineered γδ T cells in a first culture condition comprising IL-15 and IL-21, and a second culture condition comprising IL-15 and not comprising IL-21.

[0252] In embodiments, the IL-15 in the first culture condition has a concentration greater thanabout 10 ng / mL, e.g., greater than 20 ng / mL, 25 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, or 80 ng / mL. In one example, the IL-15 in the first culture condition has a concentration greater than about 25 ng / mL. In one example, the IL-15 in the first culture condition has a concentration greater than about 30 ng / mL. In some examples, the IL-15 in the first culture condition has a concentration from about 20 ng / mL to 100 ng / mL, e.g., 25 ng / mL to 80 ng / mL, 30 ng / mL to 90 ng / mL, 40 ng / mL to 80 ng / mL, 50 ng / mL to 70 ng / mL, or 30 ng / mL to 70 ng / mL. In one example, the IL-15 in the first culture condition has a concentration from about 25 ng / mL to Page 68 of 117 1104881063\1\AMERICAS70 ng / mL. In one example, the IL-15 in the first culture condition has a concentration from about 50 ng / mL to 70 ng / mL.

[0253] In embodiments, the IL-15 in the second culture condition has a concentration greaterthan about 10 ng / mL, e.g., greater than 20 ng / mL, 25 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, or 80 ng / mL. In one example, the IL-15 in the second culture condition has a concentration greater than about 25 ng / mL. In one example, the IL-15 in the second culture condition has a concentration greater than about 30 ng / mL. In some examples, the IL-15 in the second culture condition has a concentration from about 20 ng / mL to 100 ng / mL, e.g., 25 ng / mL to 80 ng / mL, 30 ng / mL to 90 ng / mL, 40 ng / mL to 80 ng / mL, 50 ng / mL to 70 ng / mL, 30 ng / mL to 70 ng / mL. In one example, the IL-15 in the second culture condition has a concentration from about 25 ng / mL to 70 ng / mL. In one example, the IL-15 in the second culture condition has a concentration from about 50 ng / mL to 70 ng / mL.

[0254] The engineered γδ T cells may be cultured in a first culture medium for about 1 to 10days, e.g., 2 to 8 days, 3 to 7 days, 1 to 3 days, 2 to 4 days, 3 to 5 days, 4 to 6 days, 5 to 7 days, 6 to 8 days, 7 to 9 days, or 8 to 10 days. In some examples, the engineered γδ T cells may be cultured in the first medium for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days. The engineered γδ T cells may be cultured in a second culture medium for about 1 to 10 days, e.g., 2 to 8 days, 3 to 7 days, 1 to 3 days, 2 to 4 days, 3 to 5 days, 4 to 6 days, 5 to 7 days, 6 to 8 days, 7 to 9 days, or 8 to 10 days. In some examples, the engineered γδ T cells may be cultured in the second medium for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. In one example, the engineered γδ T cells may be cultured in the first culture medium for about 4, 5, or 6 days and in the second culture medium for about 3, 4, or 5 days. In another example, the engineered γδ T cells may be cultured in the first culture medium for about 4 days and in the second culture medium for about 3 or 4 days. In another example, the engineered γδ T cells may be cultured in the first culture medium for about 4 days and in the second culture medium for about 3 days. In another example, the engineered γδ T cells may be cultured in the first culture medium for about 4 days and in the second culture medium for about 4 days. In another example, the engineered γδ T cells may be cultured in the first culture medium for 5 days and in the second culture medium for about 4 days.

[0255] In embodiments, the γδ T cells are be expanded in vitro without activation by APCs,or without co-culture with APCs and / or aminophosphonates. Additionally or alternatively, the γδ Page 69 of 117 1104881063\1\AMERICAST cells may be expanded in vitro with at least one expansion step that includes activation by or co- culture with APCs and / or with one or more aminophosphonates.

[0256] In embodiments, during the expansion step, the ^^^^^ T cells grow at a faster rate suchthat over a period of time, e.g., from 1 day to 90 days of culture (e.g., about 1 day to about 19, 21, or 23 days of culture) the expansion results in greater than 10-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1,000-fold, 10,000-fold, 20,000-fold, 30,000-fold, 50,000-fold, 70,000-fold, 100,000-fold or 1,000,000-fold expansion over another ^^ T cell population, such as a ^2 or ^3 population; over a starting number of ^^ T cells before the expansion; over a starting number of ^^1 T cells before the expansion; or over an αβ T cell population in the culture.

[0257] In embodiments, the expansion is performed under conditions in which culture mediumis replenished about every 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 5, 2 to 4, or 2 to 3 days. In embodiments, the expansion is performed under conditions in which the cells are diluted or adjusted to a density that supports further γδ T cell expansion 1, 2, 3, 4, 5, 6, or more times. In some cases, the cell density adjustment is performed contemporaneously with (i.e., on the same day as, or at the same time as) replenishment of the culture medium. For example, cell density may be adjusted every 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 5, 2 to 4, or 2 to 3 days in the expansion step. Exemplary cell densities that support further γδ T cell expansion include about 1 x 105cells / mL, 2 x 105cells / mL, 3 x 105cells / mL, 4 x 105cells / mL, 5 x 105cells / mL, 6 x 105cells / mL, 7 x 105cells / mL, 8 x 105cells / mL, 9 x 105cells / mL, 1 x 106cells / mL, 2 x 106cells / mL, 3 x 106cells / mL, 4 x 106cells / mL, 5 x 106cells / mL, 10 x 106cells / mL, 15 x 106cells / mL, 20 x 106cells / mL, or 30 x 106cells / mL of culture.

[0258] In embodiments, cell density is adjusted to a density of from about 0.01 x 106 to about1 x 106cells / mL, from about 0.01 x 106to about 1.5 x 106cells / mL, from about 0.01 x 106to about 2 x 106cells / mL, from about 0.05 x 106to about 1 x 106cells / mL, from about 0.05 x 106to about 1.5 x 106cells / mL, from about 0.05 x 106to about 2 x 106cells / mL, from about 0.1 x 106to about 1 x 106cells / mL, from about 0.1 x 106to about 1.5 x 106cells / mL, from about 0.1 x 106to about 2 x 106cells / mL, from about 0.5 x 106to about 1 x 106cells / mL, from about 0.5 x 106to about 1.5 x 106cells / mL, from about 0.5 x 106to about 2 x 106cells / mL, from about 0.75 x 106to about 1 x 106cells / mL, from about 0.75 x 106to about 1.5 x 106cells / mL, from about 0.75 x 106to about 2 x 106cells / mL, from about 1 x 106to about 2 x 106cells / mL, or from about 1 x 106to about 1.5 x Page 70 of 117 1104881063\1\AMERICAS106cells / mL, from about 1 x 106to about 2 x 106cells / mL, from about 1 x 106to about 3 x 106cells / mL, from about 1 x 106to about 4 x 106cells / mL, from about 1 x 106to about 5 x 106cells / mL, from about 1 x 106to about 10 x 106cells / mL, from about 1 x 106to about 15 x 106cells / mL, from about 1 x 106to about 20 x 106cells / mL, or from about 1 x 106to about 30 x 106cells / mL.

[0259] In some embodiments, the expansion is performed under conditions in which the cellsare monitored and maintained at a predetermined cell density (or density interval) and / or maintained in culture medium having a predetermined glucose content. For example, the cells can be maintained at a viable cell density of from about from about 0.01 x 106to about 1 x 106cells / mL, from about 0.01 x 106to about 1.5 x 106cells / mL, from about 0.01 x 106to about 2 x 106cells / mL, from about 0.05 x 106to about 1 x 106cells / mL, from about 0.05 x 106to about 1.5 x 106cells / mL, from about 0.05 x 106to about 2 x 106cells / mL, from about 0.1 x 106to about 1 x 106cells / mL, from about 0.1 x 106to about 1.5 x 106cells / mL, from about 0.1 x 106to about 2 x 106cells / mL, 0.5 x 106to about 1 x 106cells / mL, from about 0.5 x 106to about 1.5 x 106cells / mL, from about 0.5 x 106to about 2 x 106cells / mL, from about 0.75 x 106to about 1 x 106cells / mL, from about 0.75 x 106to about 1.5 x 106cells / mL, from about 0.75 x 106to about 2 x 106cells / mL, from about 1 x 106to about 2 x 106cells / mL, or from about 1 x 106to about 1.5 x 106cells / mL, from about 1 x 106to about 3 x 106cells / mL, from about 1 x 106to about 4 x 106cells / mL, from about 1 x 106to about 5 x 106cells / mL, from about 1 x 106to about 10 x 106cells / mL, from about 1 x 106to about 15 x 106cells / mL, from about 1 x 106to about 20 x 106cells / mL, from about 1 x 106to about 30 x 106cells / mL.

[0260] In some cases, the cells can be maintained at a higher concentration for at least a portionof the expansion. For example, for a first portion of expansion, cells viability may be enhanced at a higher cell concentration. As another example, for a final portion of expansion culture volume may be most efficiently utilized at a higher cell concentration. Thus, in embodiments, cells can be maintained at a viable cell density of from about 0.5 x 106cells / mL to about 20 x 106cells / mL for at least a portion of expansion culture or all of expansion culture.

[0261] As another example, the cells can be maintained in culture medium having a glucosecontent of from about 0.5 g / L to about 1 g / L, from about 0.5 g / L to about 1.5 g / L, from about 0.5 g / L to about 2 g / L, from about 0.75 g / L to about 1 g / L, from about 0.75 g / L to about 1.5 g / L, from about 0.75 g / L to about 2 g / L, from about 1 g / L to about 1.5 g / L, from about 1 g / L to about 2 g / L, Page 71 of 117 1104881063\1\AMERICASfrom 1 g / L to 3 g / L, or from 1 g / L to 4 g / L. In embodiments, the cells can be maintained in culture medium having a glucose content of about 1.25 g / L. In some cases, such as where a high cell density culture is maintained, cells can be maintained in culture medium having a glucose content of about 1 g / L to about 5 g / L, from about 1 g / L to about 4 g / L, from about 2 g / L to about 5 g / L, or from about 2 g / L to about 4 g / L.

[0262] In embodiments, glucose content is maintained by addition of fresh serum containingor serum free culture medium to the culture. In embodiments, the cells can be maintained at a predetermined viable cell density interval and in a culture medium having a predetermined glucose content interval, e.g., by monitoring each parameter and adding fresh media to maintain the parameters within the predetermined limits. In embodiments, glucose content is maintained by adding fresh serum containing or serum free culture medium in the culture while removing spent medium in a perfusion bioreactor while retaining the cells inside. In embodiments, additional parameters including one or more of: pH, partial pressure of O2, O2 saturation, partial pressure of CO2, CO2saturation, lactate, glutamine, glutamate, ammonium, sodium, potassium, and calcium, are monitored and / or maintained during γδ T cell expansion described herein. FURTHER ENRICHMENT AND RESTING

[0263] In embodiments, the methods herein comprise further enrichment steps after theexpansion. For example, the methods may comprise a second depletion step to deplete undesired cells (e.g., αβ T cells and / or NK cells) in the expanded population of engineered γδ T cells. In one example, the methods may comprise a first depletion performed between the activation and the transduction steps, and a second depletion performed after the expansion of the engineered γδ T cells.

[0264] In embodiments, after the further enrichment, the αβ T cells are be reduced to belowabout 5%, e.g., below about 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01%, between about 0.05% and 0.15%, between about 0% and 0.05%, between about 0.01% and 0.06%, between about 0.02% and 0.07%, between about 0.03% and 0.08%, between about 0.04% and 0.09%, between about 0.05% and 0.1%, between about 0.06% and 1.1%, between about 0.07% and 0.12%, between about 0.08% and 0.13%, between about 0.09% and 0.14%, between about 0.1% and 0.15%, between about 0% and 0.05%, between about 0% and 0.03%, between about 0% and 0.02%, between about 0% and 0.01%, between about 0.01% Page 72 of 117 1104881063\1\AMERICASand 0.03%, between about 0.02% and 0.04%, between about 0.03% and 0.05%, between about 0.04% and 0.06%, between about 0.05% and 0.07%, between about 0.06% and 0.08%, between about 0.07% and 0.09%, or between about 0.08% and 0.1% of total viable cells in the cell population. In some examples, after the further enrichment, the αβ T cells are reduced to below about 0.5% of total viable cells in the cell population. In some examples, after the further enrichment, the αβ T cells are reduced to below about 0.2% of total viable cells in the cell population. In some examples, after the further enrichment, the αβ T cells are reduced to between about 0.05% and about 0.15% of total viable cells in the cell population. In some examples, after the further enrichment, the αβ T cells are reduced to between about 0.05% and about 0.1% of total viable cells in the cell population.

[0265] In embodiments, the expanded population of engineered γδ T cells after depletion hasa ratio of γδ T cells to αβ T cells of at least about 10, e.g., at least about 20, at least about 50, at least about 100, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, or at least about 800:1.

[0266] Alternatively or additionally, in embodiments, after the further enrichment, the NKcells are be reduced to below about 30%, e.g., below about 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1.5%, 1.2%, 1.1%, 1, 0.8%, or 0.6%, or between about 0% and 0.5%, between about 0% and 0.4%, between about 0% and 0.1%, between about 0.05% and 0.15%, between about 0.1% and 0.2%, between about 0.15% and 0.25%, between about 0.2% and 0.3%, between about 0.25% and 0.35%, between about 0.3% and 0.4%, between about 0.35% and 0.45%, between about 0.4% and 0.5%, between about 0.45% and 0.55%, or between about 0.5% and 0.6% of total viable cells in the cell population.

[0267] In embodiments, the methods further comprise resting the expanded engineered γδ Tcell population or the further enriched expanded engineered γδ T cell population, thereby producing a therapeutically effective population of γδ T cells. The resting step may be performed by culturing the γδ T cells for a predetermined duration of time in the presence of one or more cytokines. In embodiments, the resting step is performed after the second depletion. For example, the resting step may be performed after the second depletion and before cryopreservation.

[0268] In embodiments, the predetermined duration is between about 4 hours and 96 hours,between about 6 hours and 72 hours, between about 8 hours and 48 hours, between about 10 hours and 36 hours, or between about 12 hours and 24 hours. In one example, the predetermined duration Page 73 of 117 1104881063\1\AMERICASmay be between about 8 hours and 48 hours. In another example, the predetermined duration may be between about 12 hours and 24 hours. In some examples, the predetermined duration may be about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, or about 30 hours.

[0269] In embodiments, the resting is performed by culturing the γδ T cells in the presence ofIL-2, IL-7, IL-15, or a combination thereof. In embodiments, the resting is performed by culturing the γδ T cells in the presence of IL-2. In embodiments, the resting is performed by culturing the γδ T cells in the presence of IL-15. In some examples, the IL-15 has a concentration greater than about 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, or 80 ng / mL. In one example, the IL-15 has a concentration greater than about 30 ng / mL. In some examples, the IL-15 has a concentration from about 20 ng / mL to 100 ng / mL, 30 ng / mL to 90 ng / mL, 40 ng / mL to 80 ng / mL, 50 ng / mL to 70 ng / mL, 30 ng / mL to 70 ng / mL. In one example, the IL-15 has a concentration from about 50 ng / mL to 70 ng / mL.

[0270] In embodiments, during the resting step, the predetermined duration maintains αβ Tcells in therapeutically effective population of γδ T cells below about 5% of total viable cells, e.g., below about 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01%, between about 0.05% and 0.15%, between about 0% and 0.05%, between about 0.01% and 0.06%, between about 0.02% and 0.07%, between about 0.03% and 0.08%, between about 0.04% and 0.09%, between about 0.05% and 0.1%, between about 0.06% and 1.1%, between about 0.07% and 0.12%, between about 0.08% and 0.13%, between about 0.09% and 0.14%, between about 0.1% and 0.15%, between about 0% and 0.05%, between about 0% and 0.03%, between about 0% and 0.02%, between about 0% and 0.01%, between about 0.01% and 0.03%, between about 0.02% and 0.04%, between about 0.03% and 0.05%, between about 0.04% and 0.06%, between about 0.05% and 0.07%, between about 0.06% and 0.08%, between about 0.07% and 0.09%, or between about 0.08% and 0.1% of total viable cells in the cell population.

[0271] Alternatively or additionally, in embodiments, during the resting step, thepredetermined duration maintains NK T cells in therapeutically effective population of γδ T cells below 30% of total viable cells, e.g., below about 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1.5%, 1.2%, 1.1%, 1, 0.8%, or 0.6%, or between about 0% and 0.5%, between about 0% and 0.4%, between about 0% and 0.1%, between about 0.05% and 0.15%, between about 0.1% and 0.2%, between about 0.15% and 0.25%, between about 0.2% and 0.3%, between about 0.25% and 0.35%, between Page 74 of 117 1104881063\1\AMERICASabout 0.3% and 0.4%, between about 0.35% and 0.45%, between about 0.4% and 0.5%, between about 0.45% and 0.55%, or between about 0.5% and 0.6% of total viable cells in the cell population. CRYOPRESERVATION

[0272] The expanded population of ^^^^^ T cells (e.g., therapeutically effective population of^^^^^ T cells) obtained from the methods herein may be cryopreserved. In embodiments, the cryopreservation is performed by formulating the ^^^^^ T cells in freezing media and placed in cryogenic storage units such as liquid nitrogen freezers (e.g., -195 °C) or ultra-low temperature freezers (e.g., -65 °C, -80 °C or -120 °C) for long-term storage of at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, or at least 5 years. The freeze media can contain dimethyl sulfoxide (DMSO), and / or sodium chloride (NaCl), and / or dextrose, and / or dextran sulfate and / or hydroxyethyl starch (HES) with physiological pH buffering agents to maintain pH between about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5, about 7.5 to about 8.0, or about 6.5 to about 7.5. The cryopreserved ^^T cells can be thawed and further processed by stimulation with antibodies, proteins, peptides, and / or cytokines as described herein. The cryopreserved ^^^^^T cells can be thawed and genetically modified with viral vectors (including retroviral and lentiviral vectors) or non-viral means (including RNA, DNA, and proteins) as described herein.

[0273] The expanded population of ^^^^^ T cells may be further cryopreserved to generate cellbanks in quantities of at least 1, 5, 10, 100, 150, 200, 500 vials at least 101, 102, 103, 104, 105, 106, 107, 108, 109, or at least 1010cells per mL in freeze media. The cryopreserved cell banks may retain their functionality and can be thawed and further stimulated and expanded. In embodiments, thawed cells are stimulated and expanded in suitable closed vessels such as cell culture bags and / or bioreactors to generate quantities of cells as allogeneic cell product. Cryopreserved ^^^^^T cells may maintain their biological functions for at least 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 15 months, 18 months, 20 months, 24 months, 30 months, 36 months, 40 months, 50 months, or at least about 60 months under cryogenic storage condition. In embodiments, no preservatives are used in the formulation. The cryopreserved ^^^^^ T cells may be thawed and administered to (e.g., infused into) multiple patients as allogeneic off- the-shelf cell product. The infused cells may be expanded and / or maintained in the administered Page 75 of 117 1104881063\1\AMERICASsubject(s) by administering one or more agents described herein that selectively expand ^^^^^ T cells. EXPANDED γδ T CELL POPULATIONS

[0274] In embodiments, the expanded population of γδ T cells prepared by the methods hereincomprise less than less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, less than 0.1%, less than 0.08%, less than 0.07%, less than 0.05%, less than less than 0.04%, less than 0.03%, less than 0.02%, less than 0.01%, or less than 0.005% αβ T cells. In one example, the expanded population of γδ T cells comprises less 0.07% αβ T cells. In one example, the expanded population of γδ T cells comprises less 0.02% αβ T cells. In one example, the expanded population of γδ T cells comprises less 0.01% αβ T cells.

[0275] In embodiments, the expanded population of γδ T cells comprises less than 30%, lessthan 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, less than 0.8%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% NK cells. In some examples, the expanded population of γδ T cells comprises less than 2.5% or 0.5% NK cells.

[0276] In embodiments, the expanded population of γδ T cells comprises at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% γδ T cells.

[0277] In embodiments, the expanded population of γδ T cells comprises at least 91% γδ Tcells, less than 0.07% αβ T cells, and less than 2.5% or 0.5% NK cells.

[0278] In embodiments, the expanded population of γδ T cells is therapeutically effective, e.g.,can be used for treating a disease, e.g., tumors, autoimmune diseases, or pathogen infections.

[0279] In embodiments, the methods herein can provide a clinically relevant number (e.g.,>108, >109, >1010, >1011, or >1012, or from 108to 1012) of expanded γδ T cells from as few as one donor. In some cases, the methods herein can provide a clinically relevant number (e.g., >108, >109, >1010, >1011, or >1012, or from about 108to about 1012) of expanded γδ T cells within less than 19 or 21 days from the time of obtaining a donor sample.

[0280] In embodiments, the expanded population of γδ T cells prepared by the methods hereincomprises clinically relevant levels of γδ T cells of >108cells, e.g., in a culture volume of less than Page 76 of 117 1104881063\1\AMERICAS10 mL, 25 mL, 50 mL, 100 mL, 150 mL, 200 mL, 500 mL, 750 mL, 1 L, 2 L, 3 L, 4 L, 5 L, 10 L, 20 L, 25 L, 30L, 40L, 50L, or 60L . For example, the expanded population of γδ T cells prepared by the methods herein comprises clinically-relevant levels of γδ T cell subsets of >108cells in a expansion culture having a volume of from 10-100 mL; from 25-100 mL; from 50-100 mL; from 75-100mL; from 10-150 mL; from 25- 150 mL; from 50-150 mL; from 75-150 mL; from 100-150 mL; from 10-200 mL; from 25-200 mL; from 50-200 mL; from 75-200 mL, from 100-200 mL; from 10-250 mL; from 25-250 mL; from 50- 250 mL; from 75-250 mL, from 100-250 mL; from 150-250 mL; from 5-1,000 mL; from 10-1,000 mL, or from 100-1,000 mL; from 150-1,000 mL; from 200-1,000 mL; from 250-1,000 mL, 400 mL to 1L, 1 L to 2 L, 2 L to 5 L, 2 L to 10 L, 4 L to 10 L, 4 L to 15 L, 4 L to 20 L, or 4 L to 25 L, 5 L to 25 L, 10 L to 30 L, 15 L to 35 L, 20 L to 40 L, 25 L to 45 L, 30 L to 50 L, 35 L to 55 L, 40 L to 60 L. PHENOTYPE OF EXPANDED ^^ T CELLS

[0281] The expanded population of γδ T cells prepared by the methods herein may home to aspecific physical location in a subject’s body. Migration and homing of the γδ T cells, can be dependent on the combined expression and actions of specific chemokines and / or adhesion molecules. Homing of the γδ T cells can be controlled by the interactions between chemokines and their receptors. For example, cytokines including CXCR3 (whose ligands are represented by IP- 10 / CXCL10 and 6Ckine / SLC / CCL21) CCR4+ CXCR5+ (receptor for RANTES, MIP-1α, MIP- 1β), CCR6+ and CCR7 may affect homing of γδ T cells. In some cases, the γδ T cells may home to sites of inflammation and injury, and to diseased cells to perform repair functions. In some cases, the γδ T cells can home to a cancer. In some cases, the γδ T cells may home to a thymus, a bone marrow, a skin, a larynx, a trachea, pleurae, a lung, an esophagus, an abdomen, a stomach, a small intestine, a large intestine, a liver, a pancreas, a kidney, a urethra, a bladder, a testis, a prostate, a ductus deferens, am ovary, a uterus, a mammary gland, a parathyroid gland, a spleen or another site in a subject’s body. The γδ T cells can express one or more homing moieties, such as particular TCR allele and / or a lymphocyte homing molecule.

[0282] The expanded population of γδ T cells prepared by the methods herein may have aparticular phenotype and a phenotype can be described in terms of cell-surface marker expression. Various types of γδ T cells can be engineered as described herein. In embodiments, the engineered Page 77 of 117 1104881063\1\AMERICASγδ T cell is derived from a human. In embodiments, the engineered γδ T cell is derived from a different source, such as a mammal or a synthetic cell.

[0283] The immunophenotype of γδ T cells may be determined using markers includingCD137, CD27, CD45RA, CD45RO, CCR7 and CD62L (Klebanoff et al., Immunol Rev.211: 214 2006). CD137, or 4-1BB, is an activation-induced costimulatory molecule and an important regulator of immune responses. Pollok et al., J. Immunol. 150, 771-81 (1993). CD45RA is expressed on naïve T lymphocytes, replaced by CD45RO upon antigen encounter, but re-expressed in late effector cells (Michie et al., Nature 360, 264 - 265 (1992); CD62L is a cell adhesion molecule that acts as a homing molecule to enter secondary lymphoid tissues and is lost after T cell activation, when T cells acquire effector functions (Sallusto et al., Nature. 401:708 (1999);. CD27 is costimulation markers that are lost during T cell differentiations (Appay et al., Nat Med.8:379 (2002); Klebanoff et al., Immunol Rev.211: 214 2006). Additional or alternative activation markers include one or more of CD25, PD-1, and CD69.

[0284] In embodiments, the engineered γδ T cells according to the present invention are CAR+and MED12-. In embodiments, the engineered γδ T cells according to the present invention are CAR+, MED12- and TGFβR2-. In embodiments, the engineered γδ T cells according to the present invention are CAR+, MED12- and dnTGFβR2+. METHODS OF TREATMENT

[0285] Pharmaceutical compositions containing the expanded population of γδ T cellsprepared by the methods herein may be administered for prophylactic and / or therapeutic treatments. In therapeutic applications, the compositions can be administered to a subject already suffering from a disease or condition in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition. The expanded population of γδ T cells can also be administered to lessen a likelihood of developing, contracting, or worsening a condition. Effective amounts of the γδ T cells, for therapeutic use can vary based on the severity and course of the disease or condition, previous therapy, the subject’s health status, weight, and / or response to the drugs, and / or the judgment of the treating physician.

[0286] The expanded population of γδ T cells may be used to treat a subject in need oftreatment for a condition. Examples of conditions include cancer, infectious disease, autoimmune disorder and sepsis. Subjects can be humans, non-human primates such as chimpanzees, and other Page 78 of 117 1104881063\1\AMERICASapes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. A subject can be of any age. Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, infants.

[0287] A method of treating a condition (e.g., ailment) in a subject herein may compriseadministering to the subject an expanded population of γδ T cells comprising a therapeutically effective amount of the γδ T cells. The expanded population of γδ T cells may be administered at various regimens (e.g., timing, concentration, dosage, spacing between treatment, and / or formulation). A subject can also be preconditioned with, for example, chemotherapy, radiation, or a combination of both, prior to receiving an expanded population of γδ T cells and / or admixtures thereof, of the disclosure. As part of a treatment, the expanded population of γδ T cells may be administered to a subject at a first regimen and the subject may be monitored to determine whether the treatment at the first regimen meets a given level of therapeutic efficacy. In some embodiments, at least one other γδ T cell can be administered to the subject in a second regimen. The second regimen may be the same as the first regimen or different than the first regimen. In some situations, the second regimen is not performed, for example, if the administration of the expanded population of γδ T cells in the first regimen is found to be effective (e.g., a single round of administration may be sufficient to treat the condition). Due to their allogeneic and universal donor characteristics, an expanded population of γδ T cells may be administrated to various subjects, with different MHC haplotypes. The expanded population of γδ T cells may be frozen or cryopreserved prior to being administered to a subject.

[0288] The expanded population of γδ T cells may also be frozen or cryopreserved prior tobeing administered to a subject and optionally further activated and expanded and / or maintained in vivo by administration of one or more agents that selectively expand the administered γδ T cells. In embodiments, an expanded population of γδ T cells can comprise two or more cells that express identical, different, or a combination of identical and different antigen recognition moieties.

[0289] For instance, an expanded population of γδ T cells can comprises several distinctengineered γδ T cells that are designed to recognize different antigens, or different epitopes of the same antigen. In one example, the antigen is PSMA.

[0290] In embodiments, the present disclosure provides a method for treating a subject with apopulation of the γδ T cells that recognizes different epitopes of the melanoma antigen NY-ESO- Page 79 of 117 1104881063\1\AMERICAS1. In a first operation, a population of the γδ T cells that recognize different epitopes of the same antigen is selected. For example, the population of the γδ T cells may comprise two or more cells that expressing different antigen recognition moieties that recognize different portions of the NY- ESO-1 protein. In a second operation, the population of the γδ T cells may be administered at a first regimen. In a second operation, the subject may be monitored, for example by a healthcare provider (e.g., treating physician or nurse). In a third operation, the subject may be administered one or more agents that selectively expand the administered γδ T cells in vivo to thereby expand and / or maintain the administered population of the γδ T cells in vivo. In a fourth operation, the subject may be monitored to determine the efficacy of the in vivo expansion and / or maintenance. In some embodiments, the second operation is not performed. In some embodiments, the fourth operation is not performed.

[0291] One or more compositions of the disclosure may be used to treat various conditions. Insome cases, a composition of the disclosure may be used to treat a cancer, including solid tumors and hematologic malignancies. Non-limiting examples of cancers include: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas, neuroblastoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancers, brain tumors, such as cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, bronchial adenomas, Burkitt lymphoma, carcinoma of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gliomas, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liposarcoma, liver cancer, lung cancers, such as non-small cell and small cell lung cancer, lymphomas, leukemias, macroglobulinemia, malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma, melanomas, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple Page 80 of 117 1104881063\1\AMERICASendocrine neoplasia syndrome, myelodysplastic syndromes, myeloid leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non- small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic cancer islet cell, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasia, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, skin cancers, skin carcinoma merkel cell, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, T cell lymphoma, throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (gestational), cancers of unknown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms tumor.

[0292] In some cases, a composition of the disclosure may be used to treat an infectiousdisease. An infectious disease may be caused, for example, by a pathogenic bacterium or by a virus. Various pathogenic proteins, nucleic acids, lipids, or fragments thereof can be expressed by a diseased cell. An antigen presenting cell can internalize such pathogenic molecules, for instance with phagocytosis or by receptor-mediated endocytosis, and display a fragment of the antigen bound to an appropriate MHC molecule. For instance, various 9 mer fragments of a pathogenic protein may be displayed by an APC. Engineered, enriched γδ T cell populations of the disclosure may be designed to recognize various antigens and antigen fragments of a pathogenic bacterium or a virus. Non-limiting examples of pathogenic bacteria can be found in the: a) Bordetella genus, such as Bordetella pertussis species; b) Borrelia genus, such Borrelia burgdorferi species; c) Brucelia genus, such as Brucella abortus, Brucella canis, Brucela meliterisis, and / or Brucella suis species; d) Campylobacter genus, such as Campylobacter jejuni species; e) Chlamydia and Chlamydophila genuses, such as Chlamydia pneumonia, Chlamydia trachomatis, and / or Chlamydophila psittaci species; f) Clostridium genus, such as Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani species; g) Corynebacterium genus, such as Corynebacterium diphtheria species; h) Enterococcus genus, such as Enterococcus faecalis, and / or Enterococcus faecium species; i) Escherichia genus, such as Escherichia coli species; j) Francisella Page 81 of 117 1104881063\1\AMERICASgenus, such as Francisella tularensis species; k) Haemophilus genus, such as Haemophilus influenza species; l) Helicobacter genus, such as Helicobacter pylori species; m) Legionella genus, such as Legionella pneumophila species; n) Leptospira genus, such as Leptospira interrogans species; o) Listeria genus, such as Listeria monocytogenes species; p) Mycobacterium genus, such as Mycobacterium leprae, mycobacterium tuberculosis, and / or mycobacterium ulcerans species; q) Mycoplasma genus, such as Mycoplasma pneumonia species; r) Neisseria genus, such as Neisseria gonorrhoeae and / or Neisseria meningitidia species; s) Pseudomonas genus, such as Pseudomonas aeruginosa species; t) Rickettsia genus, such as Rickettsia rickettsii species; u) Salmonella genus, such as Salmonella typhi and / or Salmonella typhimurium species; v) Shigella genus, such as Shigella sonnei species; w) Staphylococcus genus, such as Staphylococcus aureus, Staphylococcus epidermidis, and / or Staphylococcus saprophyticus species; x) Streptpcoccus genus, such as Streptococcus agalactiae, Streptococcus pneumonia, and / or Streptococcus pyogenes species; y) Treponema genus, such as Treponema pallidum species; z) Vibrio genus, such as Vibrio cholera; and / or aa) Yersinia genus, such as Yersinia pestis species.

[0293] In some cases, a composition of the disclosure may be used to treat an infectiousdisease, an infectious disease may be caused a virus. Non-limiting examples of viruses can be found in the following families of viruses and are illustrated with exemplary species: a) Adenoviridae family, such as Adenovirus species; b) Herpesviridae family, such as Herpes simplex type 1, Herpes simplex type 2, Varicella-zoster virus, Epstein-barr virus, Human cytomegalovirus, Human herpesvirus type 8 species; c) Papillomaviridae family, such as Human papillomavirus species; d) Polyomaviridae family, such as BK virus, JC virus species; e) Poxviridae family, such as Smallpox species; f) Hepadnaviridae family, such as Hepatitis B virus species; g) Parvoviridae family, such as Human bocavirus, Parvovirus B19 species; h) Astroviridae family, such as Human astrovirus species; i) Caliciviridae family, such as Norwalk virus species; j) Flaviviridae family, such as Hepatitis C virus (HCV), yellow fever virus, dengue virus, West Nile virus species; k) Togaviridae family, such as Rubella virus species; l) Hepeviridae family, such as Hepatitis E virus species; m) Retroviridae family, such as Human immunodeficiency virus (HIV) species; n) Orthomyxoviridaw family, such as Influenza virus species; o) Arenaviridae family, such as Guanarito virus, Junin virus, Lassa virus, Machupo virus, and / or Sabiá virus species; p) Bunyaviridae family, such as Crimean-Congo hemorrhagic fever virus species; q) Filoviridae family, such as Ebola virus and / or Marburg virus species; Paramyxoviridae family, Page 82 of 117 1104881063\1\AMERICASsuch as Measles virus, Mumps virus, Parainfluenza virus, Respiratory syncytial virus, Human metapneumovirus, Hendra virus and / or Nipah virus species; r) Rhabdoviridae genus, such as Rabies virus species; s) Reoviridae family, such as Rotavirus, Orbivirus, Coltivirus and / or Banna virus species. In some examples, a virus is unassigned to a viral family, such as Hepatitis D.

[0294] In some cases, a composition of the disclosure may be used to treat an immune disease,such as an autoimmune disease. Inflammatory diseases, including autoimmune diseases are also a class of diseases associated with B- cell disorders. Examples of immune diseases or conditions, including autoimmune conditions, include: rheumatoid arthritis, rheumatic fever, multiple sclerosis, experimental autoimmune encephalomyelitis, psoriasis, uveitis, diabetes mellitus, lupus, systemic lupus erythematosus (SLE), lupus nephritis, eczema, scleroderma, polymyositis / scleroderma, polymyositis / dermatomyositis, ulcerative proctitis, ulcerative colitis, severe combined immunodeficiency (SCID), DiGeorge syndrome, ataxia-telangiectasia, seasonal allergies, perennial allergies, food allergies, anaphylaxis, mastocytosis, allergic rhinitis, atopic dermatitis, Parkinson’s, Alzheimer’s, hypersplenism, leukocyte adhesion deficiency, X-linked lymphoproliferative disease, X-linked agammaglobulinemia, selective immunoglobulin A deficiency, hyper IgM syndrome, HIV, autoimmune lymphoproliferative syndrome, Wiskott- Aldrich syndrome, chronic granulomatous disease, common variable immunodeficiency (CVID), hyperimmunoglobulin E syndrome, Hashimoto’s thyroiditis, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenia purpura, dermatomyositis, Sydenham’a chorea, myasthenia gravis, polyglandular syndromes, bullous pemphigoid, Henoch-Schonlein purpura, poststreptococcalnephritis, erythema nodosum, erythema multiforme, gA nephropathy, Takayasu’s arteritis, Addison’s disease, sarcoidosis, ulcerative colitis, polyarteritis nodosa, ankylosing spondylitis, Goodpasture’s syndrome, thromboangitisubiterans, Sjogren’s syndrome, primary biliary cirrhosis, Hashimoto’s thyroiditis, thyrotoxicosis, chronic active hepatitis, polychondritis, pamphigus vulgaris, Wegener’s granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis, / polymyalgia, peraiciousanemia, rapidly progressive glomerulonephritis, psoriasis, fibrosing alveolitis, and cancer.

[0295] Treatment with a composition of the disclosure may be provided to the subject before,during, and after the clinical onset of the condition. Treatment may be provided to the subject after 1 day, 1 week, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment may be provided to the subject for more than 1 day, 1 week, 1 month, 6 months, 12 months, 2 years, 3 Page 83 of 117 1104881063\1\AMERICASyears, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more after clinical onset of disease. Treatment may be provided to the subject for less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment may also include treating a human in a clinical trial. A treatment can comprise administering to a subject a pharmaceutical composition comprising the γδ T cells of the disclosure. In some cases, the pharmaceutical composition comprises one or more agents of the disclosure that selectively expands the γδ T cells of the disclosure.

[0296] In some cases, administration of a composition of the disclosure to a subject modulatesthe activity of endogenous lymphocytes in a subject’s body. In some cases, administration of the composition of the disclosure to a subject provides an antigen to an endogenous T cell and may boost an immune response. In some cases, the memory T cell is a CD4+T cell. In some cases, the memory T cell is a CD8+T cell. In some cases, administration of the composition of the disclosure to a subject activates the cytotoxicity of another immune cell. In some cases, the other immune cell is a CD8+ T cell. In some cases, the other immune cell is a Natural Killer T cell. In some cases, administration of the composition to a subject suppresses a regulatory T cell. In some cases, the regulatory T cell is a Fox3+ Treg cell. In some cases, the regulatory T cell is a Fox3- Treg cell. Non-limiting examples of cells whose activity can be modulated by the γδ T cell population include: hematopioietic stem cells; B cells; CD4; CD8; red blood cells; white blood cells; dendritic cells, including dendritic antigen presenting cells; leukocytes; macrophages; memory B cells; memory T cells; monocytes; natural killer cells; neutrophil granulocytes; T-helper cells; and T- killer cells.

[0297] During most bone marrow transplants, a combination of cyclophosphamide with totalbody irradiation is conventionally employed to prevent rejection of the hematopoietic stem cells (HSC) in the transplant by the subject’s immune system. In some cases, incubation of donor bone marrow with interleukin-2 (IL-2) ex vivo is performed to enhance the generation of killer lymphocytes in the donor marrow. Interleukin-2 (IL-2) is a cytokine that is necessary for the growth, proliferation, and differentiation of wild-type lymphocytes. Current studies of the adoptive transfer of the γδ T cells into humans may include the co-administration of the γδ T cells and interleukin-2. However, both low- and high- dosages of IL-2 can have highly toxic side effects. IL-2 toxicity can manifest in multiple organs / systems, most significantly the heart, lungs, kidneys, and central nervous system. In some cases, the disclosure provides a method for administrating the Page 84 of 117 1104881063\1\AMERICASγδ T cells to a subject without the co-administration of a cytokine, such as IL-2, IL-15, IL-12, or IL-21. In some cases, the γδ T cells can be administered to a subject without co-administration with IL-2. In some cases, the γδ T cells are administered to a subject during a procedure, such as a bone marrow transplant without the co-administration of IL-2.

[0298] In some cases, the disclosure provides a method for administrating the γδ T cells to asubject with the simultaneous or sequential co-administration of a cytokine or other stimulating agent such as IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-18, IL-19, IL-21, IL 23, IL-33, IFNγ, granulocyte-macrophage colony stimulating factor (GM-CSF), or granulocyte colony stimulating factor (G-CSF), or a stem cell factor (SCF) (e.g., various forms of SCFs as described in US63 / 607808). In some cases, the cytokine is IL-2, IL-15, IL-12, or IL-21. In some cases, the cytokine is IL-2. In some cases, the cytokine is IL-15. In some cases, the cytokine is IL-4. In some cases, the cytokine is a common gamma chain cytokine selected from the group consisting of IL- 2, IL-4, IL-7, IL-9, IL-15, and IL-21, or a combination thereof. Methods of Administration

[0299] Compositions comprising the expanded population of γδ T cells prepared by themethods herein can be administered to a subject in any order or simultaneously. If simultaneously, the compositions can be provided in a single, unified form, such as an intravenous injection, or in multiple forms, for example, as multiple intravenous infusions. The compositions can be packed together or separately, in a single package or in a plurality of packages. One or all of the compositions can be given in multiple doses. If not simultaneous, the timing between the multiple doses may vary to as much as about a week, a month, two months, three months, four months, five months, six months, or about a year. In some cases, an administered γδ T cell population can expand within a subject’s body, in vivo, after administration to a subject. Pharmaceutical compositions comprising the γδ T cells and / or agents for expanding the γδ T cells can be packaged as a kit. A kit may include instructions (e.g., written instructions) on the use of the compositions, in addition to one or more of the compositions described herein.

[0300] In some cases, a method of treating a cancer comprises administering a compositiondescribed herein, wherein the administration treats the cancer. In some embodiments the therapeutically-effective amount of the composition, is administered for at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, Page 85 of 117 1104881063\1\AMERICAS12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year.

[0301] One or more compositions described herein can be administered before, during, or afterthe occurrence of a disease or condition, and the timing of administering a pharmaceutical composition can vary. For example, the one or more compositions can be used as a prophylactic and can be administered continuously to subjects with a propensity to conditions or diseases in order to lessen a likelihood of the occurrence of the disease or condition. The one or more compositions can be administered to a subject during or as soon as possible after the onset of the symptoms. The administration of the one or more compositions can be initiated immediately within the onset of symptoms, within the first 3 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, within the first 24 hours of the onset of the symptoms, within 48 hours of the onset of the symptoms, or within any period of time from the onset of symptoms. The initial administration can be via any route practical, such as by any route described herein using any formulation described herein. In some examples, the administration of the one or more compositions of the disclosure is an intravenous administration. One or multiple dosages of one or more compositions can be administered as soon as is practicable after the onset of a cancer, an infectious disease, an immune disease, sepsis, or with a bone marrow transplant, and for a length of time necessary for the treatment of the immune disease, such as, for example, from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 1 month, from about 1 month to about 3 months. For the treatment of cancer, one or multiple dosages of one or more compositions can be administered years after onset of the cancer and before or after other treatments. In some examples, one or more compositions described herein can be administered for at least about 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years at least 3 years, at least 4 years, or at least 5 years. The length of treatment can vary for each subject. Page 86 of 117 1104881063\1\AMERICASDosages

[0302] The expanded population of γδ T cells as disclosed herein may be formulated in unitdosage forms suitable for single administration of precise dosages. In some cases, the unit dosage forms comprise additional lymphocytes. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more compounds. The unit dosage can be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Multiple-dose reclosable containers can be used, for example, in combination with a preservative or without a preservative. In some examples, the pharmaceutical composition does not comprise a preservative. Formulations for parenteral injection can be presented in unit dosage form, for example, in ampoules, or in multi-dose containers with a preservative.

[0303] The γδ T cells as described herein may be present in a composition in an amount of atleast 5 cells, at least 10 cells, at least 20 cells, at least 30 cells, at least 40 cells, at least 50 cells, at least 60 cells, at least 70 cells, at least 80 cells, at least 90 cells, at least 100 cells, at least 200 cells, at least 300 cells, at least 400 cells, at least 500 cells, at least 600 cells, at least 700 cells, at least 800 cells, at least 900 cells, at least 1 x 103cells, at least 2 x 103cells, at least 3 x 103cells, at least 4 x 103cells, at least 5 x 103cells, at least 6 x 103cells, at least 7 x 103cells, at least 8 x 103cells, at least 9 x 103cells, at least 1 x 104cells, at least 2 x 104cells, at least 3 x 104cells, at least 4 x 104cells, at least 5 x 104cells, at least 6 x 104cells, at least 7 x 104cells, at least 8 x 104cells, at least 9 x 104cells, at least 1 x 105cells, at least 2 x 105cells, at least 3 x 105cells, at least 4 x 105cells, at least 5 x 105cells, at least 6 x 105cells, at least 7 x 105cells, at least 8 x 105cells, at least 9 x 105cells, at least 1 x 106cells, at least 2 x 106cells, at least 3 x 106cells, at least 4 x 106cells, at least 5 x 106cells, at least 6 x 106cells, at least 7 x 106cells, at least 8 x 106cells, at least 9 x 106cells, at least 1 x 107cells, at least 2 x 107cells, at least 3 x 107cells, at least 4 x 107cells, at least 5 x 107cells, at least 6 x 107cells, at least 7 x 107cells, at least 8 x 107cells, at least 9 x 107cells, at least 1 x 108cells, at least 2 x 108cells, at least 3 x 108cells, at least 4 x 108cells, at least 5 x 108cells, at least 6 x 108cells, at least 7 x 108cells, at least 8 x 108cells, at least 9 x 108cells, at least 1 x 109cells, or more.

[0304] The therapeutically effective dose of the γδ T cells can be from about 1 cell to about 10cells, from about 1 cell to about 100 cells, from about 1 cell to about 10 cells, from about 1 cell to Page 87 of 117 1104881063\1\AMERICASabout 20 cells, from about 1 cell to about 30 cells, from about 1 cell to about 40 cells, from about 1 cell to about 50 cells, from about 1 cell to about 60 cells, from about 1 cell about 70 cells, from about 1 cell to about 80 cells, from about 1 cell to about 90 cells, from about 1 cell to about 100 cells, from about 1 cell to about 1 x 103cells, from about 1 cell to about 2 x 103cells, from about 1 cell to about 3 x 103cells, from about 1 cell to about 4 x 103cells, from about 1 cell to about 5 x 103cells, from about 1 cell to about 6 x 103cells, from about 1 cell to about 7 x 103cells, from about 1 cell to about 8 x 103cells, from about 1 cell to about 9 x 103cells, from about 1 cell to about 1 x 104cells, from about 1 cell to about 2 x 104cells, from about 1 cell to about 3 x 104cells, from about 1 cell to about 4 x 104cells, from about 1 cell to about 5 x 104cells, from about 1 cell to about 6 x 104cells, from about 1 cell to about 7 x 104cells, from about 1 cell to about 8 x 104cells, from about 1 cell to about 9 x 104cells, from about 1 cell to about 1 x 105cells, from about 1 cell to about 2 x 105cells, from about 1 cell to about 3 x 105cells, from about 1 cell to about 4 x 105cells, from about 1 cell to about 5 x 105cells, from about 1 cell to about 6 x 105cells, from about 1 cell to about 7 x 105cells, from about 1 cell to about 8 x 105cells, from about 1 cell to about 9 x 105cells, from about 1 cell to about 1 x 106cells, from about 1 cell to about 2 x 106cells, from about 1 cell to about 3 x 106cells, from about 1 cell to about 4 x 106cells, from about 1 cell to about 5 x 106cells, from about 1 cell to about 6 x 106cells, from about 1 cell to about 7 x 106cells, from about 1 cell to about 8 x 106cells, from about 1 cell to about 9 x 106cells, from about 1 cell to about 1 x 107cells, from about 1 cell to about 2 x 107cells, from about 1 cell to about 3 x 107cells, from about 1 cell to about 4 x 107cells, from about 1 cell to about 5 x 107cells, from about 1 cell to about 6 x 107cells, from about 1 cell to about 7 x 107cells, from about 1 cell to about 8 x 107cells, from about 1 cell to about 9 x 107cells, from about 1 cell to about 1 x 108cells, from about 1 cell to about 2 x 108cells, from about 1 cell to about 3 x 108cells, from about 1 cell to about 4 x 108cells, from about 1 cell to about 5 x 108cells, from about 1 cell to about 6 x 108cells, from about 1 cell to about 7 x 108cells, from about 1 cell to about 8 x 108cells, from about 1 cell to about 9 x 108cells, or from about 1 cell to about 1 x 109cells.

[0305] In some cases, the therapeutically effective dose of the γδ T cells can be from about 1x 103cells to about 2 x 103cells, from about 1 x 103cells to about 3 x 103cells, from about 1 x 103cells to about 4 x 103cells, from about 1 x 103cells to about 5 x 103cells, from about 1 x 103cells to about 6 x 103cells, from about 1 x 103cells to about 7 x 103cells, from about 1 x 103cells to about 8 x 103cells, from about 1 x 103cells to about 9 x 103cells, from about 1 x 103cells to Page 88 of 117 1104881063\1\AMERICASabout 1 x 104cells, from about 1 x 103cells to about 2 x 104cells, from about 1 x 103cells to about 3 x 104cells, from about 1 x 103cells to about 4 x 104cells, from about 1 x 103cells to about 5 x 104cells, from about 1 x 103cells to about 6 x 104cells, from about 1 x 103cells to about 7 x 104cells, from about 1 x 103cells to about 8 x 104cells, from about 1 x 103cells to about 9 x 104cells, from about 1 x 103cells to about 1 x 105cells, from about 1 x 103cells to about 2 x 105cells, from about 1 x 103cells to about 3 x 105cells, from about 1 x 103cells to about 4 x 105cells, from about 1 x 103cells to about 5 x 105cells, from about 1 x 103cells to about 6 x 105cells, from about 1 x 103cells to about 7 x 105cells, from about 1 x 103cells to about 8 x 105cells, from about 1 x 103cells to about 9 x 105cells, from about 1 x 103cells to about 1 x 106cells, from about 1 x 103cells to about 2 x 106cells, from about 1 x 103cells to about 3 x 106cells, from about 1 x 103cells to about 4 x 106cells, from about 1 x 103cells to about 5 x 106cells, from about 1 x 103cells to about 6 x 106cells, from about 1 x 103cells to about 7 x 106cells, from about 1 x 103cells to about 8 x 106cells, from about 1 x 103cells to about 9 x 106cells, from about 1 x 103cells to about 1 x 107cells, from about 1 x 103cells to about 2 x 107cells, from about 1 x 103cells to about 3 x 107cells, from about 1 x 103cells to about 4 x 107cells, from about 1 x 103cells to about 5 x 107cells, from about 1 x 103cells to about 6 x 107cells, from about 1 x 103cells to about 7 x 107cells, from about 1 x 103cells to about 8 x 107cells, from about 1 x 103cells to about 9 x 107cells, from about 1 x 103cells to about 1 x 108cells, from about 1 x 103cells to about 2 x 108cells, from about 1 x 103cells to about 3 x 108cells, from about 1 x 103cells to about 4 x 108cells, from about 1 x 103cells to about 5 x 108cells, from about 1 x 103cells to about 6 x 108cells, from about 1 x 103cells to about 7 x 108cells, from about 1 x 103cells to about 8 x 108cells, from about 1 x 103cells to about 9 x 108cells, or from about 1 x 103cells to about 1 x 109cells.

[0306] In some cases, the therapeutically effective dose of the γδ T cells can be from about 1x 106cells to about 2 x 106cells, from about 1 x 106cells to about 3 x 106cells, from about 1 x 106cells to about 4 x 106cells, from about 1 x 106cells to about 5 x 106cells, from about 1 x 106cells to about 6 x 106cells, from about 1 x 106cells to about 7 x 106cells, from about 1 x 106cells to about 8 x 106cells, from about 1 x 106cells to about 9 x 106cells, from about 1 x 106cells to about 1 x 107cells, from about 1 x 106cells to about 2 x 107cells, from about 1 x 106cells to about 3 x 107cells, from about 1 x 106cells to about 4 x 107cells, from about 1 x 106cells to about 5 x 107cells, from about 1 x 106cells to about 6 x 107cells, from about 1 x 106cells to about 7 x 107cells, from about 1 x 106cells to about 8 x 107cells, from about 1 x 106cells to about 9 x 107cells, Page 89 of 117 1104881063\1\AMERICASfrom about 1 x 106cells to about 1 x 108cells, from about 1 x 106cells to about 2 x 108cells, from about 1 x 106cells to about 3 x 108cells, from about 1 x 106cells to about 4 x 108cells, from about 1 x 106cells to about 5 x 108cells, from about 1 x 106cells to about 6 x 108cells, from about 1 x 106cells to about 7 x 108cells, from about 1 x 106cells to about 8 x 108cells, from about 1 x 106cells to about 9 x 108cells, from about 1 x 106cells to about 1 x 109cells, from about 1 x 106cells to about 2 x 109cells, from about 1 x 106cells to about 3 x 109cells, from about 1 x 106cells to about 4 x 109cells, from about 1 x 106cells to about 5 x 109cells, from about 1 x 106cells to about 6 x 109cells, from about 1 x 106cells to about 7 x 109cells, from about 1 x 106cells to about 8 x 109cells, from about 1 x 106cells to about 9 x 109cells, from about 1 x 107cells to about 1 x 109cells, from about 1 x 107cells to about 2 x 109cells, from about 1 x 107cells to about 3 x 109cells, from about 1 x 107cells to about 4 x 109cells, from about 1 x 107cells to about 5 x 109cells, from about 1 x 107cells to about 6 x 109cells, from about 1 x 107cells to about 7 x 109cells, from about 1 x 107cells to about 8 x 109cells, from about 1 x 107cells to about 9 x 109cells, from about 1 x 108cells to about 1 x 109cells, from about 1 x 108cells to about 2 x 109cells, from about 1 x 108cells to about 3 x 109cells, from about 1 x 108cells to about 4 x 109cells, from about 1 x 108cells to about 5 x 109cells, from about 1 x 108cells to about 6 x 109cells, from about 1 x 108cells to about 7 x 109cells, from about 1 x 108cells to about 8 x 109cells, from about 1 x 108cells to about 9 x 109cells, or from about 1 x 109cells to about 1 x 1010cells.

[0307] In embodiments, the cryopreserved therapeutically effective population of engineeredγδ T cells comprises at least 1 x 109CAR+ γδ T cells with a ratio of CAR+ γδ T cells to αβ T cells of at least about 200:1. In embodiments, the cryopreserved therapeutically effective population of engineered γδ T cells comprises at least 3 x 109CAR+ γδ T cells with a ratio of CAR+ γδ T cells to αβ T cells of at least about 600:1. In embodiments, the cryopreserved therapeutically effective population of engineered γδ T cells comprises at least 1 x 108CAR+ γδ T cells with a ratio of CAR+ γδ T cells to αβ T cells of at least about 20:1.

[0308] When an antibody or other agent is administered, such as an agent that binds the sameor essentially the same epitope as, or competes with, an antibody described in any one of FIGs.1- 2, the normal dosage amounts may vary from about 10 ng / kg to up to 100 mg / kg of mammal body weight or more per day, preferably about 1 μg / kg / day to 10 mg / kg / day, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. It is anticipated Page 90 of 117 1104881063\1\AMERICASthat different formulations will be effective for different treatment compounds and different disorders, that administration targeting one organ or tissue, for example, may necessitate delivery in a manner different from that to another organ or tissue.

[0309] For the treatment or reduction in the severity of immune related disease, the appropriatedosage of a composition will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the agent is administered for preventive or therapeutic purposes, previous therapy, a patient's clinical history and response to the compound, and the discretion of the attending physician. The composition can be suitably administered to the subject at one time or over a series of treatments.

[0310] For example, depending on the type and severity of the disease, about 1 mg / kg to 15mg / kg (e.g., 0.1-20 mg / kg) of multivalent agent (e.g., polypeptide or antibody) is an initial candidate dosage for administration to the subject, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 mg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. EXAMPLES

[0311] The following examples are put forth so as to provide those of ordinary skill in the artwith a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. Example 1

[0312] This example shows expansion of δ1 γδ T cells with a method in which αβ T cells weredepleted on Day 0.

[0313] The conditions tested are shown in Table 1 below.Page 91 of 117 1104881063\1\AMERICASTable 1 Study ID Condition Donors DescriptionStudy 1 1 Donor A and Control, .

[0315] On day 0, αβ T cells were depleted under conditions 4 and 5. The rest of the conditions(1-3) were transferred to pre-coated flasks with δ1-35 antibody and supplemented with 150 IU / mL IL-2. The post depleted cells were transferred to pre-coated flasks; the δ1-35 antibody and supplemented with either the multi-cytokine cocktail (140 ng / mL anti-CD3 (clone OKT-3) + 100 ng / mL IL-4 + 70 ng / mL IFNγ + 7 ng / mL IL-21 + 15 ng / mL IL-1β) for condition 4 or with 1 µg / mL OKT-3 anti-CD3 mAb and 70ng / mL IL-15 for condition 5. All the conditions were cultured for five days in the activation flask and supplemented with the respective cytokines and media on days 3 and 4.

[0316] On day 5, all the conditions were harvested from the flasks. Conditions 2-3 for bothdonors went through a αβ T cells depletion. After the depletion all the conditions were transferred to a pre-coated bags with retronectin and γ-retroviral virus. Conditions 1 and 2 were supplemented with 150 IU / mL IL-2. Conditions 2 and 4 were supplemented with the multi-cytokine cocktail (140 ng / mL anti-CD3 (clone OKT-3) + 100 ng / mL IL-4 + 70 ng / mL IFNγ + 7 ng / mL IL-21 + 15 ng / mL IL-1β). Conditions 3 and 5 were supplemented with 1 µg / mL OKT-3 anti-CD3 mAb + 70 ng / mL IL-15. All the conditions were cultured in the transduction bags for two days and supplemented with media and the corresponding cytokines on day 6.

[0317] On day 7, all the conditions were seeded in an agitated Shake Flask for expansion.Condition 1 for both donors was supplemented with 150 IU / mL IL-2; conditions 2 and 4 were supplemented with 1 µg / mL anti-CD3 (clone OKT-3) + 70 ng / mL IL-15 + 13 ng / mL IL-21 and conditions 3 and 5 were supplemented with 70 ng / mL IL-15.

[0318] On day 9, all the conditions were supplemented with cytokines similar to day 7 for therespective conditions. Page 92 of 117 1104881063\1\AMERICAS

[0319] On days 11 and 13, all the conditions were supplemented with cytokines. Condition 1for both donors was supplemented with 150 IU / mL IL-2, conditions 2 and 4 were supplemented with 1 µg / mL anti-CD3 (clone OKT-3) + 100 ng / mL IL-15 and conditions 3 and 5 were supplemented with 70 ng / mL IL-15.

[0320] On day 14, all the conditions that grew and expanded well went through αβ T cellsdepletion and seeded in shake flasks for over-night rest with X-vivo media and cytokine similar to days 11 and 13 for the respective conditions.

[0321] On day 15 the cells were harvested and cryopreserved.

[0322] Fold Expansion (FE) of the total cells from day 0 through day 14 was calculatedaccording to Equation 1: Equation 1: Fold Expansion from day 0 calculation. ^^^^^^ ^^^^^^51.^^^^ ^^^^^^ 0 − 5 =^^^^^^ ^^^^^^^^^^^^ ^^^^ ^^^^^^ 0 × 11) ^^^^^^ ^^^^^^ 93. ^^^^ ^^^^^^ 7 − 9 =^^^^^^ ^^^^^^^^^^^^ ^^^^ ^^^^^^ 7 × (^^^^^^^^^^^^^^^^ 2)^^^^^^ ^^^^^^ ^^ + 14. ^^^^ ^^^^^^ ^^ − ^^ + 1 =^^^^^^ ^^^^^^ ^^ × (^^^^ ^^^^^^ (^^ − 1) − ^^)

[0323] FIG. 3 shows the % δ1 γδ T cells and % of αβ T cells. FIG. 4 shows the fold expansionrates of the different conditions for the two donors. In both donors, the conditions that were depleted from αβ T cells on day 0 did not expand regardless of the cytokine mixture that was supplemented. The conditions with αβ T cells depleted on day 0 or day 5 exhibited low % of αβ T cells (<10%). The δ1 γδ T cells % of cells show higher enrichment throughout the experiment in all the conditions that were depleted from αβ T cells early. All the conditions expressed CAR in within δ1 γδ T cells (FIG.5). Example 2

[0324] This example shows an exemplary method in which αβ T cells were depleted on Day5. This study also evaluated if continuous OKT3 is needed throughout the process.

[0325] The conditions tested are shown in Table 2 below.Table 2 Study ID Condition Donors DescriptionPage 93 of 117 1104881063\1\AMERICAS2 Donor C and D5 TCRab depl + Multi Cytokines +OKT3 3 Donor D D5 depl +Multi Cytokines-OKT3 D7-14ureaccording to conditions 2 and 4 in Study 1, and condition 3 supplemented the cells with the same multi-cytokine mixture as Study 2 Condition 2 but without OKT3 supplementation between days 7-14. IL-15 supplementation and OKT3 activation were provided between days 5-6, and only IL- 15 was supplemented between days 7-15 for Condition Study 2 Condition 4.

[0327] FIG. 6 shows the expansion of the different conditions in Study 2. The results showthat supplementing with OKT-3 / IL-15 (condition 4) had the lowest rate of expansion. The best expansion rate obtained by supplementing with multi-cytokines. Between the multi-cytokine conditions, supplementation with OKT3 (condition 2) or omitting OKT3 between days 7-14 (condition 3) did not affect the expansion rate of the cells during expansion stage between days 7- 14 in both donors. In this study, after αβ T cells depletion on day 5, the δ1 γδ T cells enriched and expand at higher ratios than in the control and αβ T cells ratio stayed very low (below 10%) for both donors (FIG.7). The %CAR within δ1 γδ T cell was similar for all the conditions (FIG.8). Example 3

[0328] This example shows an exemplary method in which αβ T cells were depleted on Day5. This study also evaluated if NK cells depletion had an effect in the method. The conditions were similar to Adi23-021 unless specified otherwise.

[0329] The conditions tested are shown in Table 3 below.Table 3 Study ID Condition Donors Description )

[0330] In Study 3, a double depletion of both αβ T cells and CD56 cells was evaluated for theeffect on maintaining high purity of the δ1 γδ T cells final product for two donors (conditions 3 and 5). In addition, a simpler supplementation that did not require OKT3 activation at all Page 94 of 117 1104881063\1\AMERICAS(conditions 4 and 5) was also tested. FIG.9 shows the expansion of the different conditions. The expansion rates of donor F were lower than donor E but overall the expansion rates of the day 5 depleted cells were not affected whether they were depleted only from αβ T cells or depleted from both NK cells (using CD56 as marker) and αβ T cells. In addition for donor E, the cells expanded better without OKT3 at all and for donor F, it did not affect the expansion. Therefore, OKT3 is not needed in the multi-cytokine supplementation for expansion of δ1 γδ cells.

[0331] The δ1 γδ T cells, αβ T cells, NK cells ratios, CAR expression are shown in FIG. 10.The conditions where both NK cells and αβ T cells were depleted (conditions 3 and 5) maintained lower % NK cells and lower % αβ T cells throughout the process. The cells that were not supplemented with OKT3 at all maintained high δ1 γδ T cells throughout the process. For donor E, the conditions that were supplemented with OKT3 between days 5-7 had lower ratio of δ1 γδ T cells and higher % αβ T cells than in the conditions that were not supplemented with OKT3 at all. CAR expression was lower in the conditions that were supplemented with OKT3 between days 5- 7 than the conditions that were not supplemented with OKT3 at all. Therefore, day 5 depletion can be done both for NK cells and αβ cells and OKT3 is not needed in the multi cytokine supplementation cocktail.

[0332] Below are more details about the methods for the tests in Examples 1-3.

[0333] On day -1, cryopreserved PBMCs vials were thawed using a 37oC water bath, washedand placed for overnight rest in a 37oC, 5% CO2 incubator.

[0334] On day 0, either αβ T cells under certain conditions were depleted, and then added toa pre-coated flasks with D1-35 mAb for activation or directly transferred to the pre-coated flasks for activation with different respective cytokine mixtures.

[0335] On days 3 and 4, the cells were supplemented with Growth Media (GM) and therespective cytokine mixtures.

[0336] On day 5, the cells were harvested, and either depleted from αβ T cells, or αβ T cellsand NK cells, and then transduced with a CD20 CAR γ-retrovirus vector, or directly transduced with the viral vector in retronectin pre-coated PL bags and supplemented with different respective cytokine mixtures.

[0337] On day 6, the cells were supplemented with GM (X-VIVO 15 and 10% fetal bovineserum) and different respective cytokine mixtures. Page 95 of 117 1104881063\1\AMERICAS

[0338] On day 7, the cells were transferred for expansion in shake flasks with GM and therespective cytokine mixtures.

[0339] On days 9, 11 and 13, the cells were supplemented with the respective cytokinemixtures.

[0340] On days 10 and 12, the cells were supplemented with GM. During the expansion phase(days 9-14) the cells were counted and adjusted to 1e6 cells / mL by transferring them to a bigger flask and bled cells as necessary.

[0341] On day 14, the cells went through a αβ T cells depletion and placed in an overnight restwith the respective cytokine mixtures.

[0342] On day 15, the cells were harvested, washed, formulated and cryopreserved.

[0343] Cells were incubated in a 37oC, 5% CO2 incubator. For days -1 to 7, the cells wereincubated in a stationary incubator. On days 7-15 the cells were incubated in an agitated incubator with orbital shaking speed of 85-125 rpm.

[0344] All the cell washes were done in a centrifuge at 1200rpm for 5 minutes at RoomTemperature.

[0345] Throughout the production process, the cells were counted using the ChemoMetecNucleoCounter NC200. The cells counts for viability and cells concentrations were performed on day -1 (after thaw and after washes), day 0 (after overnight rest and, if applicable, post-depletion), day 5 (upon harvest of cells from activation flasks and post-depletion, if applicable), day 7, days 10-13, day 14 (harvest and post-depletion), and day 15.

[0346] Throughout the production process samples were collected for flow cytometryimmunophenotyping.

[0347] The αβ T cells depletion was done in magnetic separation using Miltenyi’s LS columns,TCRαβ biotin antibody, and anti-biotin microbeads according to the manufacturer instructions. For the αβ T cells / NK cells double depletion the magnetic separation with LS columns, TCRαβ biotin antibody, anti-biotin microbeads, and anti-CD56 microbeads was adjusted by adding the anti-CD56 microbeads to the second labeling step together with the anti-biotin microbeads. Otherwise, all steps were followed according to the manufacturer instructions.

[0348] The activation with δ1-35 mAb was done by coating the activation flasks on day-1 withthe mAb by thawing the antibody and dilute it with DPBS to a concentration of 5 µg / mL. The working solution was added to the activation flasks at 5mL, 15mL, and 45mL for CellBIND T Page 96 of 117 1104881063\1\AMERICASflasks T25, T75 or T225 respectively. The flasks with the δ1-35 mAb were incubated for 16-32 hours at 4oC. On day 0, the activation flasks were retrieved from the refrigerator, the antibody solution was removed, the flasks were washed twice with DPBS (5mL and 15mL, and 50mL for T25 and T75, and T225, respectively) and the cells were added to the flasks (about 15e6, 45e6, 140e6 for T25, T75, and T225, respectively). Then the flasks with the cells were incubated in a 37oC, 5% CO2 incubator.

[0349] For the transduction step, PL30 bags were coated with retronectin by thawing theretronectin stock solution, diluting the retronectin to 10 µg / mL working solution with DPBS. Air bubbles were removed from the bags and the bags were incubated for 20 (±6) hours at 4oC. On day 5 the viral vector was thawed and diluted with GM.

[0350] The precoated retronectin bags were retrieved from the refrigerator and the retronectinsolution was removed from the bags. The bags were washed once with DPBS and the diluted vector with GM was added to the bags. The bags were incubated in a 37oC, 5% CO2 incubator for 2-3 hours. After the cells were harvested from the activation flasks and depleted (when needed), the cells were added to the bags (about 10e6 cells per bag) and incubated in the incubator. After 1-2 hours the bags were flipped.

[0351] For all the cytokines supplementations, the cytokines aliquots were thawed on the dayof supplementation and diluted to adequate concentration either with GM or DPBS. All the cytokines aliquots were made by diluting the stock cytokines with UltraPure distilled water to the aliquot concentration. The cytokine aliquots were stored in a -80oC freezer until used. The OKT3 anti-CD3 mAb was stored in 4oC.

[0352] On day 15 the cells were washed, resuspended with CSB first, and then an equal volumeof CS10 was added in a final ratio of CSB:CS101:1. Then, as soon as possible the cells were cryopreserved with a Controlled Rate Freezer (CRF). Example 4

[0353] This example shows a large-scale preparation of δ1 γδ T cells. The necessity of IFNγwas also evaluated.

[0354] The conditions tested are shown in Table 4 below.Table 4 Study ID Condition DescriptionPage 97 of 117 1104881063\1\AMERICASB Fed batch -IFNγ IL1525ng / mL Fed batch full cytokine cocktaily

[0356] On day -1, cryobags or cryovials were thawed, pooled together, and washed in a Sepax.Following the wash, the cells were placed either in a PL120 or PL70 bag for an overnight rest, supplemented with 150 IU / mL IL-2. In addition, T225 flasks were coated with δ1-35 mAB.

[0357] On day 0, the contents of the PL120 and PL70 bags were pooled, the activation flaskswere washed, cells were added to the coated flasks, and supplemented with 150 IU / mL IL-2.

[0358] On days 3 and 4 cells were fed with growth media and IL-2. In addition, on day 4, PL70and PL325 bags were coated with 10 µg / mL retronectin solution.

[0359] On day 5, all the conditions were harvested from the activation flasks. Prior totransduction, the transduction vector was added to the retronectin coated bags. For the control condition, about 200e6 cells were seeded in each of two vector-coated PL325 bags and supplemented with IL-2 containing growth media, within 1-2 hours of transduction the bag was flipped. For all other conditions, the remaining cells from the flask harvest were transferred to the LOVO to washout the culture medium and incubate with the antibodies and immunomagnetic microbeads for the αβ T cells and NK cell depletion. After the LOVO wash and incubations were completed, the cells were enumerated and to the CliniMACS Plus for depletion. After depletion on the CliniMACS, the about 47.6e6 cells were added to each of four vector-coated PL70 bags and supplemented with growth media and the multi cytokine mixture. Condition B was supplemented with 100 ng / mL IL-4 + 70 ng / mL IFNγ + 7 ng / mL IL-21 + 15 ng / mL IL-1β while Condition C was supplemented with 100 ng / mL IL-4 + 7 ng / mL IL-21 + 15 ng / mL IL-1β. Two of the PL70 bags were for Condition B and two were for Condition C. All conditions, including the control, were transduced at the same multiplicity of infection.

[0360] On day 6, the cells were supplemented with growth media and similar cytokine mixturethat was supplemented on day 5. In addition, to prepare for the expansion, 10L Xuri bags with a 1.2 µm pore size perfusion filter were primed with 1L GM containing either 150 IU / mL IL-2 for the control conditions or 70 ng / mL IL-15 +13 ng / mL IL-21 for Conditions B and C. Page 98 of 117 1104881063\1\AMERICAS

[0361] On day 7, the primed Xuri bags were drained, the transduction bags for each conditionwere pooled together and the cells were inoculated in the corresponding Xuris. The control condition was supplemented with 150 IU / mL IL-2 and inoculated at a 5L working volume (WV). Conditions B and C were supplemented with 70 ng / mL IL-15 + 13 ng / mL IL-21 and each inoculated at a 1L working volume. The initial Xuri rocking speed was set for 9 rpm in the 1L conditions and 12 rpm in the 5L condition. The rocking angle was set to 6° for 1L and 8° for 5L.

[0362] On day 9, perfusion blocks were started in all three Xuris. For the control condition,perfusion was initiated at 0.25 working volumes per day (WV / day) while for Conditions B and C, perfusion was initiated at 0.5 WV / day. The perfusion media for the control condition was supplemented with 750 IU / mL Il-2 while the perfusion media for Conditions B and C were supplemented with 350 ng / mL IL-15 + 65 ng / mL IL-21.

[0363] On day 10, perfusion rate for the control condition was increased to 0.5 WV / day andperfusion rate for Conditions B and C was increased to 0.75 WV / day.

[0364] On day 11, new perfusion media was prepared for all conditions. The perfusion mediafor the control condition was supplemented with 450 IU / mL IL-2 and the perfusion rate was increased to 0.75 WV / day. The rocking speed and rocking angle of the control condition were increased to 17 rpm and 10°, respectively, and remained that way until Day 14. The working volume of Condition B was increased to 2L with a batch feed of 1L GM supplemented with 70 ng / mL IL-15. The working volume of Condition C was increased to 2L with a batch feed of 1L GM supplemented with 50 ng / mL IL-15. The perfusion media for Condition B was supplemented with 210 ng / mL IL-15, the perfusion media for Condition C was supplemented with 150 ng / mL IL-15, and the perfusion rate stayed at 0.75 WV / day although the working volume had increased from 1L to 2L. The rocking speed and rocking angle of Conditions B and C were increased to 12 rpm and 8°, respectively, and remained that way until Day 13.

[0365] On day 12, new perfusion media was prepared for all conditions and supplementedwith cytokines concentrations similar to day 11. For all the conditions, the perfusion rate was increased to 1.25 WV / day.

[0366] On day 13, new perfusion media was prepared for all conditions and supplementedwith cytokines concentrations similar to day 11. Perfusion rate for the control condition was increased to 1.5 WV / day. The working volume of Condition B was increased to 5L with a batch feed of 3L GM supplemented with 70 ng / mL IL-15. The working volume of Condition C was Page 99 of 117 1104881063\1\AMERICASincreased to 5L with a batch feed of 3L GM supplemented with 25 ng / mL IL-15. The perfusion media for Condition B was supplemented with 210 ng / mL IL-15, the perfusion media for Condition C was supplemented with 75 ng / mL IL-15, and the perfusion rate stayed at 1.25 WV / day although the working volume had increased from 2L to 5L. The rocking speed and rocking angle of Conditions B and C were increased to 17 rpm and 10°, respectively.

[0367] On day 14, the Conditions B and C were split into two conditions (B2 and C2)following LOVO harvest and αβ T cell depletion. Condition B was supplemented with 70 ng / mL IL-15 for the overnight rest and Condition C was supplemented with 25 ng / mL IL-15 for the overnight rest. Conditions B2 and C2, split respectively from conditions B and C, were supplemented with 150 IU / mL IL-2 for the overnight rest.

[0368] On day 15, each condition was harvested on the LOVO to washout the X-VIVO 15containing cytokines and concentrate the cells for formulation and cryopreservation. After processing on the LOVO, the cells were formulated to target about 30e6 cells / mL with about 25% PlasmaLyte A, 0.125% human serum albumin, 25% CryoStor CSB, and 50% CryoStor CS10 and filled into AT-06 vials using the Crystal M1 filling device. The cells were cryopreserved using a controlled rate freezer.

[0369] The in-process yield of the cells and the percentages of αβ T cells and δ1 γδ T cellsthroughout the process is shown in FIG.11. In-process yield (expressed as total number of viable cells or TVC) was higher for the control process up until Day 14, but after the day 14 depletion there were similar yields in all conditions since the control condition had more cells lost on Day 14 due to the αβ depletion. Both conditions B and C had higher δ1 γδ T cell purity after Day 5 due to the early αβ and NK depletion. The results suggest that omitting INFγ supplementation from Day 5-6 and decreasing the IL-15 concentration did not affect the expansion or the phenotype of the cells.

[0370] In Conditions B and C, the purity of the δ1 γδ T cells is higher than the control due tothe αβ T cell and NK cells depletion on day 5. In the control condition, the final product had a much higher percentage of αβ T cells post-thaw (0.77%) compared to the four conditions that had undergone αβ T cell depletions at both day 5 and day 14 (0.06-0.10%).

[0371] The CD25 / CD69 activation profile of the post-thaw final product is shown in FIG. 12.In these studies, the control condition exhibited a higher percentage of %CD25-CD69- cells within the δ1 CAR+ population post-thaw compared to conditions B and C. Interestingly, conditions B2 Page 100 of 117 1104881063\1\AMERICASand C2, which were identical to conditions B and C with the exception of IL-2 instead of IL-15 for the day 14 overnight rest.

[0372] Table 5 shows yield and post-thaw phenotype of cells expanded in Study 4.Table 5 Study 4 Condition A Condition B Condition C Condition B2 Condition C2 uld

[0373] Bioactivity of the products from the various conditions from Study 4 was assessedagainst a CD20+ Raji tumor cell line in both in vitro and in vivo assays. In all the studies, tumor growth inhibition (TGI) of the Raji cells in a xenograft mouse model was similar for the all the day 5 depletion conditions when compared to the control condition. While condition C had slightly lower TGI than the others, there was no significant difference between any of the conditions and its TGI improved with terminal IL-2 supplementation on Day 14 (condition C2). In addition, an in vitro assay also using the Raji cells demonstrates that despite differences in δ1 CAR+ cell recovery and cytotoxicity of the Raji cells following CAR engagement, omission of INFγ did not impact the activity of the final product. Table 6. Study 4 potency assays results Study 4 nPage 101 of 117 1104881063\1\AMERICASExample 5

[0374] Table 7 below shows an exemplary method of expanding δ1 γδ T cells.Table 7 Process details Starting material Cryopreserved PBMCs enriched from healthy human donorsExample 6

[0375] This example demonstrates the effect of MED12 knockout on δ1 γδ T cells expansionand enrichment.

[0376] δ1 T cells were activated, transduced with a CAR and expanded with or withoutknockout of MED12, TGFBR2 or both MED12 and TGFBR2 in multiple donors using two process variants. Knockout was achieved using electroporation of Cas9 nuclease and guide RNAs directed against the MED12 and / or TGFBR2 genes. The results are shown in FIGS.13A-13C. The process variant used for the data shown in FIG.13B includes an additional isolation step to remove αβ T Page 102 of 117 1104881063\1\AMERICAScells and NK cells prior to the knockout step. The δ1 cell count as assessed by flow cytometry on day 17 of each expansion prior to final processing and cryopreservation are plotted (FIG.13A and FIG. 13B). Each line represents a unique donor. Data shown in FIG. 13C represents the fold increase in day 17 δ1 cell counts of the MED12 knockout condition compared to the day 17 δ1 count of the unedited wild type condition. Each datapoint represents a unique donor.

[0377] Expanded and cryopreserved δ1 cells from two unique donors were thawed andcultured for up to 48 hours. At various timepoints (1, 2, 3, 4, 24, and 48 hours) post-thaw, the cells were assessed for cell health using AnnexinV+DAPI staining and flow cytometry. The results are shown in FIG.14.

[0378] δ1 T cells were activated, transduced and expanded with or without knock out ofMED12 or MED12 and TGFBR2. On days 10, 14, 17 and 18 of the expansion, cells were stained with a panel of antibodies directed against surface receptors including CCR7, CD62L, CD27 and CD25 and assessed by flow cytometry. Data shown represents the percentage of δ1 CAR+cells expressing each of the receptors. The results are shown in FIG.15.

[0379] Expanded δ1 T cells expressing a CAR targeting PSMA with or without knock out ofMED12 or MED12 and TGFBR2 were cocultured with fluorescently labeled PSMA+PC3 NIR cells in a repeat stimulation assay. CAR Vδ1 T cells were stimulated with target cells for 72 hours, then transferred into a new vessel with freshly plated target cells for an additional 72 hours for a total of 3 stimulations. The results are shown in FIGS.16A-16D. Cocultures were performed in the absence (FIG.16A) or presence (FIG.16B) of TGFβ. The Cytotoxicity Index was calculated by dividing the total NIR signal of all time points by the NIR signal at the start of each stimulation. In parallel, samples were collected at the end of each 72-hour cycle and assessed by flow cytometry for the number of δ1 T cells (FIG.16C and FIG.16D). Example 7

[0380] This example shows activation of δ1 T cells using beads conjugated with a combinationof T cell mitogen antibodies. Briefly, cryopreserved PBMCs are thawed using a 37oC water bath, washed and placed for overnight rest in a 37oC, 5% CO2incubator. On the next day, the cells are cultured in a culture medium with IL-2 (150 IU / mL) and beads conjugated with both anti-δ1-35 antibody and anti-CD28 antibody. Page 103 of 117 1104881063\1\AMERICAS

[0381] Multiple batches of beads with different molar ratios between the anti-δ1-35 antibodyand anti-CD28 antibody are generated. Each batch of the beads has a different a molar ratio of anti-δ1-35 antibody to anti-CD28 antibody than other batches.

[0382] For each batch of the beads, different amounts of PBMCs are mixed to test the effectsof bead to cell ratios on δ1 T cells expansion. For each batch of bead, various bead to cell ratios are tested.

[0383] The cells are cultured with the beads in the culture medium for 5 days and the expansionof δ1 T cells is evaluated with the method described in Example 3 above. Example 8

[0384] This example shows a method of expanding δ1 γδ T cells using cell culture bags in theγδ T cell activation step. In brief, this example shows the production of enriched δ1 γδ T cell population expressing anti-CD20 CAR, where the δ1 γδ T cells were activated in VueLife AC bags in the presence of anti-CD28 antibody, which then underwent TCRαβ and CD56 depletion on day 5, transduction on day 5 post-depletion and supplementation with cytokine cocktail 1 (100 ng / mL IL-4 + 15 ng / mL IL-1β + 7 ng / mL IL-21) during the transduction phase between day 5-7, expansion with cytokine cocktail 2 (70 ng / mL IL-15 + 13 ng / mL IL-21) and cytokine cocktail 3 (50 ng / mL IL-15) between day 7-14. Following expansion, on day 14 a second depletion of TCRαβ was performed and the depleted cells were rested overnight in IL-2 (150 IU / mL). The overnight rested cells were then volume reduced on formulated on day 15 prior to fill / finish.

[0385] On Day -1, cryopreserved PBMCs bags were thawed using a Plasmatherm at 37°Cusing the PLASMA program. The cryopreserved PBMCs were thawed until only small ice crystals remained, up to a maximum of 6 minutes. Following the thaw, the cells were combined and washed using the Sepax C-Pro Culture Wash v400 program using the following settings: Input volume= 80mL (for two cryobags), Dilution ratio=2.0, Final volume=100mL, Intermediate volume= 20mL, Dilution speed=17 mL / min, 300 G-force, Sedimentation time=360s, and 2 wash cycles. Following the wash, the PBMCs were supplemented with GM and IL-2 in PL120 bags and incubated for 16- 24 hours in 37°C and 5% CO2. Activation bags were prepared by coating an appropriate number of VueLife 290-AC bags in a solution of 1.0 µg / mL antibody δ1-35 in DPBS for 16-24 hours at 4°C.

[0386] On Day 0, the cells from the overnight rest were counted, diluted down to 1e6 cells / mL,and supplemented with IL-2 and 0.75µg / mL~2.5µg / mL anti-CD28 antibody. Activation flasks Page 104 of 117 1104881063\1\AMERICASwere then washed twice with DPBS and the diluted cells were seeded into the flasks and then incubated until Day 3 in a 37°C and 5% CO2 incubator.

[0387] On Days 3 and 4, the cells were supplemented with 78 mL and 104 mL of GM and IL-2, respectively. On Day 4, two PL70 bags were coated with 85.7 mL of 10 µg / mL RetroNectin solution and incubated at 4°C for 16-24 hours.

[0388] On Day 5, cells were harvested from the bags by combining to a larger sized transferbag followed with a rinse with GM of the activation bags to obtain any residual cells. The day 5 depletion cells were then washed and prepared for depletion by using the LOVO, CliniMACS TCRαβ-biotin antibody, CD56 microbeads, and anti-biotin microbeads. After the wash, the cells were incubated with a primary anti-TCRαβ-biotin antibody. Following the incubation, primary antibody was washed twice and then the secondary incubation was performed with anti-biotin microbeads and CD56 microbeads. When the incubation completed the cells went through another wash in the LOVO. While the cells were being processed on the LOVO, RetroNectin coated bags were washed once with DPBS and incubated with 35.7 mL of diluted vector at 37°C and 5% CO2for 2-3 hours. After labeling the cells on LOVO, the cells were then transferred to a CliniMACS kit for depletion using the CliniMACS Plus. Following the completion of the CliniMACS operation the depleted cells were concentrated via centrifugation and then supplemented with cytokine cocktail 1 to prepare them for transduction.

[0389] The transduction of all the development runs was performed at an MOI of 0.6. Duringcell processing on the LOVO, the CAR-expressing vector was thawed at ambient conditions and a vector master mix was made by dilution with GM. The RetroNectin coated bags were removed from the refrigerator and the RetroNectin solution was drained from the bags which were then washed with DPBS. Following the 2-3 hours’ vector incubation, depleted cells were added to each PL70 bag and placed in a 37°C, 5% CO2incubator. After 1 to 2 hours the bags were turned over.

[0390] On Day 6, The culture transduction bags were fed with 23.8 mL of GM + cytokinecocktail 1. In addition, the Xuri Bioreactor cell bags were primed with 1L of GM supplemented with cytokine cocktail 2.

[0391] On Day 7, the cultures were inoculated in the Xuri Bioreactor system. First the primingmedia was drained from the Xuri cell bags. Then, the Day 7 GM bag was supplemented with cytokine cocktail 2 and then combined with the cells. The diluted cells were drained into the Xuri Page 105 of 117 1104881063\1\AMERICASbag by gravity flow and the Xuri was set to 37°C, rocking speed of 9 rpm, rocking angle 6°, and a gas flow rate of 0.55 L / min.

[0392] On Day 9, perfusion media was supplemented with cytokine cocktail 2 and attached tothe Xuri perfusion line. Perfusion was initiated at a rate of 125 mL every 6 hours.

[0393] On Day 10, the perfusion block was updated to increase the rate of perfusion to 125mL every 4 hours.

[0394] On Day 11, 1L of GM was supplemented with cytokine cocktail 3 and gravity drainedinto the Xuri to increase the total culture volume to 2L. Next, perfusion media was supplemented with cytokine cocktail 3 and attached to the Xuri perfusion line. Perfusion was initiated at a rate of 250 mL every 4 hours.

[0395] On Day 12, perfusion media was supplemented with cytokine cocktail 3 and attachedthe Xuri perfusion line. The perfusion rate was then updated to a rate of 250 mL every 2.4 hours.

[0396] On Day 13, 3L of GM was supplemented with IL-15 and gravity drained into the Xurito increase the total culture volume to 5L. Next, perfusion media was supplemented with cytokine cocktail 3 and attached to the Xuri perfusion line. Perfusion was initiated at a rate of 625 mL every 2.4 hours. Xuri was set to a rocking speed of 15 rpm, rocking angle 10°.

[0397] On Day 14, the cells are washed and labeled on the LOVO and then TCRαβ depletedusing the CliniMACS Plus. Following the depletion, the cells were transferred to 3L Spinner Flasks for overnight rest with the appropriate GM volumes and cytokines supplementation.

[0398] On Day 15, cells were harvested, washed with Plasmalyte-A with HSA and formulatedin PlasmaLyte-A / CSB using the LOVO. Cells if required were then diluted further using CSB / PlasmaLyte-A / HSA. Formulated cells were then filtered through a 40 µm SQ40S blood filter and then further formulated with CS10 at 1:1. The formulated cells in CS10 is then aliquoted to AT-06 vials via the M1 filling system, and cryopreserved using the CRF.

[0399] During the manufacturing process, the cells were counted using the ChemoMetecNucleoCounter NC200 to obtain cell density and viability measurments. NC-200 was performed on Day -1 (post-thaw and post-wash), Day 0 (post-overnight rest), Day 5 (following the harvest of cells, following the LOVO wash, and following the post-depletion centrifugation), Day 7 (harvest and post-Xuri inoculation), Days 9-13, Day 14 (harvest, post-LOVO and post-depletion), and Day 15 (harvest and post-LOVO). See Table 8. The expansion profiles are shown in FIGS.17A-17C. Table 8. Process parameter data for method in Example 8 Page 106 of 117 1104881063\1\AMERICASBags Bags Bags coated coated coated with Flasks with with y t - t L 897 0 8 8 999000000Table 8 (cont’d) Bags Bags coated - LPage 107 of 117 1104881063\1\AMERICASTVC post-thaw 2.72E+09TVC post-wash 2.98E+09TVC 2.71E+0989 7 0 8 8 999000000Table 9. Sequence information SEQ Notes Sequences ID F V G E NPage 108 of 117 1104881063\1\AMERICAS(amino acid) 5 dnTGFBR2 TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCD D K E E N L E C V F K G P V F P V F P VPage 109 of 117 1104881063\1\AMERICASDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAF KDFVVASETSDCVVSSTLSPEKGKAKNPPGDSSLHWAAMA M V I A E F F K

[0400] The embodiments and examples described above are intended to be merely illustrativeand non-limiting. Those skilled in the art will recognize or will be able to ascertain using no more than routine experimentation, numerous equivalents of specific compounds, materials and procedures. All such equivalents are considered to be within the scope and are encompassed by the appended claims. Page 110 of 117 1104881063\1\AMERICAS

Claims

CLAIMS: What is claimed is:

1. A method for producing an expanded population of engineered γδ T cells, the methodcomprising: (a) culturing an isolated mixed cell population comprising γδ T cells with at least one T cell mitogen to produce an activated γδ T cell population; (b) depleting αβ T cells in the activated γδ T cell population from (a) to produce an enriched γδ T cell population; (c) engineering the enriched γδ T cell population from (b) to express at least one antigen recognition moiety to produce an engineered γδ T cell population, wherein the engineering step comprises stably integrating at least one nucleic acid construct encoding said at least one antigen recognition moiety in culture conditions comprising IL-4, IL-1β, and IL-21; and (d) culturing the engineered γδ T cell population from (c) in the presence of at least one cytokine to provide the expanded population of engineered γδ T cells.

2. The method of any one of the preceding claims, wherein step (b) is performed afterculturing the isolated mixed cell population with the at least one T cell mitogen for about 4, 5, 6, or 7 days, preferably 6 days.

3. The method of any one of the preceding claims, wherein step (b) further comprisesdepleting NK cells from the activated γδ T cell population.

4. The method of claim 3, wherein the NK cells are depleted using an agent binding toCD56.

5. The method of any one of the preceding claims, wherein step (c) is between about 16 and96 hours, more preferably about 48 hours.

6. The method of any one of the preceding claims, wherein (d) comprises sequentiallyculturing the engineered γδ T cell population from (c) in culture conditions comprising IL-15 and Page 111 of 117 1104881063\1\AMERICASIL-21 for at least about 1-7 days, preferably from about 3-5 days, more preferably about 4 days; followed by culture conditions comprising IL-15 for at least about 1-7 days, preferably between about 2-5 days, more preferably about 3 or 4 days to provide the expanded population of engineered γδ T cells.

7. The method of claim 6, wherein the IL-15 in the culture condition has a concentrationgreater than about 10 ng / mL, preferably from about 25 ng / mL to 80 ng / mL, more preferably from about 50 ng / mL to 70 ng / mL.

8. The method of any one of the preceding claims, further comprising:(e) depleting αβ T cells in the expanded population of engineered γδ T cells from (d); (f) resting the expanded population of engineered γδ T cells after depletion for a predetermined duration of time in the presence of at least one cytokine to provide a therapeutically effective population of engineered γδ T cells; and (g) cryopreserving the therapeutically effective population of engineered γδ T cells.

9. The method of claim 8, wherein the predetermined duration in (f) maintains αβ T cells inthe therapeutically effective population of engineered γδ T cells below about 5% of total viable cells, preferably between about 0.2% and about 3% of total viable cells, preferably between about 0.0% and about 0.5% of total viable cells, more preferably between about 0.0% and about 0.2%, of total viable cells.

10. The method of claim 8 or 9, wherein the predetermined duration is between about 6 hoursand about 72 hours, preferably between about 8 hours and about 48 hours, more preferably between about 12 hours and about 24 hours.

11. The method of claim 8, wherein the at least one cytokine in step (f) is IL-2.

12. The method of any one of claims 8-11, wherein depleting αβ T cells in step (e) comprisesreducing αβ T cells to below about 0.5%, preferably about 0.2%, of total viable cells of the expanded population of engineered γδ T cells. Page 112 of 117 1104881063\1\AMERICAS13. The method of claim 12, wherein depleting αβ T cells in (e) comprises reducing αβ Tcells to between about 0.05% and about 0.5% of total viable cells of the expanded population of engineered γδ T cells, preferably between about 0.05% and about 0.1% of total viable cells.

14. The method of any one of the preceding claims, wherein the isolated mixed cellpopulation in (a) is a peripheral blood sample, a cord blood sample, or a tumor.

15. The method of any one of the preceding claims, wherein the culture conditions in step (c)do not comprise an antibody or a fragment thereof; optionally wherein the culture conditions in step (c) do not comprise an antibody or a fragment thereof that recognizes CD3.

16. The method of any one of the preceding claims, wherein the culture conditions in step (c)do not comprise IFNγ.

17. The method of any one of the preceding claims, wherein the at least one T cell mitogen ina) comprises, or is, an antibody or a fragment thereof; optionally wherein the antibody binds to CD3 or a fragment thereof, or to a γδ TCR or a fragment thereof.

18. The method of claim 17, wherein the antibody binds to a variable region of a γδ TCR;preferably wherein the antibody binds to a variable region of a δ1 γδ TCR.

19. The method of claim 18, wherein the variable region of a δ1 γδ TCR comprises a Bin 3δ1 epitope comprising or consisting of amino acids FKKAAKSVALTISALQ or AKSGRYSVNFKKAAKSVALTISALQ of human Vδ1.

20. The method of claim 18, wherein the variable region of a δ1 γδ TCR comprises a Bin 4δ1 epitope comprising or consisting of amino acids AQKVTQAQSSV of human Vδ1, and amino acids TDKLIFGKGTRVTVEP of human J1 or LTAQLFFGKGTQLIVEP of human J2, wherein the at least one antibody does not bind an epitope containing a K120T mutation in J1 or J2. Page 113 of 117 1104881063\1\AMERICAS21. The method of claim 19, wherein the antibody is an antibody that binds to the sameepitope as, or competes with, antibody δ1-08 or an antibody comprising the six complementarity determining regions (CDRs) of antibody δ1-08.

22. The method of claim 20, wherein the antibody is an antibody that binds to the sameepitope as, or competes with, antibody δ1-35 or an antibody comprising the six CDRs of antibody δ1-35.

23. The method of any one of the preceding claims, wherein the at least one T cell mitogen isa means for selectively activating and expanding δ1 γδ T cells by binding to an epitope comprising a variable region of a γδ TCR.

24. The method of claim 23, wherein the epitope comprising a variable region of a δ1 γδTCR is a Bin 3 δ1 epitope comprising or consisting of amino acids FKKAAKSVALTISALQ or AKSGRYSVNFKKAAKSVALTISALQ of human Vδ1.

25. The method of claim 23, wherein the epitope comprising a variable region of a δ1 γδTCR is a Bin 4 δ1 epitope comprising or consisting of amino acids AQKVTQAQSSV of human Vδ1, and amino acids TDKLIFGKGTRVTVEP of human J1 or LTAQLFFGKGTQLIVEP of human J2, wherein the at least one antibody does not bind an epitope containing a K120T mutation in J1 or J2.

26. The method of any one of the preceding claims, wherein the at least one T cell mitogen instep a) comprises an antibody comprising six CDRs of antibody δ1-35.

27. The method of any one of the preceding claims, further comprising disrupting expressionof one or more genes in the γδ T cells.

28. The method of any one of claims 18-27, wherein the at least one T cell mitogen furthercomprises an antibody that binds to CD28, CD137, CD2, CD27, and / or OX40; preferably wherein the at least one T cell mitogen comprises a combination of an antibody that binds to Page 114 of 117 1104881063\1\AMERICASvariable region of a δ1 γδ TCR and an anti-CD28 and / or anti-CD137 antibody; optionally wherein the antibodies are bound to a solid support.

29. The method of claim 28, wherein disrupting expression of the one or more genes isperformed prior to step (a).

30. The method of claim 28, wherein disrupting expression of the one or more genes isperformed after step (b).

31. The method of claim 28, wherein disrupting expression of the one or more genes isperformed after step (b) and prior to step (c).

32. The method of any one of claims 27 to 31, wherein the one or more genes comprisesMED12.

33. The method of claim 32, wherein the one or more genes comprises MED12 and TGFβR2.

34. The method of any one of the preceding claims, wherein the expanded population of γδ Tcells has less than 5% αβ T cells, preferably less than 0.07% αβ T cells.

35. The method of any one of the preceding claims, wherein the expanded population of γδ Tcells has less than 30% NK cells, preferably less than 2.5% or 0.5% NK cells.

36. The method of any one of the preceding claims, wherein the expanded population of γδ Tcells has at least 70% γδ T cells, preferably at least 91%, 95%, or 98% γδ T cells.

37. A cryopreserved therapeutically effective population of engineered γδ T cells comprisingat least 70% γδ T cells, less than 5% αβ T cells, and less than 30% NK cells, produced by the method according to any of the preceding claims.

38. The cryopreserved therapeutically effective population of engineered γδ T cells of claim36, comprising at least 91% γδ T cells, less than 0.07% αβ T cells, and less than 2.5% NK cells. Page 115 of 117 1104881063\1\AMERICAS39. A cryopreserved therapeutically effective population of engineered γδ T cells comprisingat least 1 x 109CAR+ γδ T cells with a ratio of CAR+ γδ T cells to αβ T cells of at least about 200:1, or comprising at least 3 x 109CAR+ γδ T cells with a ratio of CAR+ γδ T cells to αβ T cells of at least about 600:1, or comprising at least 1 x 108CAR+ γδ T cells with a ratio of CAR+ γδ T cells to αβ T cells of at least about 20:1; wherein said engineered γδ T cells MED12-, preferably wherein said engineered γδ T cells are MED12- and TGFβR2-, or MED12- and dnTGFβR2+.

40. The cryopreserved therapeutically effective population of engineered γδ T cells of any ofclaims 37 to 39, wherein the γδ T cells are δ1 γδ T cells.

41. The cryopreserved therapeutically effective population of engineered γδ T cells of any ofclaims 37 to 39, wherein the γδ T cells are δ2- γδ T cells. Page 116 of 117 1104881063\1\AMERICAS

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