Compositions and methods for the generation of engineered, and or engineerable, cells

Abstract

The present invention teaches compositions and methods for the generation of engineered and or engineerable cells, such as cells expressing a transmembrane polypeptide, for example a chimeric antigen receptor (CAR)-expressing cell, such as an immune cell, e.g., CD3+ immune cells, including gamma delta (γδ) T cells and the like.

Description

J&J No. JBI6843WOPCT COMPOSITIONS AND METHODS FOR THE GENERATION OF ENGINEERED, AND OR ENGINEERABLE, CELLS FIELD OF THE INVENTION

[0001] The present invention teaches compositions and methods for the generation of engineered and or engineerable cells, such as cells expressing a transmembrane polypeptide, for example a chimeric antigen receptor (CAR)-expressing cell, such as an immune cell, e.g., CD3+ immune cells, including gamma delta (γδ) T cells and the like. BACKGROUND

[0002] Immune cell therapy, such as T cell immunotherapy, including autologous chimeric antigen receptor (CAR) T cell therapies, has been reported to be an efficacious therapy for the treatment of some diseases, such as some cancers, including some hematologic malignancies. However, applications of cell therapy to treat multiple cancer types has met numerous and varied challenges (see, e.g., Advances and challenges of CAR T therapy and suitability of animal models (Review), Authors: Xavier E. Ramos-Cardona Weichuan Luo Sulma I. Mohammed; Published online on: July 12, 2022). SUMMARY OF THE INVENTION

[0003] Advances in T cell immunotherapy have been reported, such as in autologous CAR T cell therapy; however, applications of cell therapy to treat multiple cancer types has unmet challenges and the present invention, against this backdrop, meets many of the challenges that impede access to therapeutic cell products, such as manufacturing robust chimeric antigen receptor (CAR)-expressing CD3+ gamma delta (γδ) T cells in sufficient numbers. The present application addresses these and other needs.

[0004] In one aspect, the invention provides a method of generation of chimeric antigen receptor (CAR)-expressing CD3+ gamma delta (γδ) T cells, said method comprising one or more of the following steps: a) seeding an enriched γδ T cell fraction obtained from peripheral blood mononuclear cells (PBMCs) at about 1x105to about 2x106cells / well on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in an expansion medium;J&J No. JBI6843WOPCT b) culturing the CD3+ γδ T cells on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in the expansion medium for about 1.5 to about 2.5 days, preferably 2 days, to produce activated CD3+ γδ T cells; c) collecting the activated CD3+ γδ T cells produced in step (b) and re-plating the activated CD3+ γδ T cells at about 1x105to about 2x1052x106cells / well on a second tissue culture vessel in the absence of anti-CD3 antibodies; d) adding a chimeric antigen receptor (CAR)-encoding vector to the activated CD3+ γδ T cells of step (c) to achieve a multiplicity of infection (MOI) of 0.65-2, preferably a MOI of 1; e) adding Vectofusin®-1 at about a dilution of 1:50 to about a dilution of 1:200, preferably about a dilution of 1:100, to the mixture of activated CD3+ γδ T cells and CAR- encoding vector; f) culturing the mixture of step (e) for about 0.5 to about 1.5 days, preferably about 1 day to produce CAR-expressing CD3+ γδ T cells; g) adding additional expansion medium to the CAR-expressing CD3+ γδ T cells to achieve a total culture volume which is about double the culture volume of step (f); h) after about 1.5 to about 2.5 days, preferably 2 days, transferring the CAR-expressing CD3+ γδ T cells to a third tissue culture vessel and culturing for about 3.5 to about 4.5 days, preferably 4 days; and i) optionally, harvesting the CAR-expressing CD3+ γδ T cells or introducing the CAR- expressing CD3+ γδ T cells into a sorter, enrichment column, or a bioreactor.

[0005] In some embodiments of the above-described method, the method further comprises i) harvesting the CAR-expressing CD3+ γδ T cells and / or introducing the CAR-expressing CD3+ γδ T cells into a sorter, enrichment column, or a bioreactor.

[0006] In some embodiments, the harvesting is by centrifugation.

[0007] In some embodiments, the third tissue culture vessel is larger than the second tissue culture vessel or the first tissue culture vessel.

[0008] In some embodiments, the CD3+ γδ T cells in the enriched γδ T cell fraction are primary CD3+ γδ T cells.

[0009] In some embodiments, the anti-CD3 mAb is derived from clone OKT3.

[0010] In some embodiments, the collecting is by centrifugation, fluorescence activated cell sorting (FACS) or enrichment through a column.J&J No. JBI6843WOPCT

[0011] In some embodiments, the vector is a viral vector.

[0012] In some embodiments, the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, or a vaccinia virus vector.

[0013] In some embodiments, the viral vector is a retroviral vector.

[0014] In some embodiments, the vector is a non-viral vector.

[0015] In some embodiments, the method further comprises one or more of the following steps: (1) counting the total number of the activated CD3+ γδ T cells produced in step (c) or the CAR-expressing CD3+ γδ T cells produced in step (h); (2) assaying for one or more cell surface markers on the activated CD3+ γδ T cells produced in step (c) or the CAR-expressing CD3+ γδ T cells produced in step (h); and (3) calculating an expansion efficacy of the CAR-expressing CD3+ γδ T cells produced in step (h) using results obtained in steps (1) and (2).

[0016] In some embodiments, the one or more cell surface markers comprises CD3, γδ T cell receptor (TCR), Vδ1, and / or Vδ2.

[0017] In some embodiments, the assay for the one or more cell surface markers is performed via flow cytometry.

[0018] In some embodiments, the final concentration of Vectofusin®-1 in the total culture volume is 10 µg / mL.

[0019] In some embodiments, the expansion medium comprises CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ OpTmizer™ T-Cell Expansion Supplement, about 2mM (1x) GlutaMAX™ Supplement, about 5% Human Type AB Serum, about 1% antibiotic-antimycotic (anti-anti), recombinant human interleukin-2 (rhIL-2) at a concentration of about 100 IU / mL, and recombinant human interleukin-15 (rhIL-15) at a concentration of about 10 ng / mL.

[0020] In another aspect, the invention provides chimeric antigen receptor (CAR)-expressing CD3+ gamma delta (γδ) T cells obtained by a method of generation from γδ T cells, said method comprising one or more of the following steps: a) a means for seeding an enriched γδ T cell fraction obtained from peripheral blood mononuclear cells (PBMCs) at about 1x105to about 2x106cells / well on an anti-J&J No. JBI6843WOPCT CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in an expansion medium; b) a means for culturing the CD3+ γδ T cells on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in the expansion medium for about 1.5 to about 2.5 days, preferably 2 days, to produce activated CD3+ γδ T cells; c) a means for collecting the activated CD3+ γδ T cells produced in step (b) and a means for re-plating the activated CD3+ γδ T cells at about 1x105to about 2x106cells / well on a second tissue culture vessel in the absence of anti-CD3 antibodies; d) a means for adding a chimeric antigen receptor (CAR)-encoding vector to the activated CD3+ γδ T cells of step (c) to achieve a multiplicity of infection (MOI) of 0.65- 2, preferably a MOI of 1; e) a means for adding Vectofusin®-1 at about a dilution of 1:50 to about a dilution of 1:200, preferably about a dilution of 1:100, to the mixture of activated CD3+ γδ T cells and CAR-encoding vector; f) a means for culturing the mixture of step (e) for about 0.5 to about 1.5 days, preferably about 1 day to produce CAR-expressing CD3+ γδ T cells; g) a means for adding additional expansion medium to the CAR-expressing CD3+ γδ T cells to achieve a total culture volume which is about double the culture volume of step (f); h) after about 1.5 to about 2.5 days, preferably 2 days, a means for transferring the CAR-expressing CD3+ γδ T cells to a third tissue culture vessel and culturing for about 3.5 to about 4.5 days, preferably 4 days; and i) optionally, providing a means for harvesting the CAR-expressing CD3+ γδ T cells or a means for introducing the CAR-expressing CD3+ γδ T cells into a sorter, enrichment column, or a bioreactor.

[0021] In another aspect, the invention provides a system for performing a method of generating chimeric antigen receptor (CAR)-expressing CD3+ gamma delta (γδ) T cells, the system comprising:J&J No. JBI6843WOPCT a) a means for seeding an enriched γδ T cell fraction obtained from peripheral blood mononuclear cells (PBMCs) at about 1x105to about 2x106cells / well on an anti- CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in an expansion medium; b) a means for culturing the CD3+ γδ T cells on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in the expansion medium for about 1.5 to about 2.5 days, preferably 2 days, to produce activated CD3+ γδ T cells; c) a means for collecting the activated CD3+ γδ T cells produced in step (b) and a means for re-plating the activated CD3+ γδ T cells at about 1x105to about 2x106cells / well on a second tissue culture vessel in the absence of anti-CD3 antibodies; d) a means for adding a chimeric antigen receptor (CAR)-encoding vector to the activated CD3+ γδ T cells of step (c) to achieve a multiplicity of infection (MOI) of 0.65- 2, preferably a MOI of 1; e) a means for adding Vectofusin®-1 at about a dilution of 1:50 to about a dilution of 1:200, preferably about a dilution of 1:100, to the mixture of activated CD3+ γδ T cells and CAR-encoding vector; f) a means for culturing the mixture of step (e) for about 0.5 to about 1.5 days, preferably about 1 day to produce CAR-expressing CD3+ γδ T cells; g) a means for adding additional expansion medium to the CAR-expressing CD3+ γδ T cells to achieve a total culture volume which is about double the culture volume of step (f); h) after about 1.5 to about 2.5 days, preferably 2 days, a means for transferring the CAR-expressing CD3+ γδ T cells to a third tissue culture vessel and culturing for about 3.5 to about 4.5 days, preferably 4 days; and i) optionally, providing a means for harvesting the CAR-expressing CD3+ γδ T cells or a means for introducing the CAR-expressing CD3+ γδ T cells into a sorter, enrichment column, or a bioreactor.

[0022] In another aspect, the invention provides a method of generation of chimeric antigen receptor (CAR)-expressing CD3+ gamma delta (γδ) T cells, said method comprising the steps of:J&J No. JBI6843WOPCT a) seeding an enriched γδ T cell fraction obtained from peripheral blood mononuclear cells (PBMCs) at about 1x105to about 2x106cells / well on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in an expansion medium; b) culturing the CD3+ γδ T cells on an anti-CD3 monoclonal antibody (mAb)- coated first tissue culture vessel in the expansion medium for about 1.5 to about 2.5 days, preferably 2 days, to produce activated CD3+ γδ T cells; c) collecting the activated CD3+ γδ T cells produced in step (b) and re-plating the activated CD3+ γδ T cells at about 1x105to about 2x106cells / well on a second tissue culture vessel in the absence of anti-CD3 antibodies; d) adding a chimeric antigen receptor (CAR)-encoding vector to the activated CD3+ γδ T cells of step (c) to achieve a multiplicity of infection (MOI) of 0.65-2, preferably a MOI of 1; e) adding Vectofusin®-1 at about a dilution of 1:50 to about a dilution of 1:200, preferably about a dilution of 1:100, to the mixture of activated CD3+ γδ T cells and CAR-encoding vector; f) culturing the mixture of step (e) for about 0.5 to about 1.5 days, preferably about 1 day to produce CAR-expressing CD3+ γδ T cells; g) adding additional expansion medium to the CAR-expressing CD3+ γδ T cells to achieve a total culture volume which is about double the culture volume of step (f); h) after about 1.5 to about 2.5 days, preferably 2 days, transferring the CAR- expressing CD3+ γδ T cells to a third tissue culture vessel and culturing for about 3.5 to about 4.5 days, preferably 4 days; and i) optionally, harvesting the CAR-expressing CD3+ γδ T cells or introducing the CAR-expressing CD3+ γδ T cells into a sorter, enrichment column, or a bioreactor. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 displays multicolor flow cytometry gating on singlets, viable cells, cluster of differentiation 3-positive (CD3+) and gamma-delta (γδ) T cell receptor positive (TCR+) cells,J&J No. JBI6843WOPCT and alpha-beta (αβ) TCR+ cells. The data illustrates typical purity of CD3+ γδ TCR+ T cells with very limited αβ TCR+ T cell contamination. The two panels on the left-hand side represent Donor 1 and the two panels on the right-hand side represent Donor 2.

[0024] Figure 2 shows an example of a general multicolor flow cytometry gating strategy. For the gating strategy, all cells were gated. From the cells, singlets, viable cells, CD3+ and γδ TCR+ cells, and chimeric antigen receptor (CAR) expression on γδ TCR+ cells were gated. Typical plots for peripheral blood mononuclear cell (PBMC)-derived γδ T cells using CD3 (clone OKT3) monoclonal antibody (mAb) expansion methods are shown. CAR expression was analyzed on different TCR subsets, e.g., δ1+, δ2+ and δ1- / δ2- (not shown). The untransduced control is not shown.

[0025] Figure 3 depicts a graphical representation of an exemplary CAR design which may be used in accordance with the present disclosure. SP, signal peptide; TMD, transmembrane domain; Act. domain, activation domain.

[0026] Figures 4A-4B illustrate chimeric antigen receptor (CAR) expression and cytotoxicity of human primary γδ T cells. The purity of γδ T cells was determined by staining of γδ TCR on CD3+ T cells using flow cytometry (Figure 4A, left) and CAR expression was determined by R- Phycoerythrin (PE)-labeled human prostate-specific membrane antigen (PSMA; Acro Biosystems) on CD3+ γδ TCR+ cells 2 weeks post transduction (Figure 4A, right) (1: CD3 (Clone OKT3; 2: Vδ2 (Clone: REA771); 3: Dynabeads Human T-Expander CD3 / CD28). The percentage of the purity of γδ T cells and CAR expression (% positive) were analyzed. PSMA- expressing target cells (C4-2B cells) were seeded to a xCELLigence 96-well E-Plate (Agilent) to adhere. Target cell proliferation was measured as a change in relative impedance which was termed cell index (Figure 4B). The y-axis is the normalized cell index generated by the RTCA software and displayed in real time. The x-axis is the time of cell culture and treatment time in hours (hrs). After 24 hours, the effector CAR γδ T cells generated by 3 different activators (Dynabeads, CD3 (clone OKT3) or Vδ2 monoclonal antibody (mAb) (clone REA771)) were added at a ratio of effector to target cell (E:T) of approximately 1:3. Mean values of the normalized cell index were plotted. When T cells were added to the target cells, PSMA CAR γδ T cells generated by CD3 (OKT3) or Vδ2 monoclonal antibody (mAb) (clone REA771) activation induced more efficient cell cytolysis and a greater subsequent progressive decrease in normalized cell index as compared to CAR γδ T cells generated by activation with DynabeadsJ&J No. JBI6843WOPCT Human T-Expander CD3 / CD28. Tumor cells (C4-2B cells) alone showed rapid proliferation and a subsequent progressive increase in normalized Cell Index. Similarly, target cells (C4-2B cells) were not lysed by negative control untransduced (UTD) γδ T cells.

[0027] Figures 5A-5D. Activation Markers and CAR Expression on Human Primary γδ T Cells Activated by Different Anti-CD3 mAb Clones. Figure 5A. Primary human γδ T cells were activated using various anti-CD3 mAb clones (OKT3, UTCH1, and HIT3a) coated on 24-well plates at concentrations of 2.5, 5, and 10 µg / ml for 2 days. The expression levels of activation markers CD25 and CD69 on CD3+ γδ TCR+ γδ T cells were analyzed by flow cytometry, summarized from 2 representative donors. Top panel: Donor Aph24-7 and Bottom panel: Donor Aph24-12. Figure 5B. Representative purity of γδ T cells was shown six days post-transduction with CAR RV. γδ T cells activated and expanded by different anti-CD3 mAb clones achieved high purity (>90% γδ TCR+). Figure 5B shows the data for six days post-transduction with PSMA CAR1 and PSMA CAR2 with the anti-CD3 mAb clone, OKT3 from donor Aph24-7. Flow cytometry was also performed with the clones UTCH1, and HIT3a at concentrations of 2.5, 5, and 10 µg / ml with donors Aph24-7 and Aph24-12 (flow cytometry for clones UTCH1, and HIT3a from donor Aph24-7 and Aph24-12 are not shown in Figure 5B). There was no significant difference in the percentage of γδ TCR+ cells across different anti-CD3 mAb clones (OKT3, UTCH1, and HIT3a). PSMA CAR expression was analyzed at 6 days post-transduction (Figure 5C) and 12 days post-transduction (Figure 5D), respectively. γδ T cells, activated by anti-CD3 mAbs (at 2.5, 5, and 10 µg / ml) for 2 days, were transduced with PSMA CAR RV at an MOI of 1. PSMA CAR expression was detected on CD3+ γδ TCR+ γδ T cells using an AF647- labeled anti-linker antibody (1 µg / ml, internal reagent) by flow cytometry. Symbols: Circle (2.5 µg / ml), Square (5 µg / ml), Triangle (10 µg / ml). Solid and open shapes represent two different PSMA CARs.

[0028] Figures 6A-6C. CD27 expression on naïve and activated γδ T cells. Figure 6A: Primary human γδ T cells from two representative donors were thawed and stained for CD3, γδ TCR, Vδ2, and CD27 expression in duplicate. Flow cytometry was performed to show CD27 expression on CD3+ γδ TCR+ Vδ2+ and CD3+ γδ TCR+ Vδ2- cells (data not shown). Figure 6A summarizes the average percentage of CD27 expression on CD3+ γδ TCR+ Vδ2+ and CD3+ γδ TCR+ Vδ2- cells obtained from the flow cytometry methods. Figures 6B and 6C: Two representative donors’ primary human γδ T cells were activated using different clones of anti-J&J No. JBI6843WOPCT CD3 mAb (OKT3, UCHT1, and HIT3a) coated on 24-well plates at concentrations of 2.5, 5, and 10 µg / ml for 2 days. CD27 expression was stained and analyzed on activated CD3+ γδ TCR+ Vδ2+ T cells using flow cytometry (data not shown for the flow cytometry), and the percentage of expression levels were summarized (Figures 6B and 6C). The bar graphs for Figure 6B and Figure 6C show the flow cytometry data for the CD3 (OKT3), CD3 (UCHT1), and CD3 (HIT3a) clones.

[0029] Figures 7A-7B. CD3 mAb with or without CD27 mAb promotes robust activation and proliferation of γδ T cells. Different clones of anti-CD3 mAb (OKT3, UCHT1, and HIT3a) were used for this study. Figure 7A shows the analysis of γδ T cells for CTV dye dilution (proliferation) and surface expression of CD25 at five days post activation. Representative dot plots are shown. Cells were pre-gated on live, single, CD3+ γδ TCR+ cells. The top panels are from donor Aph24-7 and the bottom panels are from donor Aph24-12. The data for the CD3(OKT3) clones are shown. Plots for the clones CD3(UCHT1) and CD3 (HIT3a) are not shown. Figure 7B shows relative cell counts of live, single, CD3+ γδ TCR+ cells recorded by flow cytometry which are summarized in a bar chart. γδ T cells activated with CD27 mAb showed relatively lower cell counts compared to those activated with CD3 mAb alone, suggesting that CD27 mAb may selectively expand CD3+ and CD27+ γδ T cells, while CD3 mAb could expand all CD3+ γδ T cells. The top bar graph represents data for Donor Aph24-7, the bottom bar graph represents data for Donor Aph24-12.

[0030] Figures 8A-8B. Enhanced Cytokine Expression in γδ T Cells Activated by Combining CD3 (UCHT1 Clone) with CD27 mAb. Different doses of CD27 at 2, 5, and 10 μg / ml were combined with 10 μg / ml anti-CD3 mAb to coat 24-well plates. Anti-CD3 mAb were derived from clones OKT3, UCHT1 and HIT3a. Human γδ T cells from 2 donors were thawed and activated with the combined CD3 and CD27 mAbs. Plates without CD27 mAb served as controls to evaluate the specific role of CD27 mAb in the activation and function of γδ T cells. Figures 8A and 8B: The percentage of IFN-γ and TNF-α producing γδ T cells from two representative donors was determined by intracellular staining of stimulated γδ T cells and summarized. DETAILED DESCRIPTION

[0031] The genetic engineering of T cells to specifically engage and kill tumor cells in a target- specific manner has resulted in the establishment of new therapeutic options for cancer patients,J&J No. JBI6843WOPCT referred to as engineered T cell therapy. This targeting is typically brought about by genetically manipulating patient-derived T cells with a recombinant DNA molecule that encodes a chimeric antigen receptor (CAR). CARs are synthetic receptors comprising an extracellular targeting domain that is linked to a linker peptide, a transmembrane domain (TMD), and one or more intracellular signaling domains.

[0032] Traditionally, the extracellular domain consists of a single chain Fv fragment of an antibody (scFv) that is specific for a given tumor-associated antigen (TAA) or cell surface target. The extracellular scFv domain confers the tumor specificity of the CAR, while the signaling domains activate the T cell upon TAA / target engagement. These engineered T cells (CAR-T cells) are re-infused into cancer patients, where they specifically engage and kill cells expressing the TAA target of the CAR (Maus et al., Blood. 2014 Apr 24;123(17):2625-35; Curran and Brentjens, J Clin Oncol. 2015 May 20;33(15):1703-6).

[0033] Autologous, patient-specific CAR-T therapy has emerged as a powerful and potentially curative therapy for cancer. However, the development of CAR-T technology and its wider application is partly limited due to number of key shortcomings including a) an inefficient anti- tumor response in solid tumors, b) limited penetration and susceptibility of adoptively transferred CAR T cells to an immunosuppressive tumor microenvironment (TME), c) poor persistence of CAR-T cells in vivo, d) serious adverse events in the patients including cytokine release syndrome (CRS) and graft-versus-host disease (GVHD) mediated by the CAR-T, and e) the time required for manufacturing.

[0034] For example, the inventors of the present invention use PBMC-derived γδ T cells, which can undergo inter alia self-renewal, gene editing, multilineage differentiation, etc., to advance off- the-shelf, allogeneic, CAR-engineered T cell therapy platforms with more commercially viable, automated, energy efficient, and resource sparing manufacturing processes. For example, a PBMC-derived cell therapy platform is based on production of γδ T cells, in accordance with the present invention, which cell therapy platform possesses intrinsic antitumor activity and whose cellular tumor infiltration is associated with more favorable outcomes. There is therefore a need in the art for methods of efficiently producing PBMC-derived allogeneic γδ T cells in sufficient numbers, of sufficient purity, and having superior fold expansion properties for use with CAR-T. The present invention is directed to such improved methods and materials.J&J No. JBI6843WOPCT Definitions

[0035] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0036] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0037] The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of up to ±10% from the specified value, as such variations are appropriate to perform the disclosed methods. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0038] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0039] As used herein, the term "about" should be construed to refer to both of the numbers specified as the endpoint (s) of any range. Any reference to a range should be considered as providing support for any subset within that range. Ranges may be expressed herein as from “about” or “approximately” or “substantially” one particular value and / or to “about” or “approximately” or “substantially” another particular value. When such a range is expressed, other exemplary embodiments include from the one particular value and / or to the other particularJ&J No. JBI6843WOPCT value. Further, the term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and more preferably still up to ±1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value. Throughout this disclosure, various aspects of the invention can be presented 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 invention. 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.

[0040] The terms “treating” or “treatment” refer to any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject’s physical or mental well-being, or prolonging the length of survival. The treatment may be assessed by objective or subjective parameters; including the results of a physical examination, neurological examination, or psychiatric evaluations.

[0041] The term “subject” refers to human and non-human animals, including all vertebrates, e.g., mammals and non-mammals, such as non-human primates, monkeys, apes, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. In many embodiments of the described methods, the subject is a human.J&J No. JBI6843WOPCT

[0042] As used herein, the term “isolated” means a biological component (such as a nucleic acid, peptide, protein, or cell) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, proteins, cells, and tissues. Nucleic acids, peptides, proteins, and cells that have been “isolated” thus include nucleic acids, peptides, proteins, and cells purified by standard purification methods and purification methods described herein. “Isolated” nucleic acids, peptides, proteins, and cells can be part of a composition and still be isolated if the composition is not part of the native environment of the nucleic acid, peptide, protein, or cell. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

[0043] As used herein, the term “polynucleotide,” synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double- stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.

[0044] As used herein, the term “vector” is a replicon in which another nucleic acid segment can be operably inserted so as to bring about the replication or expression of the segment.

[0045] As used herein, the term “host cell” refers to a cell comprising a nucleic acid molecule of the disclosure. The “host cell” can be any type of cell, e.g., a primary cell (e.g., a γδ T cell), a cell in culture, or a cell from a cell line. In one embodiment, a “host cell” is a cell transfectedJ&J No. JBI6843WOPCT with a nucleic acid molecule of the disclosure. In another embodiment, a “host cell” is a progeny or potential progeny of such a transfected cell. A progeny of a cell may or may not be identical to the parent cell, e.g., due to mutations or environmental influences that can occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

[0046] The term “expression” as used herein, refers to the biosynthesis of a gene product. The term encompasses the transcription of a gene into RNA. The term also encompasses translation of RNA into one or more polypeptides, and further encompasses all naturally occurring post- transcriptional and post-translational modifications. An expressed chimeric antigen receptor (CAR) of the present disclosure can be within the cytoplasm of a host cell, into the extracellular milieu such as the growth medium of a cell culture, or anchored to the cell membrane.

[0047] As used herein, the terms “peptide,” “polypeptide,” or “protein” can refer to a molecule comprised of amino acids and can be recognized as a protein by those of skill in the art. The conventional one-letter or three-letter code for amino acid residues is used herein. The terms “peptide,” “polypeptide,” and “protein” can be used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0048] As used herein, the term “immune cell” or “immune effector cell” refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response. Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and myeloid-derived phagocytes. According to particular embodiments, the engineered immune cells are T cells (e.g., γδ T cells), and are referred to as CAR-T cells (e.g., CAR γδ T cells) because they are engineered to express CARs of the disclosure.

[0049] As used herein, the term “engineered immune cell” refers to an immune cell, also referred to as an immune effector cell, that has been genetically modified by the addition of extra genetic material in the form of DNA or RNA to the total genetic material of the cell. According toJ&J No. JBI6843WOPCT embodiments herein, the engineered immune cells have been genetically modified to express a CAR construct according to the disclosure.

[0050] The embodiments described herein are not limited to particular methods, reagents, compounds, compositions, or biological systems, which can, of course, vary. Methods for Generating CAR T cells Using Gamma-delta (γδ) T cells.

[0051] Provided herein are methods for generation of chimeric antigen receptor (CAR)- expressing CD3+ gamma delta (γδ) T cells. In various embodiments, the method can comprise one or more of the following steps. a) seeding an enriched γδ T cell fraction obtained from peripheral blood mononuclear cells (PBMCs) at about 1x105to about 2x106cells / well of a CD3 monoclonal antibody (mAb)- coated tissue culture vessel in complete expansion medium; b) culturing CD3+ γδ T cells for about 1.5 to about 2.5 days, preferably 2 days, to produce activated CD3+ γδ T cells; c) collecting the activated CD3+ γδ T cells produced in step (b) and re-plating the activated CD3+ γδ T cells at about 1x105to about 2x106cells / well of a new tissue culture vessel; d) adding a chimeric antigen receptor (CAR)-containing vector to the activated CD3+ γδ T cells of step (c) to achieve a multiplicity of infection (MOI) of 0.65-2, preferably a MOI of 1; e) adding Vectofusin®-1 at about a dilution of 1:50 to about a dilution of 1:200, preferably about a dilution of 1:100, to the mixture of activated CD3+γδ T cells and CAR- containing vector produced in step (d); f) incubating the mixture of step (e) with the Vectofusin®-1 for about 0.5 to about 1.5 days, preferably about 1 day to produce CAR-expressing CD3+ γδ T cells; g) adding additional complete expansion medium to the CAR-expressing CD3+ γδ T cells to achieve a total culture volume which is double the culture volume achieved in step (f); h) after about 1.5 to about 2.5 days, preferably 2 days, transferring the CAR-expressing CD3+ γδ T cells to a larger tissue culture vessel; i) after about 3.5 to about 4.5 days, preferably 4 days, obtaining CAR-expressing CD3+ γδ T cells; and j) optionally harvesting the CAR-expressing CD3+ γδ T cells or introducing the CAR- expressing CD3+ γδ T cells into a sorter, enrichment column, or a bioreactor.J&J No. JBI6843WOPCT

[0052] In a non-limiting example, the enriched γδ T cell fraction obtained from peripheral blood mononuclear cells (PBMCs) is enriched by any method known to one of skill in the art, such as, e.g., via a TCR γ / δ+ T Cell Isolation Kit. In some embodiments of the above-described method, the enriched γδ T cell fraction can be seeded at about 1 x 104to about 1 x 108, about 2 x 104to about 1 x 108, about 3 x 104to about 1 x 108, about 4 x 104to about 1 x 108, about 5 x 104to about 6 x 108, about 7 x 104to about 1 x 108, about 8 x 104to about 1 x 108, about 9 x 104to about 1 x 108, about 1 x 105to about 1 x 108, about 1 x 105to about 9 x 107, about 1 x 105to about 8 x 107, about 1 x 105to about 7 x 107, about 1 x 105to about 6 x 107, about 1 x 105to about 5 x 107, about 1 x 105to about 4 x 107, about 1 x 105to about 4 x 107, about 1 x 105to about 3 x 107, about 1 x 105to about 2 x 107, about 1 x 105to about 1 x 107, about 1 x 105to about 9 x 106, about 1 x 105to about 8 x 106, about 1 x 105to about 7 x 106, about 1 x 105to about 6 x 106, about 1 x 105to about 5 x 106, about 1 x 105to about 4 x 106, about 1 x 105to about 4 x 106, about 1 x 105to about 3 x 106, about 1 x 105to about 2 x 106, about 1 x 105to about 1 x 106, about 2 x 105to about 9 x 107, about 2 x 105to about 8 x 107, about 2 x 105to about 7 x 107, about 2 x 105to about 6 x 107, about 2 x 105to about 5 x 107, about 2 x 105to about 4 x 107, about 2 x 105to about 4 x 107, about 2 x 105to about 3 x 107, about 2 x 105to about 2 x 107, about 2 x 105to about 1 x 107, about 2 x 105to about 9 x 106, about 2 x 105to about 8 x 106, about 2 x 105to about 7 x 106, about 2 x 105to about 6 x 106, about 2 x 105to about 5 x 106, about 2 x 105to about 4 x 106, about 2 x 105to about 4 x 106, about 2 x 105to about 3 x 106, about 2 x 105to about 2 x 106, about 2 x 105to about 1 x 106, or about 3 x 105to about 3 x 106cells / well of a CD3 (clone OKT3) monoclonal antibody (mAb)-coated tissue culture vessel in complete expansion medium.

[0053] In some embodiments, the enriched γδ T cell fraction can be seeded at about 1x105to about 2x106cells / well of a CD3 monoclonal antibody (mAb)-coated tissue culture vessel in complete expansion medium. A non-limiting exemplary mAb to activate the γδ T cells is obtained from clone OKT3. In some embodiments, the tissue culture vessel can be a tissue culture-treated flat-bottom 96-well plate and the enriched γδ T cell fraction are seeded at about 1x105cells / well; however, it is well within the knowledge of one skilled in the art to scale up the tissue culture vessel size and to scale up the anti-CD3 mAb volume accordingly. As a non- limiting example, if the enriched γδ T cell fraction is seeded at about 2x105cells, the tissue culture vessel can be a 48-well plate coated with about 100 μL of mAb per well. As another non-J&J No. JBI6843WOPCT limiting example, if the enriched γδ T cell fraction is seeded about 5x105to about 1x106cells, the tissue culture vessel can be a 24-well plate coated with about 500 μL of mAb per well.

[0054] In some embodiments of any of the above-described methods, the culturing of the enriched γδ T cell fraction to produce activated CD3+ γδ T cells can be for about 0.5 day to about 1 day, about 0.5 day to about 1.5 days, about 0.5 day to about 2 days, about 0.5 day to about 2.5 days, about 0.5 day to about 3 days, about 0.5 day to about 3.5 days, about 0.5 day to about 4 days, about 1day to about 1.5 days, about 1 to about 2 days, about 1 to about 2.5 days, about 1 to about 3 days, about 1 to about 3.5 days, about 1 to about 4 days, about 1.5 to about 2 days, about 1.5 to about 2.5 days, about 1.5 to about 3 days, about 1.5 to about 3.5 days, about 1.5 to about 4 days, about 2 to about 2.5 days, about 2 to about 3 days, about 2 to about 3.5 days, about 2 to about 4 days, about 2.5 to about 3 days, about 2.5 to about 3.5 days, or about 2.5 to about 4 days, or more. In some embodiments, the culturing the enriched γδ T cell fraction to produce activated CD3+ γδ T cells can be for about 0.5 day, about 1 day, about 1.5 days, about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days, or more. In some embodiments, the culturing the enriched γδ T cell fraction to produce activated CD3+ γδ T cells can be for about 1.5 about 1.5 to about 2.5 days. In some embodiments, the culturing the enriched γδ T cell fraction to produce activated CD3+ γδ T cells can be for about 2 days.

[0055] In some embodiments of any of the above-described methods, the activated CD3+ γδ T cells are collected. Non-limiting exemplary methods of collecting the activated CD3+ γδ T cells include centrifugation, dilution of the activated CD3+ γδ T cells produced in step (b) with expansion medium, FACS sorting, and chromatography. In some embodiments of any of the above-described methods, the activated CD3+ γδ T cells are collected by centrifugation.

[0056] In some embodiments of any of the above-described methods, the re-plating of the activated CD3+ γδ T cells can be at about 1 x 104to about 1 x 108, about 2 x 104to about 1 x 108, about 3 x 104to about 1 x 108, about 4 x 104to about 1 x 108, about 5 x 104to about 6 x 108, about 7 x 104to about 1 x 108, about 8 x 104to about 1 x 108, about 9 x 104to about 1 x 108, about 1 x 105to about 1 x 108, about 1 x 105to about 9 x 107, about 1 x 105to about 8 x 107, about 1 x 105to about 7 x 107, about 1 x 105to about 6 x 107, about 1 x 105to about 5 x 107, about 1 x 105to about 4 x 107, about 1 x 105to about 4 x 107, about 1 x 105to about 3 x 107, about 1 x 105to about 2 x 107, about 1 x 105to about 1 x 107, about 1 x 105to about 9 x 106, about 1 x 105to about 8 x 106, about 1 x 105to about 7 x 106, about 1 x 105to about 6 x 106,J&J No. JBI6843WOPCT about 1 x 105to about 5 x 106, about 1 x 105to about 4 x 106, about 1 x 105to about 4 x 106, about 1 x 105to about 3 x 106, about 1 x 105to about 2 x 106, about 1 x 105to about 1 x 106, about 2 x 105to about 9 x 107, about 2 x 105to about 8 x 107, about 2 x 105to about 7 x 107, about 2 x 105to about 6 x 107, about 2 x 105to about 5 x 107, about 2 x 105to about 4 x 107, about 2 x 105to about 4 x 107, about 2 x 105to about 3 x 107, about 2 x 105to about 2 x 107, about 2 x 105to about 1 x 107, about 2 x 105to about 9 x 106, about 2 x 105to about 8 x 106, about 2 x 105to about 7 x 106, about 2 x 105to about 6 x 106, about 2 x 105to about 5 x 106, about 2 x 105to about 4 x 106, about 2 x 105to about 4 x 106, about 2 x 105to about 3 x 106, about 2 x 105to about 2 x 106, about 2 x 105to about 1 x 106, or about 3 x 105to about 3 x 106cells / well of a new tissue culture vessel in complete expansion medium.

[0057] In some embodiments, the methods of the present disclosure can include genetically modifying γδ T cells of the present disclosure (e.g., activated CD3+ γδ T cells) with any of various chimeric antigen receptors (CARs) described herein or otherwise known in the art, e.g., by transduction with a vector (such as, e.g., a recombinant vector) comprising a nucleic acid encoding the CAR. Any vector known to those skilled in the art in view of the present disclosure can be used. In some embodiments, the vector may be a viral vector. Non-limiting examples of a viral vector include a retroviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a lentiviral vector, a vaccinia virus vector, a herpes virus vector, or a baculoviral vector. In some embodiments, the viral vector may be a retroviral vector.

[0058] In some embodiments, the vector may be a non-viral vector. In some embodiments, the non-viral vector may be a single or double stranded DNA molecule that is used as a template for homology directed repair (HDR) based gene editing, a Sleeping Beauty transposon, a minicircle plasmid, or a piggyBac transposon.

[0059] In some embodiments of any of the above-described methods, a chimeric antigen receptor (CAR)-containing vector (such as, e.g., a viral vector or a retroviral vector) can be added to the activated CD3+ γδ T cells (e.g., for transduction with the retrovirus comprising a nucleic acid encoding the CAR). In some embodiments, the chimeric antigen receptor (CAR)- containing vector (such as, e.g., a viral vector or a retroviral vector) can be added to the activated CD3+ γδ T cells to achieve a multiplicity of infection (MOI) of about 0.65 to about 0.7, about 0.65 to about 0.75, about 0.65 to about 0.8, about 0.65 to about 0.85, about 0.65 to about 0.9, about 0.65 to about 0.95, about 0.65 to about 1, about 0.65 to about 1.05, about 0.65 to about 1.1,J&J No. JBI6843WOPCT about 0.65 to about 1.15, about 0.65 to about 1.2, about 0.65 to about 1.25, about 0.65 to about 1.3, about 0.65 to about 1.35, about 0.65 to about 1.4, about 0.65 to about 1.45, about 0.65 to about 1.5, about 0.65 to about 1.55, about 0.65 to about 1.6, about 0.65 to about 1.65, about 0.65 to about 1.7, about 0.65 to about 1.75, about 0.65 to about 1.8, about 0.65 to about 1.85, about 0.65 to about 1.9, about 0.65 to about 1.95, or about 0.65 to about 2, or more.. In some embodiments, the chimeric antigen receptor (CAR)-containing vector (such as, e.g., a viral vector or a retroviral vector) can be added to the activated CD3+ γδ T cells to achieve a multiplicity of infection (MOI) of about 0.6, about 0.61, about 0.62, about 0.63, about 0.64, about 0.65, about 0.66, about 0.67, about 0.68, about 0.69, about 0.7, about 0.71, about 0.72, about 0.73, about 0.74, about 0.75, about 0.76, about 0.77, about 0.78, about 0.79, about 0.8, about 0.81, about 0.82, about 0.83, about 0.84, about 0.85, about 0.86, about 0.87, about 0.88, about 0.89, about 0.9, about 0.91, about 0.92, about 0.93, about 0.94, about 0.95, about 0.96, about 0.97, about 0.98, about 0.99, about 1, about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.11, about 1.12, about 1.13, about 1.14, about 1.15, about 1.16, about 1.17, about 1.18, about 1.19, about 1.2, about 1.21, about 1.22, about 1.23, about 1.24, about 1.25, about 1.26, about 1.27, about 1.28, about 1.29, about 1.3, about 1.31, about 1.32, about 1.33, about 1.34, about 1.35, about 1.36, about 1.37, about 1.38, about 1.39, about 1.4, about 1.41, about 1.42, about 1.43, about 1.44, about 1.45, about 1.46, about 1.47, about 1.48, about 1.49, about 1.5, about 1.51, about 1.52, about 1.53, about 1.54, about 1.55, about 1.56, about 1.57, about 1.58, about 1.59, about 1.6, about 1.61, about 1.62, about 1.63, about 1.64, about 1.65, about 1.66, about 1.67, about 1.68, about 1.69, about 1.7, about 1.71, about 1.72, about 1.73, about 1.74, about 1.75, about 1.76, about 1.77, about 1.78, about 1.79, about 1.8, about 1.81, about 1.82, about 1.83, about 1.84, about 1.85, about 1.86, about 1.87, about 1.88, about 1.89, about 1.9, about 1.91, about 1.92, about 1.93, about 1.94, about 1.95, about 1.96, about 1.97, about 1.98, about 1.99 or about 2, or more. In some embodiments, the chimeric antigen receptor (CAR)-containing vector (such as, e.g., a viral vector such as, e.g., a retroviral vector) can be added to the activated CD3+ γδ T cells to achieve a multiplicity of infection (MOI) of 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25,J&J No. JBI6843WOPCT 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99 or 2, or more. In some embodiments, the chimeric antigen receptor (CAR)-containing vector (such as, e.g., a viral vector such as, e.g., a retroviral vector) can be added to the activated CD3+ γδ T cells to achieve a multiplicity of infection (MOI) of about 0.65 to about 1.65, or even 2. In some embodiments, the chimeric antigen receptor (CAR)- containing vector (such as, e.g., a viral vector such as, e.g., a retroviral vector) can be added to the activated CD3+ γδ T cells to achieve a multiplicity of infection (MOI) of 1. In some embodiments, the chimeric antigen receptor (CAR)-containing vector (such as, e.g., a viral vector such as, e.g., a retroviral vector) can be added to the activated CD3+ γδ T cells to achieve a multiplicity of infection (MOI) of 2.

[0060] In some embodiments, the methods of the present disclosure involve adding to the mixture of activated CD3+ γδ T cells and CAR-containing retrovirus Vectofusin®-1. Without wishing to be bound by theory, Vectofusin®-1 is a fully synthetic non-toxic cationic amphipathic peptide with a viral transduction enhancing capacity which can enable higher transduction levels with low amounts of retroviral vector than without Vectofusin®-1. Vectofusin®-1 is capable of promoting the entry of several retroviral pseudo-types into target cells when added to the culture medium. The Vectofusin®-1 which can be used in accordance with the methods described herein can be, for example, as described in Vectofusin®-1 data sheet static.miltenyibiotec.com / asset / 150655405641 / document_uv9l1cv6p54italp1ft5beu23n?content- disposition=inline, the content of which is incorporated herein by reference in its entirety for all purposes. In some embodiments, the final concentration of Vectofusin®-1 can be 1 µg / mL, 2 µg / mL, 3 µg / mL, 4 µg / mL, 5 µg / mL, 6 µg / mL, 7 µg / mL, 8 µg / mL, 9 µg / mL, 10 µg / mL, 11 µg / mL, 12 µg / mL, 13 µg / mL, 14 µg / mL, 15 µg / mL, 16 µg / mL, 17 µg / mL, 18 µg / mL, 19 µg / mL, 20 µg / mL, or more, in the total culture volume. In some embodiments, the final concentration of Vectofusin®-1 can be 10 µg / mL in the total culture volume. In some embodiments, the Vectofusin®-1 is added to the mixture of activated CD3+ γδ T cells and CAR-containing vector (such as, e.g., a viral vector such as, e.g., a retroviral vector) to achieve a ratio of about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:150, about 1:200, or more.J&J No. JBI6843WOPCT In some embodiments, the Vectofusin®-1 is added to the mixture of activated CD3+ γδ T cells and CAR-containing vector (such as, e.g., a viral vector such as, e.g., a retroviral vector) to achieve a ratio of about 1:100.

[0061] In some embodiments, the mixture of activated CD3+γδ T cells and CAR-containing retrovirus described herein can be incubated with the Vectofusin®-1 for about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, 1 day, 1.5 days, 2 days, 2.5 days, 3 days, or more. In some embodiments, the mixture of activated CD3+γδ T cells and CAR-containing retrovirus described herein can be incubated with the Vectofusin®-1 for about 0.5 to about 1.5 days. In some embodiments, the mixture of activated CD3+γδ T cells and CAR-containing retrovirus described herein can be incubated with the Vectofusin®-1 for 1 day.

[0062] In some embodiments, additional fresh complete expansion medium can be added to the CAR-expressing CD3+ γδ T cells to achieve a total culture volume which is about double the culture volume achieved when incubating the mixture of activated CD3+γδ T cells and CAR- containing retrovirus with the Vectofusin®-1. In some embodiments, after about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, 1 day, 1.5 days, 2 days, 2.5 days, 3 days, or more, the CAR-expressing CD3+ γδ T cells can be transferred to a larger tissue culture vessel. In some embodiments, after 1 day or about 2.5 days the CAR-expressing CD3+ γδ T cells can be transferred to a larger tissue culture vessel. In some embodiments, the CAR-expressing CD3+ γδ T cells can be transferred to a larger tissue culture vessel after about 2 days. In some embodiments, after about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, or more, CAR- expressing CD3+ γδ T cells are then generated. In some embodiments, the CAR-expressing CD3+ γδ T cells are then generated after about 3.5 to about 4.5 days. In some embodiments, the CAR-expressing CD3+ γδ T cells are then generated after about 4 days. In some embodiments, the CAR-expressing CD3+ γδ T cells can be harvested by, e.g., centrifugation, or by similar methodological approaches known to those skilled in the art to achieve same. In some embodiments, the harvested CAR-expressing CD3+ γδ T cells can be frozen for later use. In some embodiments, the CAR-expressing CD3+ γδ T cells are used without harvesting inJ&J No. JBI6843WOPCT subsequent processes, such as, e.g., introduction into a sorter, an enrichment column, or a bioreactor.

[0063] In some embodiments of any of the above-described methods, the CD3+ γδ T cells can be any of various subsets of γδ T cells known in the art, or any combination thereof. The methods described herein can be aimed at generating CAR engineered γδ T cells which are pan γδ TCR+ and may not be focused on generating any particular subset within γδ T cells such as δ1 or δ2 cells. In some embodiments, the CD3+ γδ T cells can be for example, without limitation, Vγ9Vδ2 T cells, Vδ1 T cells, and / or Vδ1- Vδ2- γδ TCR+ T cells, or any combination thereof. In some embodiments, the CD3+ γδ T cells can include Vδ3 and / or Vδ5 T cells. In some embodiments, the CD3+ γδ T cells can be primary CD3+ γδ T cells. In some embodiments, the CD3+ γδ T cells can be derived from peripheral blood mononuclear cells (PBMCs). In some embodiments, the CD3+ γδ T cells can be a cell in culture, or a cell from a cell line. In some embodiments, the CD3+ γδ T cells can be derived from tissues or other origins. As a non- limiting example, the CD3+ γδ T cells can be derived from secondary lymphoid organs, skin, gut, lungs, the reproductive tract, or solid or hematologic cancers.

[0064] In some embodiments, the CD3+ γδ T cells described herein are activated using a CD3 (clone OKT3) monoclonal antibody in accordance with the methods of the present disclosure. Further, it has been generally thought that cell-cell contact is necessary for the effective expansion of γδ T cells. The methods of the present disclosure use CD3 (clone OKT3) mAb as an activator for the isolated γδ T cells (CD3+ γδ TCR+), ultimately achieving a high purity of γδ T cells, CAR expression, and cytotoxicity as disclosed herein.

[0065] In some embodiments of any of the above-described methods, step (c) and step (i) further comprise (1) counting the total number of the activated CD3+ γδ T cells produced in step (c) or the CAR-expressing CD3+ γδ T cells produced in step (i); (2) assaying for one or more cell surface markers on the activated CD3+ γδ T cells produced in step (c) or the CAR-expressing CD3+ γδ T cells produced in step (i) via flow cytometry, or an equivalent thereof; and (3) calculating an expansion efficacy of the CAR-expressing CD3+ γδ T cells produced in step (i) using results obtained in steps (1) and (2).J&J No. JBI6843WOPCT

[0066] As a non-limiting example, the one or more cell surface markers can comprise CD3, γδ TCR (Gamma delta TCR antibody), Vδ1, and / or Vδ2.

[0067] In some embodiments, the CAR-expressing CD3+ γδ T cells produced by the methods of the present disclosure can be expanded by about 3-fold, about 3.1-fold, about 3.2-fold, about 3.3- fold, about 3.4-fold, about 3.5-fold, about 3.6-fold, about 3.7-fold, about 3.8-fold, about 3.9-fold, about 4-fold, about 4.1-fold, about 4.2-fold, about 4.3-fold, about 4.4-fold, about 4.5-fold, about 4.6-fold, about 4.7-fold, about 4.8-fold, about 4.9-fold, about 5-fold, about 5.1-fold, about 5.2- fold, about 5.3-fold, about 5.4-fold, about 5.5-fold, about 5.6-fold, about 5.7-fold, about 5.8-fold, about 5.9-fold, about 6-fold, about 6.1-fold, about 6.2-fold, about 6.3-fold, about 6.4-fold, about 6.5-fold, about 6.6-fold, about 6.7-fold, about 6.8-fold, about 6.9-fold, about 7-fold, about 7.1- fold, about 7.2-fold, about 7.3-fold, about 7.4-fold, about 7.5-fold, about 7.6-fold, about 7.7-fold, about 7.8-fold, about 7.9-fold, about 8-fold, about 8.1-fold, about 8.2-fold, about 8.3-fold, about 8.4-fold, about 8.5-fold, about 8.6-fold, about 8.7-fold, about 8.8-fold, about 8.9-fold, about 9.0- fold, about 9.1-fold, about 9.2-fold, about 9.3-fold, about 9.4-fold, about 9.5-fold, about 9.6-fold, about 9.7-fold, about 9.8-fold, about 9.9-fold, about 10-fold, about 10.1-fold, about 10.2-fold, about 10.3-fold, about 10.4-fold, about 10.5-fold, about 10.6-fold, about 10.7-fold, about 10.8- fold, about 10.9-fold, about 11-fold, about 11.1-fold, about 11.2-fold, about 11.3-fold, about 11.4-fold, about 11.5-fold, about 11.6-fold, about 11.7-fold, about 11.8-fold, about 11.9-fold, about 12-fold, about 12.1-fold, about 12.2-fold, about 12.3-fold, about 12.4-fold, about 12.5-fold, about 12.6-fold, about 12.7-fold, about 12.8-fold, about 12.9-fold, about 13-fold, about 13.1-fold, about 13.2-fold, about 13.3-fold, about 13.4-fold, about 13.5-fold, about 13.6-fold, about 13.7- fold, about 13.8-fold, about 13.9-fold, about 14-fold, about 14.1-fold, about 14.2-fold, about 14.3-fold, about 14.4-fold, about 14.5-fold, about 14.6-fold, about 14.7-fold, about 14.8-fold, about 14.9-fold, about 15-fold, about 15.1-fold, about 15.2-fold, about 15.3-fold, about 15.4-fold, about 15.5-fold, about 15.6-fold, about 15.7-fold, about 15.8-fold, about 15.9-fold, about 16-fold, about 16.1-fold, about 16.2-fold, about 16.3-fold, about 16.4-fold, about 16.5-fold, about 16.6- fold, about 16.7-fold, about 16.8-fold, about 16.9-fold, about 17-fold, about 17.1-fold, about 17.2-fold, about 17.3-fold, about 17.4-fold, about 17.5-fold, about 17.6-fold, about 17.7-fold, about 17.8-fold, about 17.9-fold, about 18-fold, about 18.1-fold, about 18.2-fold, about 18.3-fold, about 18.4-fold, about 18.5-fold, about 18.6-fold, about 18.7-fold, about 18.8-fold, about 18.9- fold, about 19-fold, about 19.1-fold, about 19.2-fold, about 19.3-fold, about 19.4-fold, aboutJ&J No. JBI6843WOPCT 19.5-fold, about 19.6-fold, about 19.7-fold, about 19.8-fold, about 19.9-fold, about 20-fold, or more. In some embodiments, the CAR-expressing CD3+ γδ T cells produced by the methods of the present disclosure are expanded by about 8-fold, about 8.1-fold, about 8.2-fold, about 8.3- fold, about 8.4-fold, about 8.5-fold, about 8.6-fold, about 8.7-fold, about 8.8-fold, about 8.9-fold, about 9.0-fold, about 9.1-fold, about 9.2-fold, about 9.3-fold, about 9.4-fold, about 9.5-fold, about 9.6-fold, about 9.7-fold, about 9.8-fold, about 9.9-fold, or about 10-fold. In some embodiments, the CAR-expressing CD3+ γδ T cells produced by the methods of the present disclosure are expanded by about 8.9-fold.

[0068] As described above, the CD3+ γδ T cells or the activated CD3+ γδ T cells or the CAR- expressing CD3+ γδ T cells are cultured in a complete expansion medium. The complete expansion medium can comprise CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ OpTmizer™ T-Cell Expansion Supplement, about 2mM (1x) GlutaMAX™ Supplement, about 5% Human Type AB Serum, about 1% antibiotic-antimycotic (anti-anti), recombinant human interleukin-2 (rhIL-2) to a final concentration of about 100 IU / mL, and recombinant human interleukin-15 (rhIL-15) to a final concentration of about 10 ng / mL.

[0069] In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM and CTS™ OpTmizer™ T-Cell Expansion Supplement can be mixed together in accordance within the methods set forth, for example, thermofisher.com / order / catalog / product / A1048501, the content of which is incorporated herein by reference in its entirety for all purposes. OpTmizer™ CTS™ T Cell Expansion SFM has been developed for the growth and expansion of human T lymphocytes. OpTmizer™ CTS™ T-Cell Expansion medium is a complete serum-free, xeno- free 1X medium consisting of OpTmizer™ T-Cell Expansion Basal Medium (1 L bottle) and OpTmizer™ T-Cell Expansion Supplement (26 mL), which are mixed together prior to use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM and CTS™ OpTmizer™ T-Cell Expansion Supplement can be mixed together in any volume prior to use such that the ratio achieved is about 1 L of OpTmizer™ T-Cell Expansion Basal Medium to about 26 mL of OpTmizer™ T-Cell Expansion Supplement. In some embodiments, the CTS™ OpTmizer™ T- Cell Expansion Supplement is at a percentage of approximately 2.5% when mixed together with the CTS™ OpTmizer™ T Cell Expansion SFM.

[0070] In some embodiments, the complete expansion medium described herein can contain about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, aboutJ&J No. JBI6843WOPCT 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 5.6%, about 5.7%, about 5.8%, about 5.9%, about 6%, about 6.1%, about 6.2%, about 6.3%, about 6.4%, about 6.5%, about 6.6%, about 6.7%, about 6.8%, about 6.9%, about 7%, about 7.1%, about 7.2%, about 7.3%, about 7.4%, about 7.5%, about 7.6%, about 7.7%, about 7.8%, about 7.9%, about 8%, about 8.1%, about 8.2%, about 8.3%, about 8.4%, about 8.5%, about 8.6%, about 8.7%, about 8.8%, about 8.9%, about 9%, about 9.1%, about 9.2%, about 9.3%, about 9.4%, about 9.5%, about 9.6%, about 9.7%, about 9.8%, about 9.9%, about 10%, or more, Human Type AB Serum. In some embodiments, the complete expansion medium described herein can contain about 5% Human Type AB Serum.

[0071] In some embodiments, the complete expansion medium described herein can contain about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 5.6%, about 5.7%, about 5.8%, about 5.9%, about 6%, about 6.1%, about 6.2%, about 6.3%, about 6.4%, about 6.5%, about 6.6%, about 6.7%, about 6.8%, about 6.9%, about 7%, about 7.1%, about 7.2%, about 7.3%, about 7.4%, about 7.5%, about 7.6%, about 7.7%, about 7.8%, about 7.9%, about 8%, about 8.1%, about 8.2%, about 8.3%, about 8.4%, about 8.5%, about 8.6%, about 8.7%, about 8.8%, about 8.9%, about 9%, about 9.1%, about 9.2%, about 9.3%, about 9.4%, about 9.5%, about 9.6%, about 9.7%, about 9.8%, about 9.9%, about 10%, or more, antibiotic-antimycotic (Anti-Anti). In some embodiments, the complete expansion medium described herein can contain about 1% antibiotic- antimycotic (Anti-Anti).J&J No. JBI6843WOPCT

[0072] In some embodiments, the final concentration of recombinant human interleukin-2 (rhIL- 2) within the complete expansion medium of the present disclosure is about 50 IU / mL to about 5000 IU / mL. The concentration of recombinant human interleukin-2 (rhIL-2) can, for example, be about 50 IU / mL to about 4000 IU / mL, about 50 IU / mL to about 3000 IU / mL, about 50 IU / mL to about 2000 IU / mL, about 50 IU / mL to about 1000 IU / mL, about 50 IU / mL to about 500 IU / mL, about 50 IU / mL to about 250 IU / mL, about 100 IU / mL to about 5000 IU / mL, about 100 IU / mL to about 4000 IU / mL, about 100 IU / mL to about 3000 IU / mL, about 100 IU / mL to about 2000 IU / mL, about 100 IU / mL to about 1000 IU / mL, about 100 IU / mL to about 500 IU / mL, about 250 IU / mL to about 5000 IU / mL, about 250 IU / mL to about 4000 IU / mL, about 250 IU / mL to about 3000 IU / mL, about 250 IU / mL to about 2000 IU / mL, about 250 IU / mL to about 1000 IU / mL, about 500 IU / mL to about 5000 IU / mL, about 500 IU / mL to about 4000 IU / mL, about 500 IU / mL to about 3000 IU / mL, about 500 IU / mL to about 2000 IU / mL, about 500 IU / mL to about 1000 IU / mL, about 1000 IU / mL to about 5000 IU / mL, about 1000 IU / mL to about 4000 IU / mL, about 1000 IU / mL to about 3000 IU / mL, about 1000 IU / mL to about 2000 IU / mL, about 2000 IU / mL to about 5000 IU / mL, about 2000 IU / mL to about 4000 IU / mL, about 2000 IU / mL to about 3000 IU / mL, or any concentration in between. In certain embodiments, the concentration of IL-2 is 50, 100, 250, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 IU / mL. In some embodiments, the final concentration of recombinant human interleukin-2 (rhIL-2) within the complete expansion medium of the present disclosure is about 100 IU / mL.

[0073] In certain embodiments, the final concentration of recombinant human interleukin-15 (rhIL-15) within the complete expansion medium of the present disclosure is about 1 ng / mL to about 100 ng / mL. The concentration of IL-15 can be about 1ng / mL to about 90 ng / mL, about 1 ng / mL to about 80 ng / mL, about 1 ng / mL to about 70 ng / mL, about 1 ng / mL to about 60 ng / mL, about 1 ng / mL to about 50 ng / mL, about 1 ng / mL to about 40 ng / mL, about 1 ng / mL to about 30 ng / mL, about 1 ng / mL to about 20 ng / mL, about 1 ng / mL to about 10 ng / mL, about 10 ng / mL to about 100 ng / mL, about 10 ng / mL to about 90 ng / mL, about 10 ng / mL to about 80 ng / mL, about 10 ng / mL to about 70 ng / mL, about 10 ng / mL to about 60 ng / mL, about 10 ng / mL to about 50 ng / mL, about 10 ng / mL to about 40 ng / mL, about 10 ng / mL to about 30 ng / mL, about 10 ng / mL to about 20 ng / mL, about 25 ng / mL to about 100 ng / mL, about 25 ng / mL to about 90 ng / mL, about 25 ng / mL to about 80 ng / mL, about 25 ng / mL to about 70 ng / mL, about 25 ng / mL to about 60 ng / mL, about 25 ng / mL to about 50 ng / mL, about 25 ng / mL to about 40 ng / mL, about 50J&J No. JBI6843WOPCT ng / mL to about 100 ng / mL, about 50 ng / mL to about 90 ng / mL, about 50 ng / mL to about 80 ng / mL, about 50 ng / mL to about 70 ng / mL, about 50 ng / mL to about 60 ng / mL, or any value in between. In some embodiments, the final concentration of recombinant human interleukin-15 (rhIL-15) within the complete expansion medium of the present disclosure is about 10 ng / mL. Chimeric Antigen Receptors (CARs)

[0074] As used herein, the term “chimeric antigen receptor” (CAR) refers to a recombinant polypeptide comprising at least an extracellular domain that binds specifically to an antigen or a target, a transmembrane domain and an intracellular T cell receptor-activating signaling domain. Engagement of the extracellular domain of the CAR with the target antigen on the surface of a target cell results in clustering of the CAR and delivers an activation stimulus to the CAR- containing cell. CARs redirect the specificity of immune effector cells and trigger proliferation, cytokine production, phagocytosis and / or production of molecules that can mediate cell death of the target antigen-expressing cell in a major histocompatibility (MHC)-independent manner.

[0075] As used herein, the term “signal peptide” refers to a leader sequence at the amino- terminus (N-terminus) of a nascent CAR protein, which co-translationally or post- translationally directs the nascent protein to the endoplasmic reticulum and subsequent surface expression.

[0076] As used herein, the term “extracellular antigen binding domain,” “extracellular domain,” or “extracellular ligand binding domain” refers to the part of a CAR that is located outside of the cell membrane and is capable of binding to an antigen, target or ligand.

[0077] As used herein, the term “hinge region” refers to the part of a CAR that connects two adjacent domains of the CAR protein, e.g., the extracellular domain and the transmembrane domain.

[0078] As used herein, the term “transmembrane domain” refers to the portion of a CAR that extends across the cell membrane and anchors the CAR to cell membrane.

[0079] As used herein, the term “intracellular T cell receptor-activating signaling domain,” “cytoplasmic signaling domain,” or “intracellular signaling domain” refers to the part of a CAR that is located inside of the cell membrane and is capable of transducing an effector signal.

[0080] As used herein, the term “stimulatory molecule” refers to a molecule expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of the T cell receptor (TCR) complex in a stimulatory way for at least some aspect of the T cell signaling pathway. Stimulatory molecules comprise two distinct classes of cytoplasmic signalingJ&J No. JBI6843WOPCT sequence, those that initiate antigen-dependent primary activation (referred to as “primary signaling domains”), and those that act in an antigen-independent manner to provide a secondary of co-stimulatory signal (referred to as “co-stimulatory signaling domains”).

[0081] In certain general aspects, provided herein are methods of generating a chimeric antigen receptor (CAR) γδ T cell. The methods can comprise (a) obtaining an isolated γδ T cell of the disclosure; (b) contacting the γδ T cell with a nucleic acid encoding a chimeric antigen receptor (CAR), the CAR comprising (i) an extracellular domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, wherein the CAR optionally further comprises a signal peptide at the amino terminus and a hinge region connecting the extracellular domain and the transmembrane domain, and wherein contacting the γδ T cell with the nucleic acid encoding the CAR generates a CAR γδ T cell.

[0082] In certain embodiments, the extracellular domain comprises an antigen binding domain and / or an antigen binding fragment.

[0083] In certain embodiments, the antigen binding fragment is a Fab, a Fab’, a F(ab’)2, an Fv, a single-chain variable fragment (scFv), a minibody, a diabody, a single- domain antibody (sdAb), a light chain variable domain (VL), or a variable domain (VHH) of a camelid antibody.

[0084] In certain embodiments, the antigen binding domain and / or antigen binding fragment can, for example, specifically bind a tumor antigen. Any suitable tumor antigen for binding by an antibody or antigen binding fragment can be chosen based on the type of tumor and / or cancer exhibited by the subject to be treated.

[0085] In certain embodiments, the extracellular domain of the CAR is preceded by a signal peptide at the amino-terminus. Any suitable signal peptide can be used in the disclosure. The signal peptide can, for example, be derived from a natural, synthetic, semi-synthetic, or recombinant source. Non-limiting examples of signal peptides are a human CD8 a signal peptide, a human CD3 d signal peptide, a human CD3z signal peptide, a human GMCSFR signal peptide, and a human 4-1BB signal peptide, or derivatives thereof. The signal peptide can be cleaved by a signal peptidase during or after completion of translocation of the CAR to generate a mature CAR free of the signal peptide.

[0086] In certain embodiments, the CAR can further comprise a hinge region connecting the extracellular domain and the transmembrane domain. The hinge region functions to move the extracellular domain away from the surface of the engineered immune cell to enable properJ&J No. JBI6843WOPCT cell / cell contact, binding to the target or antigen and activation (Patel et al., Gene Therapy 6:412- 9 (1999)). Any suitable hinge region can be used in a CAR of the disclosure. The hinge region can be derived from a natural, synthetic, semi-synthetic, or recombinant source. In some embodiments, a spacer region may be used in combination with a hinge region. In some embodiments, a spacer region may be used in lieu of a hinge region. In some embodiments, a spacer region can comprise an IgG hinge region, e.g., IgG1 or IgG4, and the CH2-CH3 domain of IgG Fc.

[0087] In some embodiments, a CAR of the disclosure can comprise a transmembrane domain (TMD). Any suitable transmembrane domain can be used in a CAR of the disclosure. The transmembrane domain can be derived from a natural, synthetic, semi-synthetic, or recombinant source. In some embodiments, the transmembrane domain can be a transmembrane domain from a peptide for example, without limitation, a CD8 a peptide, a CD28 peptide, a CD4 peptide, a CD3 z peptide, a CD2 peptide, a 4-1BB peptide, an OX40 peptide, an ICOS peptide, a CTLA-4 peptide, a PD-1 peptide, a LAG-3 peptide, a 2B4 peptide, a BTLA peptide, a GMCSFR peptide, and the like.

[0088] In some embodiments, a CAR of the disclosure comprises an intracellular signaling domain. Any suitable intracellular domain can be used in a CAR of the disclosure. In particular embodiments, the entire intracellular signaling domain can be used. In some embodiments, a truncated portion of the signaling domain that transduces the effector or signal is used. According to embodiments of the disclosure, the intracellular signaling domain generates a signal that promotes an immune effector function of the CAR- containing cell, e.g., a CAR-T cell, including, but not limited to, proliferation, activation, and / or differentiation. In particular embodiments, the signal promotes, e.g., cytolytic activity, helper activity, and / or cytokine secretion of the CAR-T cell. According to some embodiments, the intracellular signaling domain of the CAR comprises a signaling domain of an Fcg receptor (FcgR), an Fce receptor (FceR), an Fca receptor (FcaR), neonatal Fc receptor (FcRn), CD3, CD3z, CD3g, CD3d, CD3e, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247z, CD247h, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-kB, PLC-g, iC3b, C3dg, C3d, and Zap70. According to some embodiments, the intracellular signaling domain is selected from the group consisting of a signaling domain of CD3z, FcRg, FcRb, CD3g, CD3d, CD3e, CD5, CD22, CD79z, CD79b, andJ&J No. JBI6843WOPCT CD6d. In particular embodiments, the intracellular domain is a CD3z or 4-1BB intracellular domain.

[0089] According to particular embodiments, the intracellular signaling domain further comprises one or more co-stimulatory signaling domains. The co-stimulatory domain can, for example, comprise a signaling domain of a peptide selected from: 2B4 / CD244 / SLAMF4, 4- 1BB / TNFSF9 / CD137, B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF-R / TNFRSF13C, BAFF / BLyS / TNFSF13B, BLAME / SLAMF8, BTLA / CD272, CD100 (SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160 (BY55), CD18, CD19, CD2, CD200, CD229 / SLAMF3, CD27 Ligand / TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30 Ligand / TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40 Ligand / TNFSF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD53, CD58 / LFA-3, CD69, CD7, CD8a, CD8b, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDS, CEACAM1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1 / CLEC7A, DNAM1 (CD226), DPPIV / CD26, DR3 / TNFRSF25, EphB6, GADS, Gi24 / VISTA / B7-H5, GITR Ligand / TNFSF18, GITR / TNFRSF18, HLA Class I, HLA- DR, HVEM / TNFRSF14, IA4, ICAM- 1, ICOS / CD278, Ikaros, IL2R b, IL2R g, IL7R a, Integrin a4 / CD49d, Integrin a4b1, Integrin a4b7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNFSF14, LTBR, Ly108, Ly9 (CD229), lymphocyte function associated antigen-1 (LFA-1), Lymphotoxin-a / TNF-b, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), NTB-A / SLAMF6, OX40 Ligand / TNFSF4, OX40 / TNFRSF4, PAG / Cbp, PD-1, PDCD6, PD-L2 / B7-DC, PSGL1, RELT / TNFRSF19L, SELPLG (CD162), SLAM (SLAMF1), SLAM / CD150, SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TACI / TNFRSF13B, TCL1A, TCL1B, TIM-1 / KIM-1 / HAVCR, TIM- 4, TL1A / TNFSF15, TNF RII / TNFRSF1B, TNF-a, TRANCE / RANKL, TSLP, TSLP R, VLA1, and VLA-6. In certain embodiments, the costimulatory domain can be one or more of CD28, 4-1BB (CD137), CD27, OX40, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA- 1), CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, CD83, and a ligand that specifically binds with CD83.J&J No. JBI6843WOPCT Antigen-binding Fragments Antibodies

[0090] The disclosure generally relates to CAR constructs comprising an antigen binding fragment. The antigen binding fragment can, for example, be an antibody or antigen binding fragment thereof that specifically binds a tumor antigen. The antigen binding fragments of the disclosure possess one or more desirable functional properties, including but not limited to high- affinity binding to a tumor antigen, high specificity to a tumor antigen, the ability to stimulate complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC), and / or antibody-dependent cellular-mediated cytotoxicity (ADCC) against cells expressing a tumor antigen, and the ability to inhibit tumor growth in subjects in need thereof and in animal models when administered alone or in combination with other anti-cancer therapies.

[0091] The antigen binding fragment can, for example, be an antibody or antigen binding fragment thereof that specifically binds a tumor antigen. Any suitable tumor antigen for binding by an antibody or antigen binding fragment can be chosen based on the type of tumor and / or cancer exhibited by the subject to be treated. Suitable antigens include, but are not limited to, prostate specific membrane antigen (PSMA), prostate stem cell antigen (PCSA), mesothelin (MSLN), carbonic anhydrase IX (CAIX), B-cell maturation antigen (BCMA or BCM), G-protein coupled receptor family C group 5 member D (GPRC5D), interleukin-1 receptor accessory protein (IL1RAP), delta-like 3 (DLL3), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, epithelial glycoprotein-2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial adhesion molecule (EpCAM), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor a and b (FR a and b), ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2 / ERB2), epidermal growth factor receptor (EGFR), epidermal growth factor receptor vIII (EGFRvIII), ERB3, ERB4, human telomerase reverse transcriptase (hTERT), interleukin- 13 receptor subunit alpha-2 (IL-13R a2), k-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (LICAM), melanoma-associated antigen 1 (melanoma antigen family A1, MAGE-A1), Mucin-16 (Muc-16), Mucin 1 (Muc-1), NKG2D ligands, cancer-testis antigen NY- ESO-1, oncofetal antigen (h5T4), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor R2 (VEGF-R2),J&J No. JBI6843WOPCT Wilms tumor protein (WT-1), type 1 tyrosine-protein kinase transmembrane receptor (ROR1), B7-H3 (CD276), B7-H6 (Nkp30), chondroitin sulfate proteoglycan-4 (CSPG4), DNAX accessory molecule (DNAM-1), ephrin type A receptor 2 (EpHA2), fibroblast associated protein (FAP), Gp100 / HLA-A2, glypican 3 (GPC3), HA-1H, HERK-V, IL-11R a, latent membrane protein (LMP1), neural cell-adhesion molecule (N-CAM / CD56), and trail receptor (TRAIL R).

[0092] As used herein, the term “antibody” is used in a broad sense and includes immunoglobulin or antibody molecules including human, humanized, composite and chimeric antibodies and antibody fragments that are monoclonal or polyclonal. In general, antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. Antibody structures are well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG and IgM), depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Accordingly, the antibodies of the disclosure can be of any of the five major classes or corresponding sub-classes.

[0093] Preferably, the antibodies of the disclosure are IgG1, IgG2, IgG3 or IgG4. Antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains.

[0094] Accordingly, the antibodies of the disclosure can contain a kappa or lambda light chain constant domain. According to particular embodiments, the antibodies of the disclosure include heavy and / or light chain constant regions from rat or human antibodies. In addition to the heavy and light constant domains, antibodies contain an antigen-binding region that is made up of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., complementarity determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2, and HCDR3.

[0095] As used herein, the term an “isolated antibody” refers to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to the specific tumor antigen is substantially free of antibodies that do not bind to the tumor antigen). In addition, an isolated antibody is substantially free of other cellular material and / or chemicals.J&J No. JBI6843WOPCT

[0096] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. The monoclonal antibodies of the disclosure can be made by the hybridoma method, phage display technology, single lymphocyte gene cloning technology, or by recombinant DNA methods. For example, the monoclonal antibodies can be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, such as a transgenic mouse or rat, having a genome comprising a human heavy chain transgene and a light chain transgene.

[0097] As used herein, the term “antigen-binding fragment” refers to an antibody fragment such as, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), a single domain antibody (sdAb), a scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a minibody, a nanobody, a domain antibody, a bivalent domain antibody, a light chain variable domain (VL), a variable domain (VHH) of a camelid antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment binds.

[0098] As used herein, the term “single-chain antibody” refers to a conventional single- chain antibody in the field, which comprises a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to about 20 amino acids (e.g., a linker peptide).

[0099] As used herein, the term “single domain antibody” refers to a conventional single domain antibody in the field, which comprises a heavy chain variable region and a heavy chain constant region or which comprises only a heavy chain variable region.

[0100] As used herein, the term “human antibody” refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide.J&J No. JBI6843WOPCT

[0101] As used herein, the term “humanized antibody” refers to a non-human antibody that is modified to increase the sequence homology to that of a human antibody, such that the antigen-binding properties of the antibody are retained, but its antigenicity in the human body is reduced.

[0102] As used herein, the term “chimeric antibody” refers to an antibody wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. The variable region of both the light and heavy chains often corresponds to the variable region of an antibody derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capability, while the constant regions correspond to the sequences of an antibody derived from another species of mammal (e.g., human) to avoid eliciting an immune response in that species.

[0103] As used herein, the term “multispecific antibody” refers to an antibody that comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In an embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodiment, the first and second epitopes overlap or substantially overlap. In an embodiment, the first and second epitopes do not overlap or do not substantially overlap. In an embodiment, the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In an embodiment, a multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In an embodiment, a multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0104] As used herein, the term “bispecific antibody” refers to a multispecific antibody that binds no more than two epitopes or two antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In an embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodiment, the first and second epitopes overlap or substantially overlap. In an embodiment, the first and second epitopes are on differentJ&J No. JBI6843WOPCT antigens, e.g., the different proteins (or different subunits of a multimeric protein). In an embodiment, a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope. In an embodiment, a bispecific antibody comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In an embodiment, a bispecific antibody comprises a scFv, or fragment thereof, having binding specificity for a first epitope, and a scFv, or fragment thereof, having binding specificity for a second epitope.

[0105] As used herein, an antigen binding domain or antigen binding fragment that “specifically binds to a tumor antigen” refers to an antigen binding domain or antigen binding fragment that binds a tumor antigen, with a KD of 1×10-7 M or less, preferably 1×10-8M or less, more preferably 5×10-9M or less, 1×10-9M or less, 5×10-10M or less, or 1×10-10M or less. The term “KD” refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods in the art in view of the present disclosure. For example, the KD of an antigen binding domain or antigen binding fragment can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a Biacore® system, or by using bio-layer interferometry technology, such as an Octet RED96 system.

[0106] The smaller the value of the KDof an antigen binding domain or antigen binding fragment, the higher affinity that the antigen binding domain or antigen binding fragment binds to a target antigen.

[0107] According to particular aspects, the disclosure relates to a CAR construct comprising an antigen binding fragment, wherein the antigen binding fragment is an antibody or antigen binding fragment that specifically binds a tumor antigen. The antibody or antigen binding fragment can, for example, be a Fab, a Fab’, a F(ab’)2, an Fv, a single-chain variable fragment (scFv), a minibody, a diabody, a single-domain antibody (sdAb), a light chain variable domain (VL), or a variable domain (VHH) of a camelid antibody. Polynucleotides, Vectors, and Host cells

[0108] In another general aspect, the disclosure relates to an isolated nucleic acid encoding a chimeric antigen receptor (CAR) of the disclosure. It will be appreciated by thoseJ&J No. JBI6843WOPCT skilled in the art that the coding sequence of a CAR can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein.

[0109] Accordingly, it will be understood by those skilled in the art that nucleic acid sequences encoding CARs of the disclosure can be altered without changing the amino acid sequences of the proteins.

[0110] In another general aspect, the disclosure relates to a vector comprising a CAR of the disclosure. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. A number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of a CAR in the cell. Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments of the disclosure. EMBODIMENTS

[0111] This invention provides the following non-limiting embodiments. As contemplated herein, any of embodiments A1-A18 may be combined and / or applied to the claims described herein.

[0112] In one set of embodiments, provided are: A1. A method of generation of chimeric antigen receptor (CAR)-expressing CD3+ gamma delta (γδ) T cells, said method comprising one or more of the following steps: a) seeding an enriched γδ T cell fraction obtained from peripheral blood mononuclear cells (PBMCs) at about 1x105to about 2x106cells / well on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in an expansion medium; b) culturing the CD3+ γδ T cells on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in the expansion medium for about 1.5 to about 2.5 days, preferably 2 days, to produce activated CD3+ γδ T cells; c) collecting the activated CD3+ γδ T cells produced in step (b) and re-plating the activated CD3+ γδ T cells at about 1x105to about 2x106cells / well on a second tissue culture vessel in the absence of anti-CD3 antibodies;J&J No. JBI6843WOPCT d) adding a chimeric antigen receptor (CAR)-encoding vector to the activated CD3+ γδ T cells of step (c) to achieve a multiplicity of infection (MOI) of 0.65-2, preferably a MOI of 1; e) adding Vectofusin®-1 at about a dilution of 1:50 to about a dilution of 1:200, preferably about a dilution of 1:100, to the mixture of activated CD3+ γδ T cells and CAR- encoding vector; f) culturing the mixture of step (e) for about 0.5 to about 1.5 days, preferably about 1 day to produce CAR-expressing CD3+ γδ T cells; g) adding additional expansion medium to the CAR-expressing CD3+ γδ T cells to achieve a total culture volume which is about double the culture volume of step (f); h) after about 1.5 to about 2.5 days, preferably 2 days, transferring the CAR-expressing CD3+ γδ T cells to a third tissue culture vessel and culturing for about 3.5 to about 4.5 days, preferably 4 days; and i) optionally, harvesting the CAR-expressing CD3+ γδ T cells or introducing the CAR- expressing CD3+ γδ T cells into a sorter, enrichment column, or a bioreactor. A2. The method of item A1, further comprising i) harvesting the CAR-expressing CD3+ γδ T cells and / or introducing the CAR-expressing CD3+ γδ T cells into a sorter, enrichment column, or a bioreactor. A3. The method of item A2, wherein the harvesting is by centrifugation. A4. The method of any one of items A1-A3, wherein the third tissue culture vessel is larger than the second tissue culture vessel or the first tissue culture vessel. A5. The method of any one of items A1-A4, wherein the CD3+ γδ T cells in the enriched γδ T cell fraction are primary CD3+ γδ T cells. A6. The method of any one of items A1-A5, wherein the anti-CD3 mAb is derived from clone OKT3.J&J No. JBI6843WOPCT A7. The method of any one of items A1-A6, wherein the collecting is by centrifugation, fluorescence activated cell sorting (FACS) or enrichment through a column. A8. The method of any one of items A1-A7, wherein the vector is a viral vector. A9. The method of item A8, wherein the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, or a vaccinia virus vector. A10. The recombinant vector of item A9, wherein the viral vector is a retroviral vector. A11. The recombinant vector of any one of items A1-A7, wherein the vector is a non-viral vector. 12. The method of any one of items A1-A11, wherein the method further comprises one or more of the following steps: (1) counting the total number of the activated CD3+ γδ T cells produced in step (c) or the CAR-expressing CD3+ γδ T cells produced in step (h); (2) assaying for one or more cell surface markers on the activated CD3+ γδ T cells produced in step (c) or the CAR-expressing CD3+ γδ T cells produced in step (h); and (3) calculating an expansion efficacy of the CAR-expressing CD3+ γδ T cells produced in step (h) using results obtained in steps (1) and (2). A13. The method of item A12, wherein the one or more cell surface markers comprises CD3, γδ T cell receptor (TCR), Vδ1, and / or Vδ2. A14. The method of item A12 or item A13, wherein the assay for the one or more cell surface markers is performed via flow cytometry. A15. The method of any one of items A1-A14, wherein the final concentration of Vectofusin®- 1 in the total culture volume is 10 µg / mL.J&J No. JBI6843WOPCT A16. The method of any one of items A1-A15, wherein the expansion medium comprises CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ OpTmizer™ T-Cell Expansion Supplement, about 2nM (1x) GlutaMAX™ Supplement, about 5% Human Type AB Serum, about 1% antibiotic- antimycotic (anti-anti), recombinant human interleukin-2 (rhIL-2) at a concentration of about 100 IU / mL, and recombinant human interleukin-15 (rhIL-15) at a concentration of about 10 ng / mL. A17. Chimeric antigen receptor (CAR)-expressing CD3+ gamma delta (γδ) T cells obtained by a method of generation from γδ T cells, said method comprising one or more of the following steps: a) a means for seeding an enriched γδ T cell fraction obtained from peripheral blood mononuclear cells (PBMCs) at about 1x105to about 2x106cells / well on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in an expansion medium; b) a means for culturing the CD3+ γδ T cells on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in the expansion medium for about 1.5 to about 2.5 days, preferably 2 days, to produce activated CD3+ γδ T cells; c) a means for collecting the activated CD3+ γδ T cells produced in step (b) and a means for re-plating the activated CD3+ γδ T cells at about 1x105to about 2x106cells / well on a second tissue culture vessel in the absence of anti-CD3 antibodies; d) a means for adding a chimeric antigen receptor (CAR)-encoding vector to the activated CD3+ γδ T cells of step (c) to achieve a multiplicity of infection (MOI) of 0.65-2, preferably a MOI of 1; e) a means for adding Vectofusin®-1 at about a dilution of 1:50 to about a dilution of 1:200, preferably about a dilution of 1:100, to the mixture of activated CD3+ γδ T cells and CAR-encoding vector; f) a means for culturing the mixture of step (e) for about 0.5 to about 1.5 days, preferably about 1 day to produce CAR-expressing CD3+ γδ T cells; g) a means for adding additional expansion medium to the CAR-expressing CD3+ γδ T cells to achieve a total culture volume which is about double the culture volume of step (f); h) after about 1.5 to about 2.5 days, preferably 2 days, a means for transferring the CAR- expressing CD3+ γδ T cells to a third tissue culture vessel and culturing for about 3.5 to about 4.5 days, preferably 4 days; andJ&J No. JBI6843WOPCT i) optionally, providing a means for harvesting the CAR-expressing CD3+ γδ T cells or a means for introducing the CAR-expressing CD3+ γδ T cells into a sorter, enrichment column, or a bioreactor. A18. A system for performing a method of generating chimeric antigen receptor (CAR)- expressing CD3+ gamma delta (γδ) T cells, the system comprising: a) a means for seeding an enriched γδ T cell fraction obtained from peripheral blood mononuclear cells (PBMCs) at about 1x105to about 2x106cells / well on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in an expansion medium; b) a means for culturing the CD3+ γδ T cells on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in the expansion medium for about 1.5 to about 2.5 days, preferably 2 days, to produce activated CD3+ γδ T cells; c) a means for collecting the activated CD3+ γδ T cells produced in step (b) and a means for re-plating the activated CD3+ γδ T cells at about 1x105to about 2x106cells / well on a second tissue culture vessel in the absence of anti-CD3 antibodies; d) a means for adding a chimeric antigen receptor (CAR)-encoding vector to the activated CD3+ γδ T cells of step (c) to achieve a multiplicity of infection (MOI) of 0.65-2, preferably a MOI of 1; e) a means for adding Vectofusin®-1 at about a dilution of 1:50 to about a dilution of 1:200, preferably about a dilution of 1:100, to the mixture of activated CD3+ γδ T cells and CAR-encoding vector; f) a means for culturing the mixture of step (e) for about 0.5 to about 1.5 days, preferably about 1 day to produce CAR-expressing CD3+ γδ T cells; g) a means for adding additional expansion medium to the CAR-expressing CD3+ γδ T cells to achieve a total culture volume which is about double the culture volume of step (f); h) after about 1.5 to about 2.5 days, preferably 2 days, a means for transferring the CAR- expressing CD3+ γδ T cells to a third tissue culture vessel and culturing for about 3.5 to about 4.5 days, preferably 4 days; and i) optionally, providing a means for harvesting the CAR-expressing CD3+ γδ T cells or a means for introducing the CAR-expressing CD3+ γδ T cells into a sorter, enrichment column, or a bioreactor.J&J No. JBI6843WOPCT A19. A method of generation of chimeric antigen receptor (CAR)-expressing CD3+ gamma delta (γδ) T cells, said method comprising the steps of: a) seeding an enriched γδ T cell fraction obtained from peripheral blood mononuclear cells (PBMCs) at about 1x105to about 2x106cells / well on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in an expansion medium; b) culturing the CD3+ γδ T cells on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in the expansion medium for about 1.5 to about 2.5 days, preferably 2 days, to produce activated CD3+ γδ T cells; c) collecting the activated CD3+ γδ T cells produced in step (b) and re-plating the activated CD3+ γδ T cells at about 1x105to about 2x106cells / well on a second tissue culture vessel in the absence of anti-CD3 antibodies; d) adding a chimeric antigen receptor (CAR)-encoding vector to the activated CD3+ γδ T cells of step (c) to achieve a multiplicity of infection (MOI) of 0.65-2, preferably a MOI of 1; e) adding Vectofusin®-1 at about a dilution of 1:50 to about a dilution of 1:200, preferably about a dilution of 1:100, to the mixture of activated CD3+ γδ T cells and CAR- encoding vector; f) culturing the mixture of step (e) for about 0.5 to about 1.5 days, preferably about 1 day to produce CAR-expressing CD3+ γδ T cells; g) adding additional expansion medium to the CAR-expressing CD3+ γδ T cells to achieve a total culture volume which is about double the culture volume of step (f); h) after about 1.5 to about 2.5 days, preferably 2 days, transferring the CAR-expressing CD3+ γδ T cells to a third tissue culture vessel and culturing for about 3.5 to about 4.5 days, preferably 4 days; and i) optionally, harvesting the CAR-expressing CD3+ γδ T cells or introducing the CAR- expressing CD3+ γδ T cells into a sorter, enrichment column, or a bioreactor. EXAMPLES

[0113] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.J&J No. JBI6843WOPCT Example 1. Protocol for CAR T generation using primary γδ T cells

[0114] The scope of the procedure described in the present Example was to provide detailed written instructions on how to transduce primary γδ T cells and generate CAR γδ T cells. This protocol was intended for starting with high purity CD3+ γδ TCR+ T cells (Figure 1). Materials Primary γδ T cells

[0115] Primary γδ T cells were sourced such that αβ T cell contamination was <10%. See Figure 1, for reference. Table 1. CAR γδ T cell activation and transduction / expansion reagents ReagentVendor CatalogStorage instructions number CTS™ OpTmizer™ T Cell ThermoFisher A1048501 Store at 4°C Expansion SFM Human AB Serum Valley HP1022HI Store at -20°C Biomedical GlutaMAX™ Supplement ThermoFisher 35050061 Store at 4°C (100x) Antibiotic-Antimycotic ThermoFisher 15240062 Store at -20°C. Store (100X) at 4°C post thaw for 1 month.] Anti-human CD3 (Clone: BioXCell BE0001-2- Store at 4°C OKT3) 5MG Vectofusin®-1Miltenyi130-111- Upon reconstitution Biotec 163 store aliquots at - 80°C. Avoid repeated freeze-thaw cycles *Recombinant human Miltenyi 130-097- Upon reconstitution interleukin-2 (rhIL-2) IS, Biotec 748 store aliquots at - premium grade 20°C or below. Stock dilution 1000 international unit (IU) / µL *Recombinant human Miltenyi 130-095- Upon reconstitution interleukin-15 (rhIL-15), Biotec 765 store aliquots at - premium grade 20°C or below. Stock dilution 0.1mg / mLJ&J No. JBI6843WOPCT 1X DPBS (Dulbecco’s Gibco 14190250 Store at room Phosphate Buffered Saline), temperature no calcium, no magnesium Orflo Moxi Cyte ViabilityOrflo MXA055Store at 4°CReagent CryoStor® CS10 Cell STEMCELL07930Store at 4°CFreezing Medium (Animal Technologies component-free, defined cryopreservation medium with 10% DMSO) EasySep™ Human STEMCELL19255Store at 4°CGamma / Delta T Cell Technologies Isolation Kit Table 2. CAR γδ T cells flow cytometry analysis reagents ReagentVendor Catalog number Storageinstructions autoMACS (magnetic-Miltenyi Biotec130-091-221 Store at 4°Cactivated cell sorting)® Running Buffer LIVE / DEAD™ Fixable Invitrogen L10119 Store at - Near-IR Dead Cell Stain 20°C, in the Kit dark UltraComp eBeads™ ThermoFisher 01-2222-41 Store at 4°C Compensation Beads ArC™ Amine Reactive ThermoFisher A10346 Store at 4°C Compensation Bead Kit (for use with LIVE / DEAD™ Fixable dead cell stain kits) Alexa Fluor® 700 anti- BioLegend 300424 Store at 4°C human CD3 Antibody, Clone UCHT1 TCR V delta 1 Thermo Scientific TCR2730 Store at 4°C Monoclonal Antibody, FITC Brilliant Violet 421™ BioLegend 331428 Store at 4°C anti-human TCR Vδ2 Antibody PerCP / Cyanine5.5 anti- BioLegend 331224 Store at 4°C human TCR γ / δ AntibodyJ&J No. JBI6843WOPCT PE-Labeled HumanACROBiosystemsPSA-HP2Q3-Store at 4°C PSMA / FOLH1 Protein, 200tests His Tag APC anti-human TCR BioLegend 306718 Store at 4°C α / β Antibody Table 3. Laboratory supplies ItemVendor Catalog numberMoxi GO Cassette Type S+MoxiMXC030Pipette Tips RT LTS 20µL F 960A / 10Rainin30389225Pipette Tips RT LTS 200µL F 960A / 10Rainin30389239Pipette Tips RT LTS 1000µL F 768A / 8Rainin30389212Fisherbrand™ Sterile Polystyrene DisposableThermoFisher13-676-10H,Serological Pipets with Magnifier Stripe 13-678-11E, (various sizes) 13-678-11, 13-678-11F Corning™ Costar™ 96-Well, Clear RoundCorning3799Bottom TC-treated Microplate, Individually Wrapped, with Lid, Sterile Corning™ Costar™ 96-well Clear Flat BottomCorning3596Polystyrene TC-treated Microplates, Individually Wrapped, with Lid, Sterile Corning™ Costar™ 48-well Clear TC-treatedCorning3548Multiple Well Plates, Individually Wrapped, Sterile Corning™ Costar™ 24-well Clear TC-treatedCorning3526Multiple Well Plates, Individually Wrapped, SterileG-Rex®24 Well PlateWilson Wolf80192MFalcon® 15 mL Polystyrene Centrifuge Tube,Corning352095Conical Bottom, with Dome Seal Screw Cap, SterileJ&J No. JBI6843WOPCT Table 4. Equipment EquipmentModel / Catalognumber Moxi GO II Cell CounterMXG102BD LSRFortessa™ - Inverted microscope - Tissue culture incubator - Biosafety cabinet (BSC) - Water bath- Centrifuge- Fridge- Freezer (-20 °C)- Liquid nitrogen (LN2) - storage Table 5. Software SoftwareVendor VersionFlowJo™BD 10.8.1PrismGraphPad Software 8.3.0Procedure Reagent preparation a. Complete expansion medium

[0116] Complete expansion medium was prepared prior to the start of each expansion. Expansion medium could be stored for no longer than 1 week at 4°C. The complete medium was prepared as needed, rather than wasting large preparations.

[0117] Complete expansion medium contained: i. CTS™ OpTmizer™ T Cell Expansion SFM ii. CTS™ OpTmizer™ T-Cell Expansion Supplement iii. GlutaMAX™ Supplement 1xJ&J No. JBI6843WOPCT iv. Human AB Serum, 5% v. Antibiotic-antimycotic (Anti-Anti), 1% vi. rhIL-2, 100 IU / mL vii. rhIL-15, 10 ng / mL

[0118] CTS™ OpTmizer™ T-Cell Expansion Basal Medium and CTS™ OpTmizer™ T- Cell Expansion Supplement were combined according to manufacturer instructions (~2.5% Expansion Supplement) prior to the addition of the remaining components. b. Cytokines

[0119] All stock cytokines were aliquoted and stored at -80°C. Once cytokines were thawed, they were stored at 4 °C and consumed within 7 days. i. Preparation of rhIL-2 stock: Lyophilized recombinant human interleukin-2 (rhIL-2), premium grade, was stored at -20 °C until reconstituted. Reconstitution was performed with deionized sterile-filtered water to a final biological activity of 1000 IU / μL. Upon reconstitution, aliquots of 50 μL were stored at -80°C. Repeated freeze-thaw cycles were avoided. ii. Preparation of rhIL-15 stock: Lyophilized rhIL-15, premium grade, was stored at -80°C. Reconstitution was performed with deionized sterile-filtered water to a final concentration of 100 μg / mL. Upon reconstitution, aliquots of 50 μL were stored at -80°C. Repeated freeze-thaw cycles were avoided. c. Vectofusin®-1 reconstitution i. For reconstitution of the lyophilized peptide, the vial was taken from –20°C and warmed up to room temperature (warming was conducted in a biosafety hood for 15 minutes). The vial was not opened by removing the rubber plug. ii. To dissolve the Vectofusin®-1, a sterile syringe (2 mL) was filled with 1000 μL of sterile water. iii. The water was slowly injected with a sterile needle through the center of the rubber plug into the vial containing the lyophilized Vectofusin®-1.J&J No. JBI6843WOPCT iv. The solution was vortexed to completely dissolve the lyophilized peptide. The concentration of the stock solution of Vectofusin®-1 was 1 mg / mL. v. The rubber plug was removed and the stock solution was aspirated with a pipette. vi. To avoid repeated freeze-thaw cycles, working aliquots were prepared from the stock solution. vii. The working aliquots were stored at –80°C. Thawing isolated γδ T cells i. The Complete expansion medium was warmed completely in a 37°C water bath. ii. The cells were quickly thawed in a 37°C water bath by gently swirling the vial. This took approximately 1-2 minutes. The cells were not vortexed. iii. The vial was transferred to a biosafety hood and the outside of the vial wiped with 70% ethanol or isopropanol. iv. 0.5-1 mL of the pre-warmed Complete expansion medium was slowly added to the cryotube. The cells were resuspended and transferred from the cryotube to a 15 mL tube. v. The cryotube was rinsed with 1 mL of Complete expansion medium and transferred to the 15 mL tube. The tube was filled with Complete expansion medium to the 10 mL mark. vi. A 10 µL aliquot of cells was removed for counting on a Moxi FlowTMFlow Cytometer. vii. The cells were washed by centrifugation for 5 minutes at 300 x g. viii. The supernatant was discarded and the tube was tapped to disrupt the pellet. The cells were resuspended in 1 mL of Complete expansion medium by slowly pipetting up and down. The cell concentration was adjusted to 1E6 / mL using pre-warmed Complete expansion medium. The cells were then ready for use in downstream applications, e.g., activation. Anti-CD3 mAb based activation and CAR retroviral transduction

[0120] For PBMC-derived γδ T cells, the tested starting vessel size was a tissue culture (TC)-treated flat-bottom 96-well plate (plated at 1x105cells / well). Wells were coated with 50 μL of Phosphate Buffered Saline (PBS) diluted anti-CD3 (clone OKT3) mAb per well (96-well plate)J&J No. JBI6843WOPCT at 4°C overnight. The vessel size and mAb volume were scaled up accordingly (e.g., if plating was 2x105cells, a 48-well plate was used and coated with 100 μL of mAb per well; if plating was 5x105-1x106cells, a 24-well plate was used and coated with 500 μL of mAb per well).

[0121] An enriched γδ T cell fraction obtained from PBMCs using a TCR γ / δ+ T Cell Isolation Kit could contain multiple subsets of γδ T cells, including Vγ9Vδ2 T cells, Vδ1 T cells, as well as minor populations of Vδ1- Vδ2- γδ TCR+ T cells (potentially Vδ3, Vδ5 T cells).

[0122] As an alternative strategy for plate coating, the plate could be incubated for 2 hours at 37°C for a same-day experiment. a. Monoclonal antibody (mAb) dilutions and coating were performed in sterile conditions (in a biosafety cabinet (BSC) and sealed using adhesive film for microplates during coating incubation). b. The plate size needed for expansion was determined based on the available number of cells. Recommendations for selecting plate size: c. CD3 (clone OKT3) mAb stock was diluted to a final concentration of 10 μg / mL in 1X DPBS (Dulbecco’s Phosphate Buffered Saline). d. The plates were coated with mAb on Day -1 of activation by adding an appropriate volume of the mAb into the chosen vessel and sealing the plate with adhesive film. The plates were incubated with mAb at 4°C overnight. Use of edge wells was avoided. e. On Day 0 of activation, the mAb solution was removed from the plate(s) and washed 3 times with 1X DPBS. PBMC-derived γδ T cells were immediately plated at 1E6 / mL on mAb-coated plates using the suggested cell densities set forth in Table 6 below. Table 6. Suggested cell densities for plating of PBMC-derived γδ T cells Cell number to be plated Plate size PBMC-derived Medium γδ T cells / well volume, µL 96-well flat bottom plate1x105 100 48-well flat bottom plate2x105 200J&J No. JBI6843WOPCT f. On day 2 (2 days post activation), activated γδ T cells were counted and collected. The cell concentration was adjusted to the desired cell concentration (e.g., 1x106 / mL) by adding Complete expansion medium (>1x106 / mL) or spinning down [300g; 5 mins] to remove medium (<1x106 / mL). Cells were replated at the desired cell concentration, e.g., 1x105cells / well (100 µl) in a new TC-treated 96-well flat bottom plate. g. The CAR retrovirus (RV) volume needed to achieve a multiplicity of infection (MOI) = 1 was calculated. h. The RV was thawed in a Biosafety Level Two Plus (BSL-2+) BSC at room temperature and a sufficient volume of RV was added such that a MOI = 1 was achieved. i. An aliquot of Vectofusin®-1 was thawed at room temperature and vortexed thoroughly before use. The required final concentration of Vectofusin®-1 for transduction was 10 µg / mL in the total culture volume. j. The Vectofusin®-1 (1:100) was added to the mixture of cells and RV. One well was left as an untransduced control. No virus or Vectofusin®-1 was added to this control. k. If multiple wells were being transduced, the sample was pipetted up and down using a multichannel pipette. The transduction plate was re-incubated using an incubator set to 37°C and 5% CO2. l. On day 3 (1 day post CAR RV transduction), fresh Complete expansion medium was added to double the volume. • If ≥50µL of RV was utilized (i.e., a larger volume of viral supernatant was added), the plate was spun down [300 x g; 5 mins] and a volume of supernatant equivalent to the volume that was added with the RV was removed. Then fresh Complete expansion medium was added to double the well volume. As a non-limiting example: if 75 µL of RV was added during transduction, the plate was spun down, 75 µL of supernatant was removed (restoring the well back to ~100 µL) and ~100µL of fresh, pre-warmed Complete expansion medium was added for a final well volume of ~200 µL. m. On day 5, (3 days post CAR RV transduction), cells were transferred to a larger vessel, e.g., a 24-well plate, using 2 mL of Complete expansion medium or a 24-well G-Rex® using 5 mL of Complete expansion medium, as needed.J&J No. JBI6843WOPCT n. On day 9, (7 days post CAR RV transduction), CAR γδ T cells were ready to be harvested and used for functional assays or cryopreserved using CryoStor® CS10 freezing medium.

[0123] A graphical representation of an exemplary CAR design which may be used in accordance with the present disclosure is depicted in Figure 3. Cryopreservation of CAR γδ T cells in CryoStor® CS10 freezing medium

[0124] Freezing CAR γδ T cells at 10E6 / mL of CryoStor® CS10 was recommended, using 0.5mL CryoStor® CS10 for less than 5E6 cells per vial. a. The γδ T cells for cryopreservation were counted and the number of vials for cryopreservation was calculated [total cell count ÷ size of preferred vial (e.g., 5E6,10E6 cells) = number of vials]. b. Labels were printed or written with the appropriate vial and donor information on each cryogenic vial. c. The cryogenic vials were placed in the cryogenic vial holder and placed in a 4°C refrigerator for at least 30 minutes. d. The volume of freezing medium needed was calculated and a bottle of CryoStor® CS10 was chilled on ice in the BSC. e. The cells were spun down in 50 mL conical tubes at 400 x g for 10 minutes at 4°C. f. The supernatant was discarded, the cell pellet was loosened by gently tapping the tube, the tube was sprayed with 70% alcohol, and chilled on ice. g. The cryogenic vials were removed from the 4°C refrigerator, sprayed with 70% alcohol, and transferred to the BSC. h. The cell pellet was resuspended with the appropriate amount of CryoStor® CS10 freezing medium. i. The suspension was mixed well by gently pipetting up and down to ensure an even concentration solution of the cells and CryoStor® CS10 freezing medium. j. The cell solution was aliquoted into each cryogenic vial and the cryogenic vials were placed into designated freezing containers. k. The freezing container (e.g., Cool Cell®) was transferred to a -80°C freezer overnight. l. The next day, the cryogenic vials were transferred to a liquid nitrogen (LN2) tank. • Long-term storage at -80˚C was not recommended.J&J No. JBI6843WOPCT Flow cytometry analysis Cell surface markers staining i. Antibody staining mix, single color controls, and compensation controls (using beads) were prepared. ii. The Staining mix per 100 µL reaction contained: 1:100 each surface antigen antibody; 1:1000 Near-IR viability dye; and autoMACS® Running Buffer, to bring volume up to 100 µL. The solution was kept on ice in the dark. Compensation controls were set up on beads and live / dead compensation were set up on single stained ArC amine beads. iii. The cell number was determined and it was planned to stain ~1x105cells per condition per panel. iv. The sample was spun down at 400 x g for 3 minutes and the supernatant removed. v. 100 µL of the antibody staining mix was added to the cell pellet. The sample was gently pipetted up and down to create a single-cell suspension, which was incubated at 4°C in the dark for 30 minutes (e.g., in a refrigerator). vi. The sample was spun down at 400 x g for 3 minutes, the supernatant removed and the cells washed by adding autoMACS® Running Buffer. The washing step was repeated 2 times. vii. The samples were stained in 150 µL autoMACS® Running Buffer and were then ready for multicolor flow cytometry. Example data were analyzed using FlowJo™ (Figure 2) and plotted using GraphPad Prism software. Data analyses Gating strategy

[0125] For a general gating strategy, refer to Figure 2.J&J No. JBI6843WOPCT Expansion fold i. To calculate expansion efficiency, the following parameters were required for baseline (Day 2 timepoint prior to RV transduction; Day 2 post activation) and for expanded PBMC- derived γδ T cells (Day 9; 7 days post transduction): 1) Total cell count (e.g. Moxi GO II counts) 2) Cell viability (based on Flow cytometry frequency values) 3) CD3 purity (based on Flow cytometry frequency values) 4) γδ TCR cell purity (based on Flow cytometry frequency values) ii. The number of viable CD3+ γδ TCR+ T cells was calculated in the starting population. The total Moxi GO II cell count (or similar counting machine known to a person of ordinary skill in the art) was used as a reference to ensure accuracy of cell counting. • The transduced CAR γδ T cells were expanded roughly 9-fold (with a range of approximately 3- to 14-fold expansion) based on experience generating CAR γδ T cells from 5 different healthy donors. Example 2. Materials and methods for evaluating cytotoxicity activity of prostate-specific membrane antigen (PSMA) CAR γδ T cells with different activators

[0126] In order to evaluate the cytotoxicity activity of PSMA CAR γδ T cells with different activators, the xCELLigence real-time cell analysis (RTCA) system for immune cell- mediated cytolysis of target cells was used.

[0127] Primary human γδ T cells were isolated from healthy donors following leukapheresis by negative selection for γδ TCR+ cells. Frozen γδ T cells were thawed and cultured in OpTmizer medium containing 5% human AB serum, interleukin-2 (IL-2 ; Miltenyi) at 100 IU / ml and interleukin-15 (IL-15; Miltenyi) at 10 ng / ml and were activated by 3 different activators respectively, including 10 µg / ml CD3 monoclonal antibody (mAb) (Clone: OKT3, BioXCell),^ Vδ2 (Clone: REA771, custom reagent from Miltenyi Biotec) monoclonal antibody (mAb) for 2 days, and Dynabeads Human T-Expander CD3 / CD28 (Thermo Fisher Scientific) for 1 day at bead to T cell ratio of 3:1. Post activation, human γδ T cells were transduced in the presence of 10 μg / ml VectoFusin-1. Briefly, 1x105γδ T cells were transduced at a multiplicity of infection (MOI) of 1 with the prostate-specific membrane antigen (PSMA) CAR retrovirus and expanded for 2^weeks. Fresh media containing IL-2 and IL-15 was added every 2–3^days, and a cell density of 0.5^×^106^to 1^×^106^cells / ml was maintained for the duration of culture. PSMAJ&J No. JBI6843WOPCT CAR expression was checked by using R-Phycoerythrin (PE)-Labeled Human PSMA (Acro Biosystems) on CD3+ γδ TCR+ cells.^

[0128] CAR engineered γδ T cells were harvested 2 weeks post expansion, and then used for functional analysis using the xCELLigence real-time cell analysis (RTCA) system. PSMA expressing prostate cancer cell line C4-2B were adjusted to a concentration of 2x105^cells / mL (2x104 / well) in RPMI containing 10% FBS complete medium (R10) and seeded to adhere to xCELLigence 96-well E-Plate (Agilent). After 24^hours, the effector CAR γδ T cells generated by stimulating with 3 different activators added at an effector to target cell (E:T) ratio of approximately 1:3 in a total volume of 200^μl. The target cell proliferation was measured as a change in relative impedance which was termed normalized cell index (CI). The CAR γδ T cell mediated cytotoxicity was then monitored by measuring changes in impedance as normalized CI values recorded by the xCELLigence RTCA device. Untransduced (UTD) γδ T cells activated and expanded by Dynabeads Human T-Expander CD3 / CD28 were used as a negative control for CAR expression flow cytometric gating and in cytotoxicity assays. Example 3. CAR expression and cytotoxicity of human primary γδ T cells

[0129] Primary human γδ T cells were transduced with prostate-specific membrane antigen (PSMA) CAR γ-retrovirus (MOI = 1). The purity of γδ T cells was determined by staining of γδ TCR on CD3+ T cells using flow cytometry (Figure 4A, left) and CAR expression was determined by R-Phycoerythrin (PE)-labeled human PSMA (Acro Biosystems) on CD3+ γδ TCR+ cells 2 weeks post transduction (Figure 4A, right). The percentage of the purity of γδ T cells and CAR expression (% positive) were analyzed. The data showed that CAR γδ T cells maintained more than 95% purity and different activators showed similar CAR expression levels on γδ T cells. All CAR T cells were normalized to the equal CAR+ T cells for subsequent functional assays.

[0130] PSMA expressing target cells (C4-2B cells) were seeded to a xCELLigence 96- well E-Plate (Agilent) to adhere. The target cell proliferation was measured as a change in relative impedance which was termed cell index (Figure 4B). After 24 hours, the effector CAR γδ T cells generated by 3 different activators (Dynabeads, CD3 (clone OKT3) or Vδ2 monoclonal antibody (mAb) (clone REA771)) were added at a ratio of effector to target cell (E:T) of approximately 1:3. Mean values of the normalized cell index were plotted. When T cellsJ&J No. JBI6843WOPCT were added to the target cells, PSMA CAR γδ T cells generated by CD3 (clone OKT3) or Vδ2 monoclonal antibody (mAb) (clone REA771) activation induced more efficient cell cytolysis and a greater subsequent progressive decrease in normalized cell index as compared to CAR γδ T cells generated by activation with Dynabeads Human T-Expander CD3 / CD28. Tumor cells (C4- 2B cells) alone showed rapid proliferation and a subsequent progressive increase in normalized Cell Index. Similarly, target cells (C4-2B cells) were not lysed by negative control untransduced (UTD) γδ T cells. Example 4. Activation and CAR transduction efficiency on γδ T cells

[0131] To evaluate the activation and CAR transduction efficiency on γδ T cells using different clones of anti-CD3 mAbs, the activation markers CD25 and CD69 were examined two days after activation (before transduction), and CAR expression was examined six and twelve days after transduction.

[0132] Primary human γδ T cells were isolated from healthy donors following leukapheresis by negative selection for γδ TCR+ cells. Frozen γδ T cells were thawed and cultured in Complete expansion medium and were activated by 3 different clones of anti-CD3 mAb, including OKT3 (Bio X Cell, CATALOG#BE0001-2);UCHT1 (Leu-4) (T3))( Bio X Cell; CATALOG# BE0231) and HIT3a (eBioscience; CATALOG# 16-0039-81) at 2.5, 5 and 10μg / ml (pre-coated 24-well plate, 4oC, 24hours) for 2 days. Post activation, human γδ T cells were transduced in the presence of 10ug / ml Vectofusin-1. Briefly, 1x105^γδ T cells were transduced at a multiplicity of infection (MOI) of 1 with the anti-Prostate-specific membrane antigen (PSMA) CAR retrovirus and expanded for up to 2^weeks. Fresh media containing IL-2 and IL-15 was added every 2–3^days, and a cell density of 0.5^×^106^to 1^×^106^cells / ml was maintained for the duration of culture. PSMA CAR expression was quantified using Alexa Fluor 647-labeled anti- linker (linker used between VH and VL of scFv) antibody (internal reagent) on CD3+ γδ TCR+ cells.^

[0133] Cell labeling protocol using CellTrace™ Violet Cell Proliferation Kit, for flow cytometry (Thermo Fisher; Catalog number: C34571). 20 µL of DMSO was added to a vial of CellTrace Violet staining solution to make a stock solution. This stock solution was then diluted into 20 mL of PBS (previously warmed to 37°C) for a 5 µM staining solution. 5x 106(5ml) of cells was then added to a 50 mL centrifuge tube and centrifuged for 5 minutes at 300 x g and the supernatant was carefully poured off. The cells wereJ&J No. JBI6843WOPCT then resuspended in 5 mL of CellTrace Violet staining solution. Afterwards the cells were incubated for 20 minutes in a 37°C water bath. 40 mL OpTmizer T Cell Expansion SFM was added to the cells to absorb any unbound dye and the cells were incubated for 5 minutes. The cells were then centrifuged for 5 minutes at 300 x g and the cell pellet was resuspended in pre- warmed OpTmizer T Cell Expansion SFM.

[0134] Precoated-plate for γδ T cell activation:

[0135] For γδ T cell cultures, 24-well flat-bottom plates were coated with 500µl anti-CD3 (clone OKT3, BioXCell; clone UCHT1, and clone HIT3a) at 2.5 μg / ml, 5 μg / ml and 10 μg / ml with or without anti-CD27 mAb indicated concentration in PBS at 4°C for 24 hours. Wells were washed three times with PBS before adding γδ T cell cells. T cells were plated at 2x 106cells per well (1x106 / ml) in complete T cells culture media (OpTmizer™ T Cell Expansion media, 5% heat- inactivated human AB serum, 1% Antibiotic-Antimycotic, 2mM GlutaMAX™ Supplement (1x); all Life Technologies), supplemented with IL-2 100IU / ml and IL-15 10ng / ml. Post 2 days activation, γδ T cell cultures were analyzed for activation markers CD25 and CD69 and 1x105γδ T cell were collected from above activation wells and transduced with anti-PSMA CAR gRV at MOI 1 in the presence of Vectofusion-1 at 10 µg / mL in the total culture volume.

[0136] Flow Cytometry

[0137] Surface staining

[0138] For staining for CD25 and CD69, 1x 105activated γδ T cells were washed with FACS buffer (PBS / 0.1%BSA / 0.1% sodium azide) and then stained with antibodies for CD25, CD29, γδ TCR, delta 2, delta1, CD3, and a fixable viability dye. Antibodies against CD3, γδ TCR, TCR V delta 1, VDelta2, CD25, and CD69 were purchased from BioLegend and TCR V Delta1 was purchased from ThermoFisher. Anti-linker antibody for CAR detection was produced in- house.

[0139] Intracellular cytokine staining protocol

[0140] To stain intracellular cytokines IFN-γ, TNF-α, the Foxp3 Fixation / Permeabilization Concentrate and Diluent kit from eBiosciences (Catalog number: 00-5521-00) was used according to manufacturer directions. A single-cell suspension was prepared by staining cells with a Fixable Viability Dye and cell surface markers. After a final wash with FACS buffer, the supernatant was discarded and the sample was pulse vortexed to completely dissociate the pellet.200 µL of Foxp3 Fixation / Permeabilization working solution was then added to each well. The samples were thenJ&J No. JBI6843WOPCT incubated for 60 minutes at room temperature and protected from light. Afterwards samples were centrifuged at 400 x g for 5 minutes at room temperature and the supernatant was discarded. 200 µL 1X Permeabilization Buffer was then added to each well and the samples were centrifuged samples at 400x g for 5 minutes at room temperature and the supernatant was discarded. The step of adding 200 µL 1X Permeabilization Buffer to each well and centrifuging samples at 400x g for 5 minutes at room temperature was repeated and the supernatant was discarded. The pellet formed was then resuspended in residual volume and the volume adjusted to about 100 µL with 1X Permeabilization Buffer containing conjugated antibody for detection of intracellular antigen(s) IFN-g, TNF-a to cells and incubated for 40 minutes at room temperature. This step was protected from light. After 40 minutes, 1X Permeabilization Buffer was added to each well and the samples were centrifuged at 400x g for 5 minutes at room temperature with the supernatant then discarded. The step of adding 1X Permeabilization Buffer to each well and centrifuging the samples at 400x g for 5 minutes at room temperature was repeated and the supernatant was discarded. The stained cells were then resuspended in 150µl FACS buffer and analyzed by flow cytometry.

[0141] Results

[0142] The percent co-expression of CD25 and CD69 from two donors, Donor Aph24-7 and Donor Aph24-12, after primary human γδ T cells were activated using various anti-CD3 mAb clones (OKT3, UTCH1, and HIT3a), and was analyzed after two days using flow cytometry (Figure 5A, top and bottom panels). As shown, both donors had a high co-expression of activation markers. The purity of the γδ T cells was also analyzed using flow cytometry six days post- transduction with CAR RV and the data is represented in Table 7 below. As shown, γδ T cells activated and expanded by different anti-CD3 mAb clones achieved high purity (>90% γδ TCR+). There was no significant difference in the percentage of γδ TCR+ cells across different anti-CD3 mAb clones.J&J No. JBI6843WOPCT PSMA CAR 1 PSMA CAR 2 UTD 2.5 5 10ug / ml 2.5 5 10ug / ml 10ug / ml ug / ug / ml ug / ml ug / ml ml Donor CD3 (OKT3) 93.4 93.2 90.7 95.1 94.6 94.1 98.2 Aph24- 7 CD3 93.8 94.1 93.9 95.7 95.8 95.7 98.3 (UCHT1) CD3(HIT3a) 93.6 93.3 92.1 95.7 94.9 94.5 98.3 Donor CD3(OKT3) 92.4 91.5 90.3 93.4 91.9 92.5 95.8 Aph24- 12 CD3(UCHT1) R 92.9 92.2 93.2 94.6 94.1 94.5 97.0 C CD3(HIT3a)Tδ 93.2 92.8 93.1 94.7 93.6 94.1 97.4 γ CD3

[0143] Table 7. Representative percent purity of γδ T cells six days post transduction with CAR RV.

[0144] The expression levels of CAR were also analyzed 6 days post-transduction and 12 days post transduction, as shown in Figures 5C-5D, respectively. As shown, CAR expression for two different CARs in both donors was between 50% to 80% expression at 6 days post transduction and decreased slightly at 12 days post transduction.

[0145] CD27 expression

[0146] CD27 expression was analyzed on naïve and activated γδ T cells from two donors. Human γδ T cells from two representative donors were thawed and stained for CD3, γδ TCR, Vδ2, and CD27 expression in duplicate. CD27 expression on CD3+ γδ TCR+ Vδ2+ and CD3+ γδ TCR+ Vδ2- cells as represented on flow cytometry data (not shown) and is shown in Figure 6A.

[0147] CD27 expression was also analyzed using two representative donors’ primary human γδ T cells that were activated using different clones of anti-CD3 mAb (OKT3, UCHT1, and HIT3a) coated on 24-well plates at concentrations of 2.5, 5, and 10 µg / ml for 2 days. CD27 expression was stained and analyzed on activated CD3+ γδ TCR+ Vδ2+ T cells using flow cytometry (not shown), and the percentage of expression levels were summarized (Figures 6B-6C, donor 1 and donor 2 respectively).J&J No. JBI6843WOPCT

[0148] CD3 mAb with or without CD27 mAb promotes robust activation and proliferation of γδ T cells.

[0149] To evaluate the proliferative activity of primary γδ T cells activated by CD3 mAb in combination with or without CD27 mAb, a CellTrace Violet (CTV) dilution assay was used. A total of 5^×^106γδ T cells from 2 donors were thawed and labeled with 5 µM CellTrace Violet (CTV) dye (Thermo Fisher Scientific) according to the manufacturer’s instructions. γδ T cells were then cultured in 24-well plates pre-coated with 10 μg / ml anti-CD3 mAb and varying concentrations of CD27 mAb (2, 5, and 10 μg / ml; Invivogen, Catalog #: hcd27-mab1). Plates without CD27 mAb served as controls to evaluate the specific role of CD27 mAb in the activation and proliferation of γδ T cells. After 2 days of activation, 1×105cells were collected and transferred into a 96-well plate for further culture. After an additional 5 days, the γδ T cells were stained with Near IR Live / Dead dye, CD3, γδ TCR, and anti-CD25 (all from BioLegend) antibodies. CTV dilution and CD25 expression on γδ TCR+ T cells were assessed by flow cytometry. Five days post activation, γδ T cells were analyzed for CTV dye dilution (proliferation) and surface expression of CD25. Representative dot plots are shown. Cells were pre-gated on live, single, CD3+ γδ TCR+ cells as shown in Figure 7A.

[0150] γδ T cells activated with CD27 mAb showed relatively lower cell counts compared to those activated with CD3 mAb alone, suggesting that CD27 mAb may selectively expand CD3+ and CD27+ γδ T cells, while CD3 mAb could expand all CD3+ γδ T cells (Figure 7B).

[0151] Enhanced Cytokine Expression in γδ T Cells Activated by Combining CD3 (UCHT1 Clone) with CD27 mAb.

[0152] Different doses of CD27 (Invivogen, Catalog #: hcd27-mab1) at 2, 5, and 10 μg / ml were combined with 10 μg / ml anti-CD3 mAb to coat 24-well plates. Human γδ T cells from 2 donors were thawed and activated with the combined CD3 and CD27 mAbs. Plates without CD27 mAb served as controls to evaluate the specific role of CD27 mAb in the activation and function of γδ T cells.

[0153] For intracellular cytokine staining, 2x105human primary γδ T cells (6 days post activation) were incubated for 16 hours with 2 μl CD3-based activator TransAct and GolgiPlug (1:1000; BD Biosciences, San Jose, CA, USA) in a tissue culture incubator at 37°C. LIVE / DEAD™ Fixable Near-IR Dead Cell Stain (1:1000; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was added to exclude dead cells. Surface antibodies CD3 and γδ TCR wereJ&J No. JBI6843WOPCT also stained, and cells were then permeabilized and fixed using the Foxp3 / Transcription Factor Fixation / Permeabilization Concentrate and Diluent kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s instructions. Intracellular cytokines IFN-γ and TNF- α were stained with fluorescent antibodies. For quantification of cytokines in ICS analysis, γδ T cells were gated as Live, CD3+ γδTCR+, and viable cells among them were further gated according to negative control (non-activated; not shown). Fluorescence-activated cell sorting (FACS) was performed on a LSRFortessa (BD Biosciences, San Jose, CA, USA) cell sorter. All antibodies were purchased from BioLegend (San Diego, CA, USA). The percentage of IFN-γ and TNF-α producing γδ T cells from two representative donors was determined by intracellular staining of stimulated γδ T cells and is summarized as shown in Figures 8A-8B. References Impact of culture medium on the expansion of T cells for immunotherapy. Cytotherapy. 2009;11(7):936-46. Vectofusin®-1 data sheet. static.miltenyibiotec.com / asset / 150655405641 / document_uv9l1cv6p54italp1ft5beu23n?content- disposition=inline * * *

[0154] The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the disclosure in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0155] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.

Claims

J&J No. JBI6843WOPCT CLAIMS What is claimed is:

1. A method of generation of chimeric antigen receptor (CAR)-expressing CD3+ gamma delta (γδ) T cells, said method comprising one or more of the following steps: a) seeding an enriched γδ T cell fraction obtained from peripheral blood mononuclear cells (PBMCs) at about 1x105to about 2x106cells / well on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in an expansion medium; b) culturing the CD3+ γδ T cells on an anti-CD3 monoclonal antibody (mAb)- coated first tissue culture vessel in the expansion medium for about 1.5 to about 2.5 days, preferably 2 days, to produce activated CD3+ γδ T cells; c) collecting the activated CD3+ γδ T cells produced in step (b) and re-plating the activated CD3+ γδ T cells at about 1x105to about 2x106cells / well on a second tissue culture vessel in the absence of anti-CD3 antibodies; d) adding a chimeric antigen receptor (CAR)-encoding vector to the activated CD3+ γδ T cells of step (c) to achieve a multiplicity of infection (MOI) of 0.65-2, preferably a MOI of 1; e) adding Vectofusin®-1 at about a dilution of 1:50 to about a dilution of 1:200, preferably about a dilution of 1:100, to the mixture of activated CD3+ γδ T cells and CAR-encoding vector; f) culturing the mixture of step (e) for about 0.5 to about 1.5 days, preferably about 1 day to produce CAR-expressing CD3+ γδ T cells; g) adding additional expansion medium to the CAR-expressing CD3+ γδ T cells to achieve a total culture volume which is about double the culture volume of step (f); h) after about 1.5 to about 2.5 days, preferably 2 days, transferring the CAR- expressing CD3+ γδ T cells to a third tissue culture vessel and culturing for about 3.5 to about 4.5 days, preferably 4 days; and i) optionally, harvesting the CAR-expressing CD3+ γδ T cells or introducing the CAR-expressing CD3+ γδ T cells into a sorter, enrichment column, or a bioreactor.J&J No. JBI6843WOPCT 2. The method of claim 1, further comprising i) harvesting the CAR-expressing CD3+ γδ T cells and / or introducing the CAR-expressing CD3+ γδ T cells into a sorter, enrichment column, or a bioreactor.

3. The method of claim 2, wherein the harvesting is by centrifugation.

4. The method of any one of claims 1-3, wherein the third tissue culture vessel is larger than the second tissue culture vessel or the first tissue culture vessel.

5. The method of any one of claims 1-4, wherein the CD3+ γδ T cells in the enriched γδ T cell fraction are primary CD3+ γδ T cells.

6. The method of any one of claims 1-5, wherein the anti-CD3 mAb is derived from clone OKT3.

7. The method of any one of claims 1-6, wherein the collecting is by centrifugation, fluorescence activated cell sorting (FACS) or enrichment through a column.

8. The method of any one of claims 1-7, wherein the vector is a viral vector.

9. The method of claim 8, wherein the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, or a vaccinia virus vector.

10. The recombinant vector of claim 9, wherein the viral vector is a retroviral vector.J&J No. JBI6843WOPCT 11. The recombinant vector of any one of claims 1-7, wherein the vector is a non-viral vector.

12. The method of any one of claims 1-11, wherein the method further comprises one or more of the following steps: (1) counting the total number of the activated CD3+ γδ T cells produced in step (c) or the CAR-expressing CD3+ γδ T cells produced in step (h); (2) assaying for one or more cell surface markers on the activated CD3+ γδ T cells produced in step (c) or the CAR-expressing CD3+ γδ T cells produced in step (h); and (3) calculating an expansion efficacy of the CAR-expressing CD3+ γδ T cells produced in step (h) using results obtained in steps (1) and (2).

13. The method of claim 12, wherein the one or more cell surface markers comprises CD3, γδ T cell receptor (TCR), Vδ1, and / or Vδ2.

14. The method of claim 12 or claim 13, wherein the assay for the one or more cell surface markers is performed via flow cytometry.

15. The method of any one of claims 1-14, wherein the final concentration of Vectofusin®-1 in the total culture volume is 10 µg / mL.

16. The method of any one of claims 1-15, wherein the expansion medium comprises CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ OpTmizer™ T-Cell Expansion Supplement, about 2nM (1x) GlutaMAX™ Supplement, about 5% Human Type AB Serum, about 1% antibiotic- antimycotic (anti-anti), recombinant human interleukin-2 (rhIL-2) at a concentration of about 100 IU / mL, and recombinant human interleukin-15 (rhIL-15) at a concentration of about 10 ng / mL.J&J No. JBI6843WOPCT 17. Chimeric antigen receptor (CAR)-expressing CD3+ gamma delta (γδ) T cells obtained by a method of generation from γδ T cells, said method comprising one or more of the following steps: a) a means for seeding an enriched γδ T cell fraction obtained from peripheral blood mononuclear cells (PBMCs) at about 1x105to about 2x106cells / well on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in an expansion medium; b) a means for culturing the CD3+ γδ T cells on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in the expansion medium for about 1.5 to about 2.5 days, preferably 2 days, to produce activated CD3+ γδ T cells; c) a means for collecting the activated CD3+ γδ T cells produced in step (b) and a means for re-plating the activated CD3+ γδ T cells at about 1x105to about 2x106cells / well on a second tissue culture vessel in the absence of anti-CD3 antibodies; d) a means for adding a chimeric antigen receptor (CAR)-encoding vector to the activated CD3+ γδ T cells of step (c) to achieve a multiplicity of infection (MOI) of 0.65-2, preferably a MOI of 1; e) a means for adding Vectofusin®-1 at about a dilution of 1:50 to about a dilution of 1:200, preferably about a dilution of 1:100, to the mixture of activated CD3+ γδ T cells and CAR-encoding vector; f) a means for culturing the mixture of step (e) for about 0.5 to about 1.5 days, preferably about 1 day to produce CAR-expressing CD3+ γδ T cells; g) a means for adding additional expansion medium to the CAR-expressing CD3+ γδ T cells to achieve a total culture volume which is about double the culture volume of step (f); h) after about 1.5 to about 2.5 days, preferably 2 days, a means for transferring the CAR- expressing CD3+ γδ T cells to a third tissue culture vessel and culturing for about 3.5 to about 4.5 days, preferably 4 days; and i) optionally, providing a means for harvesting the CAR-expressing CD3+ γδ T cells or a means for introducing the CAR-expressing CD3+ γδ T cells into a sorter, enrichment column, or a bioreactor.J&J No. JBI6843WOPCT 18. A system for performing a method of generating chimeric antigen receptor (CAR)-expressing CD3+ gamma delta (γδ) T cells, the system comprising: a) a means for seeding an enriched γδ T cell fraction obtained from peripheral blood mononuclear cells (PBMCs) at about 1x105to about 2x106cells / well on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in an expansion medium; b) a means for culturing the CD3+ γδ T cells on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in the expansion medium for about 1.5 to about 2.5 days, preferably 2 days, to produce activated CD3+ γδ T cells; c) a means for collecting the activated CD3+ γδ T cells produced in step (b) and a means for re-plating the activated CD3+ γδ T cells at about 1x105to about 2x106cells / well on a second tissue culture vessel in the absence of anti-CD3 antibodies; d) a means for adding a chimeric antigen receptor (CAR)-encoding vector to the activated CD3+ γδ T cells of step (c) to achieve a multiplicity of infection (MOI) of 0.65-2, preferably a MOI of 1; e) a means for adding Vectofusin®-1 at about a dilution of 1:50 to about a dilution of 1:200, preferably about a dilution of 1:100, to the mixture of activated CD3+ γδ T cells and CAR-encoding vector; f) a means for culturing the mixture of step (e) for about 0.5 to about 1.5 days, preferably about 1 day to produce CAR-expressing CD3+ γδ T cells; g) a means for adding additional expansion medium to the CAR-expressing CD3+ γδ T cells to achieve a total culture volume which is about double the culture volume of step (f); h) after about 1.5 to about 2.5 days, preferably 2 days, a means for transferring the CAR- expressing CD3+ γδ T cells to a third tissue culture vessel and culturing for about 3.5 to about 4.5 days, preferably 4 days; and i) optionally, providing a means for harvesting the CAR-expressing CD3+ γδ T cells or a means for introducing the CAR-expressing CD3+ γδ T cells into a sorter, enrichment column, or a bioreactor.J&J No. JBI6843WOPCT 19. A method of generation of chimeric antigen receptor (CAR)-expressing CD3+ gamma delta (γδ) T cells, said method comprising the steps of: a) seeding an enriched γδ T cell fraction obtained from peripheral blood mononuclear cells (PBMCs) at about 1x105to about 2x106cells / well on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in an expansion medium; b) culturing the CD3+ γδ T cells on an anti-CD3 monoclonal antibody (mAb)-coated first tissue culture vessel in the expansion medium for about 1.5 to about 2.5 days, preferably 2 days, to produce activated CD3+ γδ T cells; c) collecting the activated CD3+ γδ T cells produced in step (b) and re-plating the activated CD3+ γδ T cells at about 1x105to about 2x106cells / well on a second tissue culture vessel in the absence of anti-CD3 antibodies; d) adding a chimeric antigen receptor (CAR)-encoding vector to the activated CD3+ γδ T cells of step (c) to achieve a multiplicity of infection (MOI) of 0.65-2, preferably a MOI of 1; e) adding Vectofusin®-1 at about a dilution of 1:50 to about a dilution of 1:200, preferably about a dilution of 1:100, to the mixture of activated CD3+ γδ T cells and CAR- encoding vector; f) culturing the mixture of step (e) for about 0.5 to about 1.5 days, preferably about 1 day to produce CAR-expressing CD3+ γδ T cells; g) adding additional expansion medium to the CAR-expressing CD3+ γδ T cells to achieve a total culture volume which is about double the culture volume of step (f); h) after about 1.5 to about 2.5 days, preferably 2 days, transferring the CAR-expressing CD3+ γδ T cells to a third tissue culture vessel and culturing for about 3.5 to about 4.5 days, preferably 4 days; and i) optionally, harvesting the CAR-expressing CD3+ γδ T cells or introducing the CAR- expressing CD3+ γδ T cells into a sorter, enrichment column, or a bioreactor.

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