Method for the production of TCR gamma delta+ t cells

Abstract

The present disclosure provides methods for expanding γδ T cells, wherein said method comprises the steps of: (1) culturing a population of cells comprising one or more γδ T cells in a first culture medium comprising a T cell mitogen and interleukin-4 (IL-4) and not comprising interleukin-15 (IL-15); and (2) culturing the population of cells from (1) in a second culture medium comprising IL-15 and not comprising IL-4; wherein the population of cells is restimulated before (2), concurrently with (2) or after (2). In some aspects, the cells are engineered, e.g., to express a chimeric antigen receptor. Further provided are populations of expanded and / or engineered γδ T cells and methods of using the same.

Description

METHOD FOR THE PRODUCTION OF TCR GAMMA DELTA+ T CELLS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This PCT application claims the priority benefit of U.S. Provisional Application No. 63 / 753,839, filed February 4, 2025, which is incorporated herein by reference in its entirety.FIELD OF THE DISCLOSURE

[0002] The disclosure relates to methods for expanding y5 T cells and optionally engineering said cells. Expanded and engineered y5 T cells produced according to the methods described herein find use in the treatment of cancer, in particular solid tumors as well as in autoimmune and inflammatory diseases. The present disclosure also relates to both individual cells and populations of cells produced by the methods described herein.BACKGROUND OF THE DISCLOSURE

[0003] Gamma delta T cells (y5 T cells) represent a subset of T cells that express on their surface a distinct, defining y5 T-cell receptor (TCR). This TCR is made up of one gamma (y) and one delta (5) chain. Human y5 TCR chains are selected from three main 5 chains, V51, V52 and V53 and six y chains. Human y5 T cells can be broadly classified based on their TCR chains, as certain y and 5 types are found on cells more prevalently, though not exclusively, in one or more tissue types. For example, most blood-resident y5 T cells express a V52 TCR, for example Vy9V52, whereas this is less common among tissue-resident y5 T cells, which more frequently use V51 in skin and Vy4 in the gut.

[0004] V51 y5 T cells are a subset of innate T cells defined by expression of T cell receptors composed of a y chain paired to a V51 chain. In mice, V51 y5 T cells are predominantly tissue resident where they are highly protective against a broad spectrum of carcinomas by mediating anti-tumor responses via pattern and natural cytotoxicity receptor recognition. Similarly, in humans, V51 y5 T cells predominantly reside within epithelial tissues, mediate target cell recognition that is not MHC restricted and are not allo-HLA reactive. HLA matching of patients is therefore not required for y5 T cell adoptive cell therapies. The innate V51 y5 T cell biology which enables antigen independent tumor recognition, lack of necessity for HLA matching,and inherent migration to and residence in human tissues makes V51 y5 T cells an attractive platform for cellular therapy.

[0005] There is therefore a need for methods to efficiently expand y5 T cells to allow their adaptation as therapies, e.g., as adoptive T cell therapies, and for methods which have the potential to provide allogeneic "off-the-shelf1chimeric antigen receptor-expressing y5 T cell therapies, such as for the treatment of solid tumors.

[0006] While there are certain expansion methods in the field, there remains a need for improved methods of expanding y5 T cells.SUMMARY OF THE DISCLOSURE

[0007] Some aspects of the present disclosure are directed to a method for expanding y5 T cells, wherein said method comprises the steps of: (1) culturing a population of cells comprising one or more y5 T cells in a first culture medium, wherein the first culture medium comprises a T cell mitogen and interleukin-4 (IL-4), wherein the first culture medium does not comprise interleukin- 15 (IL-15); and (2) culturing the population of cells from (1) in a second culture medium, wherein the second culture medium comprises IL-15, and wherein the second culture medium does not comprise IL-4; wherein the population of cells is restimulated before (2), concurrently with (2), or after (2).

[0008] In some aspects, the population of cells is cultured in the first culture medium for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In some aspects, the population of cells is cultured in the first culture medium for about 4 days.

[0009] In some aspects, the population of cells is cultured in the second culture medium for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days or about 17 days. In some aspects, the population of cells is cultured in the second medium for about 6 days. In some aspects, the population of cells in cultured in the second medium for about 9 days. In some aspects, the population of cells is restimulated on day 1 of the culture in the second culture medium.

[0010] In some aspects, the population of cells is restimulated by contacting the population of cells with a T cell mitogen, a CD28 agonist, or both. In some aspects, the population of cells is restimulated by contacting the population of cells with a T cell mitogen and a CD28 agonist.

[0011] In some aspects, the T cell mitogen and the CD28 agonist are soluble.

[0012] In some aspects, the T cell mitogen and the CD28 agonist are associated with each other. In some aspects, the T cell mitogen is linked to the CD28 agonist by a covalent bond. In some aspects, the T cell mitogen is linked to the CD28 agonist by a peptide bond.

[0013] In some aspects, the population of cells is restimulated by contacting the population of cells with a matrix, wherein the T cell mitogen, the CD28 agonist, or both, are associated with the matrix. In some aspects, both the T cell mitogen and the CD28 agonist are associated with the matrix.

[0014] In some aspects, the first culture medium further comprises IL-21, interferon gamma (IFNy), IL1-P, or any combination thereof. In some aspects, the first culture medium further comprises IL-21, interferon gamma (IFNy), and IL-ip. In some aspects, the second culture medium comprises a CD30 agonist, IL-2, IL-18, or any combination thereof. In some aspects, the second culture medium comprises a CD30 agonist, IL-2, and IL- 18.

[0015] In some aspects, after the cells are cultured in the second culture medium for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days following restimulation, the population of cells is contacted with fresh second culture medium. In some aspects, fresh second culture medium is added to the population of cells about 6 days after restimulation.

[0016] In some aspects, the population of cells is obtained from a biological sample obtained from a human subject. In some aspects, the biological sample is a haematopoietic sample or a fraction thereof. In some aspects, the biological sample is selected from peripheral blood, umbilical cord blood, lymphoid tissue, thymus, bone marrow, spleen, lymph node tissue or fractions thereof. In some aspects, the biological sample comprises peripheral blood or a fraction thereof. In some aspects, the biological sample consists of peripheral blood mononuclear cells (PBMCs) or low density mononuclear cells (LDMCs). In some aspects, the biological sample is a non-haematopoietic tissue.

[0017] In some aspects, the method comprises culturing the population of cells in the first culture medium and the second culture medium for between 7 and 21 days. In some aspects, the method comprises culturing the composition in the first culture medium and the second culture medium for about 10, about 11, about 12, about 13, or about 14 days.

[0018] In some aspects, the T cell mitogen comprises a CD3 agonist, an antibody or an antigen-binding portion thereof that specifically binds TCRyS, or an antibody or antigenbinding portion thereof that specifically binds TCRV51. In some aspects, the CD3 agonistcomprises an antibody or antigen-binding portion thereof the specifically binds CD3. In some aspects, the antibody or antigen-binding portion thereof that specifically binds CD3 is 0KT3.

[0019] In some aspects, the number of y5 T cells in the population of cells following the culture in the second culture medium is expanded by at least 5-fold, at least 10-fold, or at least 20-fold relative to the number of y5 T cells in the population of cells at the start of the culture.

[0020] In some aspects, the population of cells following the culture in the second culture medium comprises y5 T cells that express NKG2D, DNAM1, NKp30, NKp80, NKp46, CD57, GITR, TIGIT, CCR6, CCR2, CCR5, CXCR3 and / or CXCR6.

[0021] In some aspects, the y5 T cells are derived from a single donor. In some aspects, the y5 T cells are derived from multiple donors.

[0022] In some aspects, the method further comprises freezing the population of cells comprising the y5 T cells following culture in the second culture medium.

[0023] Some aspects of the present disclosure are directed to a method for engineering y5 T cells, said method comprising the steps of: (i) preparing a composition enriched for y5 T cells using the method of any one of claims 1 to 38; (ii) transducing the y5 T cells with an exogenous nucleic acid for expression in the y5 T cells; and (iii) culturing the transduced y5 T cells to expand the engineered y5 T cells.

[0024] In some aspects, the exogenous nucleic acid encodes a chimeric antigen receptor (CAR), a chimeric costimulatory receptor (CCR), a chimeric switch receptor (CSR), a chimeric TCR or other chimeric recombinant protein recognizing a tumour antigen, a stromal antigen, a costimulatory receptor, an inhibitory receptor, a cytokine or other cell surface antigen. In some aspects, the tumour antigen is a tumour specific antigen that is not expressed by normal somatic cells from the subject tissue. In some aspects, the tumour antigen is a tumour associated antigen which is preferentially overexpressed on cancer cells compared to healthy somatic cells. In some aspects, the tumour antigen is an antigen expressed by a cell experiencing oxidative stress, DNA damage, UV radiation, EGF receptor stimulation, or any combination thereof.

[0025] In some aspects, the tumour antigen is an antigen expressed by a solid tumour. In some aspects, the composition is transduced using a viral vector or electroporation. In some aspects, the viral vector comprises a retroviral vector. In some aspects, the viral vector comprises a gammaretroviral vector or a lentiviral vector. In some aspects, the gammaretroviral vector is a murine stem cell virus (MSCV) or Moloney Murine Leukaemia Virus (MLV). In some aspects, the viral vector is pseudotyped with an envelope other than vesicular stomatitis virus-G (VSV-G). In some aspects, the envelope comprises a betaretroviral envelope. In someaspects, the envelope comprises a baboon endogenous virus (BaEV) or RD114. In some aspects, the y5 T cells are restimulated before, concurrently with, or after transduction.

[0026] In some aspects, the y5 T cells are restimulated by contacting the y5 T cells with a T cell mitogen, a CD28 agonist, or both. In some aspects, the y5 T cells are restimulated by contacting the y5 T cells with a T cell mitogen and a CD28 agonist.

[0027] In some aspects, the T cell mitogen and the CD28 agonist are soluble.

[0028] In some aspects, the T cell mitogen and the CD28 agonist are associated with each other. In some aspects, the T cell mitogen is linked to the CD28 agonist by a covalent bond. In some aspects, the T cell mitogen is linked to the CD28 agonist by a peptide bond.

[0029] In some aspects, the y5 T cells are restimulated by contacting the y5 T cells with a matrix, and wherein the T cell mitogen, the CD28 agonist, or both, are associated with the matrix. In some aspects, both the T cell mitogen and the CD28 agonist are associated with the matrix.

[0030] In some aspects, step (iii) comprises culturing the transduced composition in the absence of feeder cells. In some aspects, step (iii) comprises culturing the transduced composition in the presence of feeder cells.

[0031] Some aspects of the present disclosure are directed to an expanded y5 T cell population obtained by a method disclosed herein. Some aspects of the present disclosure are directed to a pharmaceutical composition comprising the expanded y5 T cell population. In some aspects, the expanded y5 T cell population or the pharmaceutical composition is for use as a medicament. In some aspects, the expanded y5 T cell population or the pharmaceutical composition is for use in the treatment of cancer. In some aspects, the cancer is a solid tumor. In some aspects, the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, or at least about 21 days. In some aspects, the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 14 days.BRIEF DESCRIPTION OF THE FIGURES

[0032] FIG. 1 pictorially represents Method 1, Method 2, and Method 3 activation and expansion protocols described in the Examples.

[0033] FIGs. 2A-2B are graphical representations of the impact of Method 1 on the expression of various biomarkers including CD28 on Pan y5, V51, and non-V51 y5T cells (V52-3) at days 0, 4, and 14 of the expansion phase.

[0034] FIGs. 3A-3B are graphical representations of raw (FIG. 3A) and normalized (FIG.3B) fold expansion of y5 T-cells mediated by restimulation with TransAct™ (TxA) as shown using Method 1, Method 1 with the addition of TxA at Day 4 (Method 1 + TxA), Method 2 with IL-18 supplementation (Method 2 + IL- 18), andMethod3 (i.e., Method 2 with the addition of IL- 18 and TxA at Day 4).

[0035] FIG. 4 is a graphical representation of the effect of restimulation with TransAct (TxA) on the expansion and purity of V51 T-cells as measured by the relative (%) population of aP T-cells, NK cells, V51 T-cells (Vdl), Non-V51 V5 T-cells (Non-Vdl V5), and triplenegative populations (TNP). Bar represents the mean of four donors.

[0036] FIGs. 5A-5F are graphical representations of the effect of restimulation with TransAct™ (TxA) on the frequency of T-cell subsets including effector (EFF), effector memory (EM), central memory (CM), and naive T-cells. FIGs. 5A-5E represent the frequency of EFF, EM, CM, and naive T-cells on day 14 as assessed by flow cytometry for cells cultured according to Method 1 (FIG. 5 A), Method 1 + TxA (FIG. 5B), Method 2 + IL- 18 (FIG. 5C), Method 3 (FIG. 5D), or for a skin expanded sample (FIG. 5E). FIG. 5F shows the (%) of V51 T-cells that have EFF, EM, CM, or naive phenotypes for each culture condition.

[0037] FIG. 6 is a graphical representation of the post-thaw viability of V51 T-cells generated by each method.

[0038] FIGs. 7A-7D are graphical representations of the impact of 0 ng / mL and 10 ng / mL IL- 15 concentrations on post-thaw viability (FIG. 7A, 7C, respectively) and total live fold expansion (FIG. 7B, 7D, respectively).

[0039] FIGs. 8A-8D are graphical representations of the expansion of thawed expanded y5 T-cells in the presence of 10 ng / mL IL- 15 or absence thereof. FIG. 8 A and FIG. 8C represent the total live cell fold expansion for 0 ng / mL and 10 ng / mL IL- 15, respectively. FIG. 8B and FIG. 8D represent the (%) cell viability of cells cultured with 0 ng / mL and 10 ng / mL IL-15, respectively.

[0040] FIG. 9 is a graphical representation of the stimulation of CellTrace labeled y5 T- cells with plate-bound antibody OKT3 in the presence or absence of ligand-Fc for 7 days.

[0041] FIGs. 10A-10D are graphical representations of the effect of cytokine combinations (i.e., IL-15 and IL-21; IL-15, IL-21, and IL-2; IL-15, IL-21, IL-2, and IL-18) and restimulationwith TransAct™ (TxA), ImmunoCult™, or Dynabeads™ on the expansion of ybT-cells. FIG.10A, FIG. 10B, and FIG. IOC represent the fold expansion, total yield, and (%) cell viability of expanded cells at Day 14, respectively. FIG. 10D represents the post-thaw viability of cells generated by each method. Three dots represent three donors, and the data are presented as Mean ± SEM.

[0042] FIGs. 11 is a graphical representation of the effect of cytokine combination (i.e., IL-15 and IL-21; IL-15, IL-21, and IL-2; IL-15, IL-21, IL-2, and IL-18) and restimulation with TransAct™ (TxA), ImmunoCult™, or Dynabeads™ on the frequency of T-cell subsets including CD45RA+CD27 V51 effector T-cells (EFF), CD45RA CD27 V51 effector memory T-cells (EM), CD45RA CD27+ V51 central memory T-cells (CM), and CD45RA+CD27+V51 naive T-cells. Each bar represents the mean of three donors.

[0043] FIGs. 12A-12B are graphical representations of the expression of activation, inhibitory, chemokine, and functional markers on V51 T-cells generated using Method 1, Method 1 with TransAct™ (TxA), Method 2 with IL- 18 supplementation, or Method 3. FIG.12A represents the expression of activation and inhibitory markers, and FIG. 12B represents the expression of chemokine and functional markers. Results are the mean of three donors.

[0044] FIGs. 13A-13E are graphical representations of the innate killing capacity of y5 T- cells expanded using Method 1, Method 1 with TransAct™ (TxA), Method 2 with IL- 18 supplementation, or Method 3 compared to y5 T-cells expanded from skin. Innate killing capacity was assessed using effector Target (E:T) ratios of 10:1, 3.3:1, 1.1:1 and 0.33:1, where the effector is expanded y5 T-cells and the target is either OVCAR3, A549, LS1034, or GSU cancer cells. FIG. 13A graphically represents the innate killing capacity. FIG. 13B, FIG. 13C, FIG. 13D, and FIG. 13E represents the (%) cytolysis of OVCAR3, A549, GSU, and LS1034, respectively, over a range of E:T ratios.

[0045] FIGs. 14A-14C are graphical representations of long-term innate tumor control as assessed in a Repeated Antigen Stimulation (RAS) assay with A549 cells as the target. V51 y5 T-cells were activated and expanded using Method 1, Method 1 with TransAct (TxA), Method 2 with IL-18 supplementation, or Method 3, and RAS assay performed with A549 cells alone as a control. FIG. 14 A, FIG. 14B, and FIG. 14C represent independent experiments with y5 T- cells derived from three different donors.

[0046] FIGs. 15A-15B are graphical representations of post Repeated Antigen Stimulation (RAS) proliferation of V51 y5 T-cells. FIG. 15A represents the V51 y5 T-cell fold expansion relative to Day 0 seeding with A549 cells as the target. FIG. 16B represents the V51 y5 T-cellfold expansion relative to Day 0 seeding with OVCAR-3 cells as the target. Each dot represents V51 y5 T-cells derived from a different donor (N=3).

[0047] FIGs. 16A-16B are graphical representations of the fold expansion of y5 T-cells with or without an anti-GCC CAR where TransAct™ restimulation was performed either 24 hrs or 48 hrs before transduction after Day 4 activation or directly after transduction on Day 5 or 24 hrs after the end of the transduction step (Day 6) during the expansion phase. FIG. 16A represents the fold expansion of y5 T-cells as measured by Day 14 cell count divided by Grex® seeding day. FIG. 16B represents the theoretical fold expansion post-stimulation if all the cells were used at each step. In this experiment, cell numbers were normalized relative to Method 1 (i.e., number of cells used for transduction, number of cells used for expansion).

[0048] FIG. 17 is a graphical representation of the vector copy number (VCN) per CAR+ y5 T-cells derived from each different restimulation / transduction process.

[0049] FIGs. 18A-18B are graphical representations of the cell viability and purity postexpansion. FIG. 18A shows the total viability and FIG. 18B shows the composition of cells subtypes within the expanded cells including aP T-cells (abT), V51 y5 T-cells (Vdl gdT), Non- V51 y5 T-cells (Non-VDl gdT), Natural Killer cells (NK), and triple negative cells (abT- gdT- NK-).

[0050] FIGs. 19A-19B are graphical representations of the expression of activation, inhibitory, chemokine, and functional markers on V51 y5 T-cells that express an anti-GCC CAR. FIG. 19A represents the expression of chemokine and functional markers, and FIG. 19B represents the expression of activation and inhibitory markers.

[0051] FIGs. 20A-20D are graphical representations of the effect of TransAct™ (TxA) restimulation 24 hr before transduction (TxA 24hr pre-TD) or after transduction (TxA post- TD) of anti-GCC CAR on V51 y5 T-cells. The subsequent effect on fold expansion (FIG. 20A), total yield (FIG. 20B), post-thaw viability (FIG. 20C), and CAR expression (FIG. 20D) is shown.

[0052] FIGs. 21A-21B are graphical representations of the effect of TransAct™ (TxA) restimulation 24 hrs before transduction (TxA 24hr pre-TD) or after transduction (TxA post- TD) of anti-GCC CAR on the expression of activation, inhibitory, chemokine, and functional markers on V51 y5 T-cells. FIG. 21A represents the expression of chemokine, and functional markers; and FIG. 2 IB represents the expression of activation and inhibitory markers.

[0053] FIG. 22 is a graphical representation of the effect of TransAct™ (TxA) restimulation 24 hrs before transduction (TxA 24hr pre-TD) or after transduction (TxA post-TD) of anti-GCC CAR on the cell killing activity of y5 T-cells. Target-mediated and innate killing capacity was assessed using effectortarget (E:T) ratios of 10:1, 3:1, 1:1 and 1:3, where the target cells were either GCC expressing (GCC+) cancer cell lines (i.e., LS1034, GSU) or GCC negative (GCC-) cancer cell lines (i.e., HT-29, A549).

[0054] FIGs. 23A-23B are graphical representations of the cytotoxic activity and proliferation of y5 T-cells as assessed in a RAS assay. y5 T-cells were either restimulated with TransAct™ (TxA) at 24 hrs pre-transduction with an anti-GCC CAR, or restimulated with TxA after transduction with an anti-GCC CAR. Mock conditions were also tested where the restimulation remained the same, but without the anti-GCC CAR transduction. FIG. 23A represents innate killing by RAS assay at an effectortarget ratio of 1:1, wherein the target is LSI 034; and FIG. 23B represents post-RAS assay fold expansion of y5 T-cells.

[0055] FIGs. 24A-24B are graphical representations of the cytotoxic activity and proliferation of y5 T-cells as assessed in a RAS assay. y5 T-cells were either restimulated with TransAct™ (TxA) at 24 hrs pre-transduction with an anti-GCC CAR, or restimulated with TxA after transduction with an anti-GCC CAR. Mock conditions were also tested where the restimulation remained the same, but without the anti-GCC CAR transduction. FIG. 24A represents innate killing by RAS assay at an effectortarget ratio of 1:1, wherein the target is HT55; and FIG. 24B represents post-RAS assay fold expansion of y5 T-cells.

[0056] FIGs. 25A-25D are graphical representations of bioreactor expansion of y5 T-cells using Method 3. Cells were transferred to an Ambr® bioreactor either after transduction (e.g., TxA 24 hrs pre-TD) or two days after TxA restimulation (e.g., TxA post-TD). FIG. 25A represents the fold expansion, FIG. 27B represents the (%) viability, and FIG. 25C represents the (%) CAR+ V51 y5 T-cells using this process. FIG. 25D represents the cell yield generated by Method 3 in an Ambr bioreactor.

[0057] FIGs. 26A-26B are graphical representations of cell differentiation and phenotype using Method 1 and Method 3 and expansion in either a G-Rex® or anAmbr® bioreactor. FIG.26A represents the (%) V51 y5 T-cells that have a phenotype of naive (Tnaive), central memory (Tcm), effector memory (Tern), or effector T-cell (Teff). FIG. 26B represents the phenotype of the V51 y5 T-cells generated using Method 1 or Method 3 and expansion in either a G-Rex® or an Ambr® Bioreactor, where the cells were transduced with either tEGFR or anti-GCC CAR.DETAILED DESCRIPTION OF THE DISCLOSURE

[0058] Some aspects of the present discslosure are directed to methods for expanding y5 T cells. In some aspects, the method comprises stimulating the y5 T cells.

[0059] Some aspects of the present disclosure are directed to methods for expanding y5 T cells, wherein said method comprises the steps of: (1) culturing a population of cells comprising one or more y5 T cells in a first culture medium, wherein the first culture medium comprises a T cell mitogen and interleukin-4 (IL-4), wherein the first culture medium does not comprise interleukin- 15 (IL-15); and (2) culturing the population of cells from (1) in a second culture medium, wherein the second culture medium comprises IL-15, and wherein the second culture medium does not comprise IL-4; wherein a restimulation agent is added to the population of cells before (2) or concurrently with (2).

[0060] Some aspects of the present disclosure are directed to methods for expanding y5 T cells, wherein said method comprises the steps of: (1) culturing a population of cells comprising one or more y5 T cells in a first culture medium, wherein the first culture medium comprises a T cell mitogen and interleukin-4 (IL-4), wherein the first culture medium does not comprise interleukin- 15 (IL-15); and (2) culturing the population of cells from (1) in a second culture medium, wherein the second culture medium comprises IL-15, and wherein the second culture medium does not comprise a T cell mitogen or IL-4; and wherein the population of cells is optionally restimulated before (2) or after (2). In some aspects, the population of cells is not restimulated. In some aspects, the population of cells is restimulated before (2). In some aspects, the population of cells is restimulated after (2).

[0061] In some aspects, the population of cells is restimulated by contacting the population of cells with a restimulation agent comprising a recombinant CD3 and CD28 agonist (+ / - CD2). In some aspects, the population of cells is restimulated by contacting the population of cells with a matrix, wherein the matrix comprises a T cell mitogen (e.g., a CD3 agonist), T cell mitogen, a CD28 agonist, or both, wherein the T cell mitogen and / or the CD28 agonist are associated with the matrix. In some instances this could also comprise a CD3, CD28 and CD2 agonist. In some aspects, the T cell mitogen (e.g., a CD3 agonist) and the CD28 agonist are soluble.I. Terms

[0062] It is to be understood that aspects and aspects of the disclosure described herein include "comprising," "consisting," and "consisting essentially of aspects and aspects. As used herein, the singular form "a," "an," and "the" includes plural references unless indicated otherwise.

[0063] The term "about" as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to "about" a value or parameter herein includes (and describes) aspects that are directed to that value or parameter per se. In some instances, "about" encompass variations of +20%, in some instances +10%, in some instances +5%, in some instances +1%, or in some instances +0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0064] As used herein, the term "T cell mitogen" refers to any agent that can stimulate T cells through TCR signaling. Nonlimiting examples of T cell mitogens include plant lectins (such as phytohemagglutinin (PHA) and concanavalin A (ConA)), lectins of non-plant origin, antibodies that activate T cells, and other non-lectin / non-antibody mitogens. In some aspects, the T cell mitogen comprises a CD3 agonist. In some aspects, the T cell mitogen comprises an antibody or antigen-binding portion thereof that binds CD3. In some aspects, the anti-CD3 antibody comprises OKT-3. In some aspects, the anti-CD3 antibody comprises UCHT-1. In some aspects, the T cell mitogen comprises a TCRyS agonist. In some aspects, the T cell mitogen comprises an antibody or antigen-binding portion thereof that binds TCRyS. In some aspects, the anti-TCRyS antibody comprises Bl. In some aspects, the anti-TCRyS antibody comprises IMMU510. In some aspects, the T cell mitogen comprises a TCRV61 agonist. In some aspects, the T cell mitogen comprises an antibody or antigen-binding portion thereof that binds TCRV61. In some aspects, the anti-TCRVSl antibody comprises 5TCS1.

[0065] As used herein, the term "TransAct™" or "TxA" refers to a T cell activation agent, which comprises a CD3 agonist and a CD28 agonist, associated with a polymeric nanomatrix. TransAct™ consists of a colloidal polymeric nanomatrix conjugated to humanized CD3 and CD28 agonists providing primary and co-stimulatory signals for T cell activation and expansion. TransAct™ is a commercially available reagent, which is marketed by Miltenyi Biotec.

[0066] As used herein, the term "ImmunoCult™" refers to a media comprising soluble antibody complexes that bind to and cross-link CD3, CD28, and CD2 cell surface ligands,thereby providing the primary and co-stimulatory signals for T cell activation. Immunocult™ is a commercially available reagent, which is marketed by STEMCELL Technologies.

[0067] Within the context of the present disclosure, antibodies are understood to include monoclonal antibodies (mAbs), polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab')2), single chain antibodies, single chain variable fragments (ScFv) and recombinantly produced binding partners. In some aspects, the antibody is an anti-CD3 monoclonal antibody (mAb). Other mitogens include phorbol 12-myri state- 13 -acetate (TPA) and its related compounds, such as mezerein, or bacterial compounds (e.g., Staphylococcal enterotoxin A (SEA) and Streptococcal protein A). The T cell mitogen can be soluble or immobilized and more than one T cell mitogen can be used in the method of the disclosure.

[0068] The term "antibody" includes any antibody protein construct comprising at least one antibody variable domain comprising at least one antigen binding site (ABS). Antibodies include, but are not limited to, immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). The overall structure of Immunoglobulin G (IgG) antibodies assembled from two identical heavy (H)-chain and two identical light (L)-chain polypeptides is well established and highly conserved in mammals (Padlan (1994) Mol. Immunol. 31:169-217).

[0069] A fragment of the antibody (which can also be referred to as "antibody fragment", "immunoglobulin fragment", "antigen-binding fragment" or "antigen-binding polypeptide") as used herein refers to a portion of an antibody (or constructs that contain said portion) that specifically binds to the target, the CD3 protein that is part of the T cell receptor (TCR) complex (e.g. a molecule in which one or more immunoglobulin chains is not full length, but which specifically binds to the target). Examples of binding fragments encompassed within the term antibody fragment include:(i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL and CHI domains);(ii) a F(ab')2 fragment (a bivalent fragment consisting of two Fab fragments linked by a disulphide bridge at the hinge region);(iii) a Fd fragment (consisting of the VH and CHI domains);(iv) a Fv fragment (consisting of the VL and VH domains of a single arm of an antibody);(v) a single chain variable fragment, scFv (consisting of VL and VH domains joined, using recombinant methods, by a synthetic linker that enables them to be made asa single protein chain in which the VL and VH regions pair to form monovalent molecules);(vi) a VH (an immunoglobulin chain variable domain consisting of a VH domain); (vii) a VL (an immunoglobulin chain variable domain consisting of a VL domain); (viii) a domain antibody (dAb, consisting of either the VH or VL domain);(ix) a minibody (consisting of a pair of scFv fragments which are linked via CH3 domains); and(x) a diabody (consisting of a noncovalent dimer of scFv fragments that consist of a VH domain from one antibody connected by a small peptide linker to a VL domain from another antibody).

[0070] "Specificity" refers to the number of different types of antigens or antigenic determinants to which a particular antibody or fragment thereof can bind. The specificity of an antibody is the ability of the antibody to recognize a particular antigen as a unique molecular entity and distinguish it from another. An antibody that "specifically binds" to an antigen or an epitope is a term well understood in the art. A molecule is said to exhibit "specific binding" if it reacts more frequently, more rapidly, with greater duration and / or with greater affinity with a particular target antigen or epitope, than it does with alternative targets. An antibody "specifically binds" to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. An antibody (or fragment thereof) can be considered to specifically bind to a target if the binding is statistically significant compared to a non-relevant binder.

[0071] As used herein, the term "stimulated," "stimulation," or "stimulating," when used in the context of cell culture, refers to contacting the cells with a T cell mitogen (e.g., CD3 agonist), a CD28 agonist, a CD2 agonist, or any combination thereof, as disclosed herein. The term "restimulated," "restimulation," or "restimulating" refers to the act of stimulating the cells again, in particular by contacting the cells with a T cell mitogen, a CD28 agonist, or both, as disclosed herein.

[0072] As used herein, the term "engineered y5 T cell" refers to a y5 T cell that expresses a transgene (i.e., a gene that has been transduced into the engineered y5 T cell or a parental cell thereof).

[0073] As used herein, the term "primed y5 T cell" refers to a starting population (e.g., an endogenous population of y5 T cells) that has been affected by a culture condition. In some instances, a primed y5 T cell has a different functional viral entry receptor profile relative to itsunprimed counterpart before experiencing the culture condition. In some aspects, a population of primed y5 T cells is an expanded population of y5 T cells.

[0074] As used herein, an "expanded population of y5 cells" refers to a population of haematopoietic cells including y5 T cells that has been cultured in a condition and for a duration that has induced the expansion of y5 cells, i.e., increased y5 cell number. Likewise, an "expanded population of V51 T cells," as used herein, refers to a population of haematopoietic cells including V51 T cells that has been cultured in a condition and for a duration that has induced the expansion of V51 T cells, i.e., increased V51 cell number. Similarly, an "expanded population of V52 T cells," as used herein, refers to a population of haematopoietic cells including V52 T cells that has been cultured in a condition and for a duration that has induced the expansion of V52 T cells, i.e., increased V52 cell number.

[0075] As used herein, a "population" of y5 T cells refers to a group of three or more y5 T cells (e.g., at least 10, at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 1010, at least 1011, at least 1012, or at least 1013) y5 T cells (e.g., engineered y5 T cells). A population of a particular cell type (e.g., a population of endogenous y5 T cells, a population of primed y5 T cells, or a population of engineered y5 T cells) refers to the cells of that type and not to cells of a different type within a broader population. For example, if 10% of the cells of a starting population of 108T cells are y5 T cells, the starting population of y5 T cells is 107.

[0076] The term "marker" herein to refers to a DNA, RNA, protein, carbohydrate, glycolipid, or cell-based molecular marker, the expression or presence of which in a patient's sample can be detected by standard methods (or methods disclosed herein). In some aspects, the marker comprises CD56. In some aspects the marker comprises NKp30, CD57, GITR, TIGIT, CCR6, CCR2, CCR5, and / or CXCR6.

[0077] A cell or population of cells that "expresses" a marker of interest is one in which mRNA encoding the protein, or the protein itself, including fragments thereof, is determined to be present in the cell or the population. Expression of a marker can be detected by various means. For example, in some aspects, expression of a marker refers to a surface density of the marker on a cell. Mean fluorescence intensity (MFI), for example, as used as a readout of flow cytometry, is representative of the density of a marker on a population of cells. A person of skill in the art will understand that MFI values are dependent on staining parameters (e.g., concentration, duration, and temperature) and fluorochrome composition. However, MFI can be quantitative when considered in the context of appropriate controls. For instance, apopulation of cells can be said to express a marker if the MFI of an antibody to that marker is significantly higher than the MFI of an appropriate isotype control antibody on the same population of cells, stained under equivalent conditions. Additionally, or alternatively, a population of cells can be said to express a marker on a cell-by-cell basis using a positive and negative gate according to conventional flow cytometry analytical methods (e.g., by setting the gate according to isotype or "fluorescence-minus-one" (FMO) controls). By this metric, a population can be said to "express" a marker if the number of cells detected positive for the marker is significantly higher than background (e.g., by gating on an isotype control).

[0078] As used herein, when a population's expression is stated as a percentage of positive cells and that percentage is compared to a corresponding percentage of positive cells of a reference population, the percentage difference is a percentage of the parent population of each respective population. For example, if a marker is expressed on 10% of the cells of population A, and the same marker is expressed on 1% of the cells of population B, then population A is said to have a 9% greater frequency of marker-positive cells than population B (i.e., 10%-l%, not 10%M%). When a frequency is multiplied through by the number of cells in the parent population, the difference in absolute number of cells is calculated. In the example given above, if there are 100 cells in population A, and 10 cells in population B, then population A has 100- fold the number of cells relative to population B, i.e., (10% x 100) (1% x 10).

[0079] An expression level of a marker can be a nucleic acid expression level (e.g., a DNA expression level or an RNA expression level, e.g., an mRNA expression level). Any suitable method of determining a nucleic acid expression level can be used. In some aspects, the nucleic acid expression level is determined using qPCR, rtPCR, RNA-seq, multiplex qPCR or RT- qPCR, microarray analysis, serial analysis of gene expression (SAGE), MASSARRAY® technique, in situ hybridization (e.g., FISH), or combinations thereof.

[0080] As used herein, a "reference population" of cells refers to a population of cells corresponding to the cells of interest, against which a phenotype of the cells of interest are measured. For example, a level of expression of a marker on a population of haematopoietic- derived y5 cells cultured according to the methods disclosed herein can be compared to the level of expression of the same marker on a haematopoietic-derived y5 T cell expanded under different conditions. A population can also be compared to itself at an earlier state. For example, a reference population can be a population of haematopoietic-derived y5 T cells prior to its expansion. In this case, the expanded population is compared to its own composition prior to the expansion step, i.e., its past composition, in this case, is the reference population.

[0081] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide construct comprising an extracellular antigen binding domain, a transmembrane domain, and, optionally, an intracellular domain that propagates an activation signal that activates the cell and / or a costimulatory signal. In some aspects, the CAR includes an optional leader sequence at the N-terminus of the CAR fusion protein. In some aspects, the CAR lacks an intracellular (e.g., signaling) domain.

[0082] An "immune response" is as understood in the art, and generally refers to a biological response within a vertebrate against foreign agents or abnormal, e.g., cancerous cells, which response protects the organism against these agents and diseases caused by them. An immune response is mediated by the action of one or more cells of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and / or elimination from the vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. An immune reaction includes, e.g., activation or inhibition of a T cell, e.g., an effector T cell, a Th cell, a CD4+ cell, a CD8+ T cell, or a Treg cell, or activation or inhibition of any other cell of the immune system, e.g., NK cell.

[0083] "Immunotherapy" refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying the immune system or an immune response.

[0084] As used herein, the terms "treat," "treatment," or "treatment of when used in the context of treating a disease or condition in a subject, e.g., a cancer, refer to reducing disease pathology, reducing or eliminating disease symptoms, promoting increased survival rates, and / or reducing discomfort. For example, treating can refer to the ability of a therapy when administered to a subject, to reduce one or more disease symptoms, signs, or causes. Treating also refers to mitigating or decreasing one or more clinical symptom and / or inhibition or delay in the progression of the condition and / or prevention or delay of the onset of a disease or illness.

[0085] As used herein, "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can result in the formation of malignant tumors or cells that invade neighboring tissues and can metastasize to distant parts of the body through the lymphatic system or bloodstream.

[0086] As used herein, the term an "effective amount" or a "therapeutically effective amount" of an administered therapeutic substance, such as an immune cell comprising a polynucleotide encoding a CAR, is an amount sufficient to carry out a specifically stated or intended purpose, such as treating or treatment of cancer. An "effective amount" can be determined empirically in a routine manner in relation to the stated purpose.

[0087] As used herein, the terms "subject," "individual," or "patient," refer to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, and so on.

[0088] As used herein, the terms "ug" and "uM" are used interchangeably with "pg" and "pM," respectively.

[0089] Various aspects described herein are described in further detail in the following subsections.II. Methods of the Disclosure

[0090] Some aspects of the present disclosure are directed to methods for expanding y5 T cells, wherein said method comprises the steps of: (1) culturing a population of cells comprising one or more y5 T cells in a first culture medium, wherein the first culture medium comprises a T cell mitogen and interleukin-4 (IL-4), wherein the first culture medium does not comprise interleukin- 15 (IL-15); and (2) culturing the population of cells from (1) in a second culture medium, wherein the second culture medium comprises IL-15, and wherein the second culture medium does not comprise IL-4; wherein the population of cells is restimulated before (2), concurrently with (2), or after (2).

[0091] Some aspects of the present disclosure are directed to methods for expanding y5 T cells, wherein said method comprises the steps of: (1) culturing a population of cells comprising one or more y5 T cells in a first culture medium, wherein the first culture medium comprises a T cell mitogen and interleukin-4 (IL-4), wherein the first culture medium does not comprise interleukin- 15 (IL-15); and (2) culturing the population of cells from (1) in a second culture medium, wherein the second culture medium comprises IL-15, and wherein the second culture medium does not comprise a T cell mitogen or IL-4; and wherein the population of cells is optionally restimulated before (2) or after (2).

[0092] In some aspects, the population of cells is restimulated by contacting the population of cells with a T-cell mitogen (e.g., a CD3 agonist) and a CD28 agonist. In some aspects, the population of cells is restimulated by contacting the population of cells with a matrix, wherein the T cell mitogen (e.g., CD3 agonist), the CD28 agonist, or both, are associated with the matrix. In some aspects, the T cell mitogen (e.g., CD3 agonist) and the CD28 agonist are soluble. In some aspects, the population of cells is restimulated by contacting the population of cells with a matrix, wherein the T cell mitogen (e.g., CD3 agonist), the CD28 agonist, and CD2 agonist are associated with the matrix. In some aspects, the population of cells is restimulated by contacting the population of cells with ImmunoCult™.

[0093] In some aspects, the population of cells is cultured in the first culture medium for about 1 day to about 14 days. In some aspects, the population of cells is cultured in the first culture medium for about 1 day to about 13 days, about 1 day to about 12 days, about 1 day to about 11 days, about 1 day to about 10 days, about 1 day to about 9 days, about 1 day to about 8 days, about 1 day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, or about 1 day to about 3 days. In some aspects, the population of cells is cultured in the first culture medium for about 2 days to about 13 days, about 2 days to about 12 days, about 2 days to about 11 days, about 2 days to about 10 days, about 2 days to about 9 days, about 2 days to about 8 days, about 2 days to about 7 days, about 2 days to about 6 days, about 2 days to about 5 days, or about 2 days to about 4 days. In some aspects, the population of cells is cultured in the first culture medium for about 3 days to about 13 days, about 3 days to about 12 days, about 3 days to about 11 days, about 3 days to about 10 days, about 3 days to about 9 days, about 3 days to about 8 days, about 3 days to about 7 days, about 3 days to about 6 days, or about 3 days to about 5 days. In some aspects, the population of cells is cultured in the first culture medium for about 1 day to about 7 days. In some aspects, the population of cells is cultured in the first culture medium for about 1 day to about 6 days. In some aspects, the population of cells is cultured in the first culture medium for about 1 day to about 5 days. In some aspects, the population of cells is cultured in the first culture medium for about 1 day to about 4 days.

[0094] In some aspects, the population of cells is cultured in the first culture medium for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In some aspects, the population of cells is cultured in the first culture medium for about 1 day. In some aspects, the population of cells is cultured in the first culture medium for about 2 days. In some aspects, the population of cells is cultured in the first culture medium for about3 days. In some aspects, the population of cells is cultured in the first culture medium for about 4 days. In some aspects, the population of cells is cultured in the first culture medium for about 5 days. In some aspects, the population of cells is cultured in the first culture medium for about 6 days. In some aspects, the population of cells is cultured in the first culture medium for about 7 days.

[0095] In some aspects, the population of cells is cultured in the second culture medium for about 1 day to about 14 days. In some aspects, the population of cells is cultured in the second culture medium for about 1 day to about 13 days, about 1 day to about 12 days, about 1 day to about 11 days, about 1 day to about 10 days, about 1 day to about 9 days, about 1 day to about 8 days, about 1 day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, or about 1 day to about 3 days. In some aspects, the population of cells is cultured in the second culture medium for about 2 days to about 13 days, about 2 days to about 12 days, about 2 days to about 11 days, about 2 days to about 10 days, about 2 days to about 9 days, about 2 days to about 8 days, about 2 days to about 7 days, about 2 days to about 6 days, about 2 days to about 5 days, or about 2 days to about 4 days. In some aspects, the population of cells is cultured in the second culture medium for about 3 days to about 13 days, about 3 days to about 12 days, about 3 days to about 11 days, about 3 days to about 10 days, about 3 days to about 9 days, about 3 days to about 8 days, about 3 days to about 7 days, about 3 days to about 6 days, or about 3 days to about 5 days. In some aspects, the population of cells is cultured in the second culture medium for about 4 days to about 13 days, about 4 days to about 12 days, about 4 days to about 11 days, about 4 days to about 10 days, about 4 days to about 9 days, about 4 days to about 8 days, about 4 days to about 7 days, or about 4 days to about 6 days. In some aspects, the population of cells is cultured in the second culture medium for about 5 days to about 13 days, about 5 days to about 12 days, about 5 days to about 11 days, about 5 days to about 10 days, about 5 days to about 9 days, about 5 days to about 8 days, about 5 days to about 7 days, or about 5 days to about 6 days. In some aspects, the population of cells is cultured in the second culture medium for about 6 days to about 13 days, about 6 days to about 12 days, about 6 days to about 11 days, about 6 days to about 10 days, about 6 days to about 9 days, about 6 days to about 8 days, or about 6 days to about 7 days. In some aspects, the population of cells is cultured in the second culture medium for about 7 days to about 13 days, about 7 days to about 12 days, about 7 days to about 11 days, about 7 days to about 10 days, about 7 days to about 9 days, or about 7 days to about 8 days. In some aspects, the population of cells is cultured in the second culture medium for about 8days to about 13 days, about 8 days to about 12 days, about 8 days to about 11 days, about 8 days to about 10 days, or about 8 days to about 9 days. In some aspects, the population of cells is cultured in the second culture medium for about 3 days to about 7 days. In some aspects, the population of cells is cultured in the second culture medium for about 6 days to about 9 days. In some aspects, the population of cells is cultured in the second culture medium for about 7 days to about 10 days.

[0096] In some aspects, the population of cells is cultured in the second culture medium for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days. In some aspects, the population of cells is cultured in the second culture medium for about 4 days. In some aspects, the population of cells is cultured in the second culture medium for about 5 days. In some aspects, the population of cells is cultured in the second culture medium for about 6 days. In some aspects, the population of cells is cultured in the second culture medium for about 7 days. In some aspects, the population of cells is cultured in the second culture medium for about 8 days. In some aspects, the population of cells is cultured in the second culture medium for about 9 days. In some aspects, the population of cells is cultured in the second culture medium for about 10 days.

[0097] In some aspects, fresh second culture medium is added to the population of cells about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days after the population of cells is restimulated. In some aspects, fresh second culture medium is added to the population of cells about 3 days after the population of cells is restimulated. In some aspects, fresh second culture medium is added to the population of cells about 4 days after the population of cells is restimulated. In some aspects, fresh second culture medium is added to the population of cells about 5 days after the population of cells is restimulated. In some aspects, fresh second culture medium is added to the population of cells about 6 days after the population of cells is restimulated. In some aspects, fresh second culture medium is added to the population of cells about 7 days after the population of cells is restimulated.

[0098] In some aspects, the method comprises culturing the population of cells in the first culture medium and the second culture medium for between 2 and 30 days. In some aspects, the method comprises culturing the population of cells in the first culture medium and the second culture medium for between 7 and 21 days. In some aspects, the method comprises culturing the population of cells in the first culture medium and the second culture medium for between 7 and 18 days. In some aspects, the method comprises culturing the population of cellsin the first culture medium and the second culture medium for between 10 and 18 days. In some aspects, the method comprises culturing the population of cells in the first culture medium and the second culture medium for between 10 and 14 days. In some aspects, the method comprises culturing the population of cells in the first culture medium and the second culture medium for between 12 and 16 days.

[0099] In some aspects, the method comprises culturing the population of cells in the first culture medium and the second culture medium for about 10 days. In some aspects, the method comprises culturing the population of cells in the first culture medium and the second culture medium for about 11 days. In some aspects, the method comprises culturing the population of cells in the first culture medium and the second culture medium for about 12 days. In some aspects, the method comprises culturing the population of cells in the first culture medium and the second culture medium for about 13 days. In some aspects, the method comprises culturing the population of cells in the first culture medium and the second culture medium for about 14 days. In some aspects, the method comprises culturing the population of cells in the first culture medium and the second culture medium for about 15 days. In some aspects, the method comprises culturing the population of cells in the first culture medium and the second culture medium for about 16 days. In some aspects, the method comprises culturing the population of cells in the first culture medium and the second culture medium for about 17 days. In some aspects, the method comprises culturing the population of cells in the first culture medium and the second culture medium for about 18 days.

[0100] In some aspects, the method comprises (i) culturing the population of cells in the first culture medium for about 4 days, (ii) restimulating the population of cells on about day 5, wherein day 0 is the initiation of the culture, (iii) culturing the population of cells in the second culture medium from about day 5 to about day 14. In some aspects, the method further comprises adding fresh second culture medium on about day 11, wherein day 0 is the initiation of the culture.

[0101] In some aspects, the method comprises (i) culturing the population of cells in a first culture medium comprising a T cell mitogen and IL-4 for about 4 days, wherein the first culture medium does not comprise IL-15; (ii) optionally restimulating the population of cells on about day 5, wherein day 0 is the initiation of the culture; (iii) culturing the population of cells in a second culture medium comprising IL-15 from about day 5 to about day 14, wherein the second culture medium does not comprise IL-4. In some aspects, the method further comprises adding fresh second culture medium on about day 11, wherein day 0 is the initiation of the culture.

[0102] In some aspects, the method comprises (i) culturing the population of cells in a first culture medium comprising a T cell mitogen and IL-4 for about 4 days, wherein the first culture medium does not comprise IL-15; (ii) culturing the population of cells in a second culture medium comprising IL-15 from about day 5 to about day 14, wherein the second culture medium does not comprise a T cell mitogen or IL-4.

[0103] In some aspects, the method comprises (i) culturing the population of cells in a first culture medium comprising a T cell mitogen and IL-4 for about 4 days, wherein the first culture medium does not comprise IL-15; (ii) optionally restimulating the population of cells on about day 5, wherein day 0 is the initiation of the culture; (iii) culturing the population of cells in a second culture medium comprising IL-15 from about day 5 to about day 14, wherein the second culture medium does not comprise a T cell mitogen or IL-4.

[0104] In some aspects, the method comprises (i) culturing the population of cells in a first culture medium comprising a T cell mitogen and IL-4 for about 4 days, wherein the first culture medium does not comprise IL-15; (ii) culturing the population of cells in a second culture medium comprising IL-15 from about day 5 to about day 14, wherein the second culture medium does not comprise a T cell mitogen or IL-4; and (iii) optionally restimulating the population of cells on about day 14, wherein day 0 is the initiation of the culture. In some aspects, the method further comprises adding fresh second culture medium comprising IL- 15 on about day 11, wherein day 0 is the initiation of the culture.

[0105] In some aspects, the number of y5 T cells in the population of cells is increased following the culture in the first culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 2-fold, at least about 3-fold, at least about 4- fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, or at least about 100-fold relative to the number of y5 T cells in the population of cells prior to culture in the first culture medium (i.e., at the start of the culture). In some aspects, number of y5 T cells in the population of cells is increased by at least about 2-fold relative to the number of y5 T cells in the population of cells prior to culture in the first culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 3-fold relative to the number of y5 T cells in the population of cells prior to culture in the first culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 4-fold relative to the number of y5 T cells in the population of cells prior toculture in the first culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 5-fold relative to the number of y5 T cells in the population of cells prior to culture in the first culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 6-fold relative to the number of y5 T cells in the population of cells prior to culture in the first culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 7-fold relative to the number of y5 T cells in the population of cells prior to culture in the first culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 8-fold relative to the number of y5 T cells in the population of cells prior to culture in the first culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 9-fold relative to the number of y5 T cells in the population of cells prior to culture in the first culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 10-fold relative to the number of y5 T cells in the population of cells prior to culture in the first culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 15-fold relative to the number of y5 T cells in the population of cells prior to culture in the first culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 20-fold relative to the number of y5 T cells in the population of cells prior to culture in the first culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 25-fold relative to the number of y5 T cells in the population of cells prior to culture in the first culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 30-fold relative to the number of y5 T cells in the population of cells prior to culture in the first culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 35-fold relative to the number of y5 T cells in the population of cells prior to culture in the first culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 40-fold relative to the number of y5 T cells in the population of cells prior to culture in the first culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 50-fold relative to the number of y5 T cells in the population of cells prior to culture in the first culture medium.

[0106] In some aspects, the number of y5 T cells in the population of cells is increased following the culture in the second culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, atleast about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, or at least about 100-fold relative to the number of y5 T cells in the population of cells prior to culture in the second culture medium (i.e., following culture in the first culture medium). In some aspects, number of y5 T cells in the population of cells is increased by at least about 2-fold relative to the number of y5 T cells in the population of cells prior to culture in the second culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 3-fold relative to the number of y5 T cells in the population of cells prior to culture in the second culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 4-fold relative to the number of y5 T cells in the population of cells prior to culture in the second culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 5-fold relative to the number of y5 T cells in the population of cells prior to culture in the second culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 6-fold relative to the number of y5 T cells in the population of cells prior to culture in the second culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 7-fold relative to the number of y5 T cells in the population of cells prior to culture in the second culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 8-fold relative to the number of y5 T cells in the population of cells prior to culture in the second culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 9-fold relative to the number of y5 T cells in the population of cells prior to culture in the second culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 10-fold relative to the number of y5 T cells in the population of cells prior to culture in the second culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 15-fold relative to the number of y5 T cells in the population of cells prior to culture in the second culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 20-fold relative to the number of y5 T cells in the population of cells prior to culture in the second culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 25-fold relative to the number of y5 T cells in the population of cells prior to culture in the second culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 30-fold relative to the number of y5 T cells in the population of cells prior to culture in the second culture medium.In some aspects, number of y5 T cells in the population of cells is increased by at least about 35-fold relative to the number of y5 T cells in the population of cells prior to culture in the second culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 40-fold relative to the number of y5 T cells in the population of cells prior to culture in the second culture medium. In some aspects, number of y5 T cells in the population of cells is increased by at least about 50-fold relative to the number of y5 T cells in the population of cells prior to culture in the second culture medium.

[0107] In some aspects, the number of y5 T cells in the population of cells is increased by at least about 10-fold following culture in the second culture medium relative to the number of y5 T cells in the population of cells prior to culture in the first medium (i.e., at the start of the culture). In some aspects, the number of y5 T cells in the population of cells is increased by at least about 20-fold following culture in the second culture medium relative to the number of y5 T cells in the population of cells prior to culture in the first medium (i.e., at the start of the culture). In some aspects, the number of y5 T cells in the population of cells is increased by at least about 30-fold following culture in the second culture medium relative to the number of y5 T cells in the population of cells prior to culture in the first medium (i.e., at the start of the culture). In some aspects, the number of y5 T cells in the population of cells is increased by at least about 40-fold following culture in the second culture medium relative to the number of y5 T cells in the population of cells prior to culture in the first medium (i.e., at the start of the culture). In some aspects, the number of y5 T cells in the population of cells is increased by at least about 50-fold following culture in the second culture medium relative to the number of y5 T cells in the population of cells prior to culture in the first medium (i.e., at the start of the culture). In some aspects, the number of y5 T cells in the population of cells is increased by at least about 60-fold following culture in the second culture medium relative to the number of y5 T cells in the population of cells prior to culture in the first medium (i.e., at the start of the culture). In some aspects, the number of y5 T cells in the population of cells is increased by at least about 70-fold following culture in the second culture medium relative to the number of y5 T cells in the population of cells prior to culture in the first medium (i.e., at the start of the culture). In some aspects, the number of y5 T cells in the population of cells is increased by at least about 80-fold following culture in the second culture medium relative to the number of y5 T cells in the population of cells prior to culture in the first medium (i.e., at the start of the culture). In some aspects, the number of y5 T cells in the population of cells is increased by at least about 90-fold following culture in the second culture medium relative to the number ofy5 T cells in the population of cells prior to culture in the first medium (i.e., at the start of the culture). In some aspects, the number of y5 T cells in the population of cells is increased by at least about 100-fold following culture in the second culture medium relative to the number of y5 T cells in the population of cells prior to culture in the first medium (i.e., at the start of the culture).

[0108] The methods described herein are performed outside the human or animal body, i.e.they are in vitro and / or ex vivo. Thus, in some aspects the methods described herein are in vitro methods. In some aspects, the methods described herein are ex vivo methods.

[0109] In some aspects, the method further comprises freezing the expanded y5 T cells.Such frozen expanded y5 T cells can subsequently be thawed for downstream processing (such as further culturing and expansion steps) and / or use (such as a therapeutic use).ILA. Restimulation

[0110] In some aspects, the population of cells is restimulated before the being contacted with the second culture medium. In some aspects, the population of cells is restimulated while the population of cells are being cultured in the first culture medium. In some aspects, the population of cells is restimulated on the final day of culture in the first culture medium. In some aspects, the population of cells is restimulated on day 1 of the culture in the second culture medium.[OHl] In some aspects, the population of cells is restimulated by contacting the population of cells with a T cell mitogen (e.g., a CD3 agonist), a CD28 agonist, or both. In some aspects, the population of cells is restimulated by contacting the population of cells with a T cell mitogen (e.g., a CD3 agonist). In some aspects, the population of cells is restimulated by contacting the population of cells with a CD28 agonist. In some aspects, the population of cells is restimulated by contacting the population of cells with a T cell mitogen and a CD28 agonist. In some aspects, the population of cells is restimulated by contacting the population of cells with a T cell mitogen (e.g., a CD3 agonist), a CD28 agonist, and a CD2 agonist.

[0112] In some aspects, the T cell mitogen is soluble. As such, in some aspects, the population of cells is restimulated by contacting the population of cells with a soluble T cell mitogen (e.g., CD3 agonist). In some aspects, the CD28 agonist is soluble. As such, in some aspects, the population of cells is restimulated by contacting the population of cells with a soluble CD28 agonist. In some aspects, the T cell mitogen and the CD28 agonist are soluble. As such, in some aspects, the population of cells is restimulated by contacting the populationof cells with a soluble T cell mitogen and a soluble CD28 agonist. In some aspects, the population of cells is restimulated by contacting the population of cells with ImmunoCult™ (StemCellTechnologies).

[0113] In some aspects, the population of cells is restimulated by contacting the population of cells with a T cell mitogen and a CD28 agonist, wherein the T cell mitogen and the CD28 agonist are associated with each other. In some aspects, the T cell mitogen is linked to the CD28 agonist by a covalent bond. In some aspects, the T cell mitogen is linked to the CD28 agonist by a peptide bond. In some aspects, the T cell mitogen is linked to the CD28 agonist by a peptide linker comprising one or more amino acids.

[0114] In some aspects, the population of cells is restimulated by contacting the population of cells with a matrix, wherein the T cell mitogen, the CD28 agonist, or both, are associated with the matrix. In some aspects, the T cell mitogen, the CD28 agonist, or both, are covalently linked to the matrix. In some aspects, the T cell mitogen, the CD28 agonist, or both, are covalently linked to the matrix through one or more peptide bonds. In some aspects, the T cell mitogen, the CD28 agonist, or both, are covalently linked to the matrix by a peptide linker. In some aspects, the T cell mitogen, the CD28 agonist, or both, are associated with the matrix through one or more non-covalent interactions. In some aspects, the matrix comprises a nanomatrix. In some aspects, the matrix comprises a polymeric nanomatrix.

[0115] In some aspects, the population of cells is restimulated by contacting the population of cells with a matrix, wherein the T cell mitogen is associated with the matrix. In some aspects, the population of cells is restimulated by contacting the population of cells with a matrix, wherein the CD28 agonist is associated with the matrix. In some aspects, the population of cells is restimulated by contacting the population of cells with a matrix, wherein the T cell mitogen and the CD28 agonist are associated with the matrix. In some aspects, the population of cells is restimulated by contacting the population of cells with TransAct™ (Miltenyi Biotech). In some aspects, the population of cells is restimulated by contacting the population of cells with ImmunoCult™ (StemCell Biosciences).

[0116] In some aspects, the T cell mitogen comprises a CD3 agonist. In some aspects, the CD3 agonist comprises an antibody or an antigen-binding portion thereof that specifically binds CD3. In some aspects, the anti-CD3 antibody comprises OKT-3. In some aspects, the anti-CD3 antibody comprises UCHT-1. In some aspects, the cells are contacted with the anti- CD3 antibody, e.g., OKT-3, in combination with retronectin.

[0117] In some aspects, the T cell mitogen comprises a TCRyS agonist. In some aspects, the T cell mitogen comprises an antibody or antigen-binding portion thereof that binds TCRyS. In some aspects, the anti-TCRyS antibody comprises Bl. In some aspects, the anti-TCRyS antibody comprises IMMU510. In some aspects, the T cell mitogen comprises a TCRV61 agonist. In some aspects, the T cell mitogen comprises an antibody or antigen-binding portion thereof that binds TCRV61. In some aspects, the anti-TCRVSl antibody comprises 5TCS1.

[0118] In some aspects, the T cell mitogen, e.g., the anti-CD3 antibody or fragment thereof, is in a soluble or immobilized form. For example, the T cell mitogen can be added to the culture in a soluble form. Alternatively, the T cell mitogen can be added to the culture, wherein the T cell mitogen is bound to or covalently linked to a surface, such as a matrix, bead, or plate (i.e., in an immobilized form). In some aspects, the T cell mitogen is immobilized on a surface, such as Fc-coated wells. Alternatively, the T cell mitogen can be bound to the surface of a cell (e.g., immobilized on the surface of an antigen presenting cell (APC)).

[0119] In some aspects, the fresh second culture medium is added to the cells. In some aspects, after the cells are cultured in the second culture medium for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days following restimulation, the population of cells is contacted with fresh second culture medium. In some aspects, fresh second culture medium is added to the population of cells about 2 days after restimulation. In some aspects, fresh second culture medium is added to the population of cells about 3 days after restimulation. In some aspects, fresh second culture medium is added to the population of cells about 4 days after restimulation. In some aspects, fresh second culture medium is added to the population of cells about 5 days after restimulation. In some aspects, fresh second culture medium is added to the population of cells about 6 days after restimulation. In some aspects, fresh second culture medium is added to the population of cells about 7 days after restimulation. In some aspects, fresh second culture medium is added to the population of cells about 8 days after restimulation. In some aspects, fresh second culture medium is added to the population of cells about 9 days after restimulation. In some aspects, fresh second culture medium is added to the population of cells about 10 days after restimulation. In some aspects, fresh second culture medium is added to the population of cells about 11 days after restimulation. In some aspects, fresh second culture medium is added to the population of cells about 12 days after restimulation. In some aspects, fresh second culture medium is added to the population of cells about 13 days after restimulation. In some aspects, fresh second culture medium is added to the population of cells about 14 days after restimulation.

[0120] In some aspects, after the cells are cultured in the second culture medium for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days following restimulation, the population of cells is contacted with fresh second culture medium. In some aspects, fresh second culture medium is added to the population of cells about 2 days after restimulation, wherein the population of cells is stimulated on day one of the culture in the second culture medium. In some aspects, fresh second culture medium is added to the population of cells about 3 days after restimulation, wherein the population of cells is stimulated on day one of the culture in the second culture medium. In some aspects, fresh second culture medium is added to the population of cells about 4 days after restimulation, wherein the population of cells is stimulated on day one of the culture in the second culture medium. In some aspects, fresh second culture medium is added to the population of cells about 5 days after restimulation, wherein the population of cells is stimulated on day one of the culture in the second culture medium. In some aspects, fresh second culture medium is added to the population of cells about 6 days after restimulation, wherein the population of cells is stimulated on day one of the culture in the second culture medium. In some aspects, fresh second culture medium is added to the population of cells about 7 days after restimulation, wherein the population of cells is stimulated on day one of the culture in the second culture medium. In some aspects, fresh second culture medium is added to the population of cells about 8 days after restimulation, wherein the population of cells is stimulated on day one of the culture in the second culture medium. In some aspects, fresh second culture medium is added to the population of cells about 9 days after restimulation, wherein the population of cells is stimulated on day one of the culture in the second culture medium. In some aspects, fresh second culture medium is added to the population of cells about 10 days after restimulation, wherein the population of cells is stimulated on day one of the culture in the second culture medium. In some aspects, fresh second culture medium is added to the population of cells about 11 days after restimulation, wherein the population of cells is stimulated on day one of the culture in the second culture medium. In some aspects, fresh second culture medium is added to the population of cells about 12 days after restimulation, wherein the population of cells is stimulated on day one of the culture in the second culture medium. In some aspects, fresh second culture medium is added to the population of cells about 13 days after restimulation, wherein the population of cells is stimulated on day one of the culture in the second culture medium. In some aspects, fresh second culture medium is added to the population of cells about14 days after restimulation, wherein the population of cells is stimulated on day one of the culture in the second culture medium.

[0121] In some aspects, the method comprises (i) culturing the population of cells in the first culture medium from day 0 to about day 4, wherein day 0 is the initiation of the culture; (ii) restimulating the population of cells on about day 5; (iii) culturing the population of cells in the second culture medium from about day 5 to about day 14. In some aspects, the method further comprises adding fresh second culture medium on about day 11, wherein day 0 is the initiation of the culture.II.B. Culture Conditions

[0122] In some aspects, the first culture medium, the second culture medium, or both is supplemented with additional factors to support T cell growth and / or expansion. In some aspects, the first culture medium, the second culture medium, or both comprises one or more additional growth factors, one or more cytokine, or any combination thereof.

[0123] In some aspects, the first culture medium further comprises IL-21, interferon gamma (IFNy), IL-ip, or any combination thereof. In some aspects, the first culture medium comprises IL-21. In some aspects, the first culture medium comprises IFNy. In some aspects, the first culture medium comprises IL-ip. In some aspects, the first culture medium comprises IL-21 and IFNy. In some aspects, the first culture medium comprises IL-21 and IL-ip. In some aspects, the first culture medium comprises IL-ip and IFNy. In some aspects, the first culture medium comprises IL-21, IL-ip, and IFNy.

[0124] As such, in some aspects, the first culture medium comprises a T cell mitogen (e.g., a CD3 agonist or a TCRyS agonist, e.g., a TCRV51 agonist), IL-4, and IL-21. In some aspects, the first culture medium comprises a T cell mitogen (e.g., a CD3 agonist or a TCRyS agonist, e.g., a TCRV61 agonist), IL-4, and IFNy. In some aspects, the first culture medium comprises a T cell mitogen (e.g., a CD3 agonist or a TCRyS agonist, e.g., a TCRV61 agonist), IL-4, and IL-ip. As such, in some aspects, the first culture medium comprises a T cell mitogen (e.g., a CD3 agonist or a TCRyS agonist, e.g., a TCRV61 agonist), IL-4, IL-21, and IFNy. In some aspects, the first culture medium comprises a T cell mitogen (e.g., a CD3 agonist or a TCRyS agonist, e.g., a TCRV61 agonist), IL-4, IL-21, and IL-ip. In some aspects, the first culture medium comprises a T cell mitogen (e.g., a CD3 agonist or a TCRyS agonist, e.g., a TCRV61 agonist), IL-4, IL-ip, and IFNy. In some aspects, the first culture medium comprises a T cellmitogen (e.g, a CD3 agonist or a TCRyS agonist, e.g, a TCRV51 agonist), IL-4, IL-21, IL-ip, and IFNy.

[0125] In some aspects, the second culture medium further comprises a CD30 agonist, IL- 2, IL- 18, or any combination thereof. In some aspects, the second culture medium comprises a CD30 agonist. In some aspects, the second culture medium comprises IL-2. In some aspects, the second culture medium further comprises IL- 18. In some aspects, the second culture medium comprises a CD30 agonist and IL-2. In some aspects, the second culture medium comprises a CD30 agonist and IL-18. In some aspects, the second culture medium comprises IL-2 and IL- 18. In some aspects, the second culture medium comprises a CD30 agonist, IL-2, and IL-18.

[0126] As such, in some aspects, the second culture medium comprises IL- 15 and a CD30 agonist. In some aspects, the second culture medium comprises IL-15 and IL-2. In some aspects, the second culture medium comprises IL- 15 and IL-21. In some aspects, the second culture medium comprises IL- 15 and IL-21, wherein the second culture medium does not comprise a T cell mitogen. In some aspects, the second culture medium further comprises IL- 15 and IL-18. In some aspects, the second culture medium comprises IL-15, a CD30 agonist, and IL-2. In some aspects, the second culture medium comprises IL-15, a CD30 agonist, and IL- 18. In some aspects, the second culture medium comprises IL- 15, IL-2, and IL-18. In some aspects, the second culture medium comprises IL- 15, a CD30 agonist, IL-2, and IL- 18.

[0127] In some aspects, the second culture medium comprises IL- 15, IL-21, and a CD30 agonist. In some aspects, the second culture medium comprises IL- 15, IL-21, and IL-2. In some aspects, the second culture medium further comprises IL- 15, IL-21, and IL- 18. In some aspects, the second culture medium comprises IL-15, IL-21, a CD30 agonist, and IL-2. In some aspects, the second culture medium comprises IL-15, IL-21, a CD30 agonist, and IL-18. In some aspects, the second culture medium comprises IL- 15, IL-21, IL-2, and IL- 18. In some aspects, the second culture medium comprises IL- 15, IL-21, a CD30 agonist, IL-2, and IL-18. In some aspects, the first culture medium comprises a T cell mitogen (e.g., a CD3 agonist), IL-4, IL-21, IL-ip, and IFNy; and the second culture medium comprises IL- 15, IL-21, a CD30 agonist, IL- 2, and IL-18.

[0128] In some aspects, the second culture medium comprises IL- 15, IL-21. In some aspects, the second culture medium comprises IL- 15, IL-21, wherein IL- 15 and IL-21 are the only cytokines in the second culture medium. In some aspects, the second culture medium further comprises IL- 15, IL-21, and IL- 18. In some aspects, the second culture mediumcomprises IL-15, IL-21, a CD30 agonist, and IL-2. In some aspects, the second culture medium comprises IL-15, IL-21, a CD30 agonist, and IL-18. In some aspects, the second culture medium comprises IL- 15, IL-21, IL-2, and IL- 18. In some aspects, the second culture medium comprises IL-15, IL-21, a CD30 agonist, IL-2, and IL-18.

[0129] In some aspects, the first culture medium comprises a T cell mitogen (e.g., a CD3 agonist), IL-4, IL-21, IL-ip, and IFNy; and the second culture medium comprises IL-15 and IL-21 (e.g., wherein IL-15 and IL-21 are the only cytokines in the second culture medium). In some aspects, the first culture medium comprises a T cell mitogen (e.g., a CD3 agonist), IL-4, IL-21, IL-ip, and IFNy; and the second culture medium comprises IL-15 and IL-21 (e.g., wherein IL- 15 and IL-21 are the only cytokines in the second culture medium), and wherein a restimulation agent is added to the population of cells before (2) or concurrently with (2).

[0130] In some aspects, the second culture medium comprises TGF[3. In some aspects, the second culture medium comprises at least about 0.5 ng / mL, at least about 1 ng / mL, at least about 1.5 ng / mL, at least about 2 ng / mL, at least about 2.5 ng / mL, at least about 3 ng / mL, at least about 3.5 ng / mL, at least about 4 ng / mL, at least about 4.5 ng / mL, at least about 5 ng / mL, at least about 5.5 ng / mL, at least about 6 ng / mL, at least about 6.5 ng / mL, at least about 7 ng / mL, at least about 7.5 ng / mL, at least about 8 ng / mL, at least about 8.5 ng / mL, at least about 9 ng / mL, at least about 9.5 ng / mL, or at least about 10 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 0.5 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 1 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 1.5 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 2 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 2.5 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 3 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 3.5 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 4 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 4.5 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 5 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 5.5 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 6 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 6.5 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 7 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 7.5 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 8 ng / mL TGFp. In some aspects, the second culture medium comprisesat least about 8.5 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 9 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 9.5 ng / mL TGFp. In some aspects, the second culture medium comprises at least about 10 ng / mL TGFp.

[0131] In some aspects, the transduction step comprises culturing the cells in a medium comprising TGFp. In some aspects, the transduction step comprises culturing the cells in a medium comprising about 0.5 ng / mL, at least about 1 ng / mL, at least about 1.5 ng / mL, at least about 2 ng / mL, at least about 2.5 ng / mL, at least about 3 ng / mL, at least about 3.5 ng / mL, at least about 4 ng / mL, at least about 4.5 ng / mL, at least about 5 ng / mL, at least about 5.5 ng / mL, at least about 6 ng / mL, at least about 6.5 ng / mL, at least about 7 ng / mL, at least about 7.5 ng / mL, at least about 8 ng / mL, at least about 8.5 ng / mL, at least about 9 ng / mL, at least about 9.5 ng / mL, or at least about 10 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 0.5 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 1 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 1.5 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 2 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 2.5 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 3 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 3.5 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 4 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 4.5 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 5 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 5.5 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 6 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 6.5 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 7 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 7.5 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 8 ng / mL TGFp. In some aspects, thetranduction step comprises culturing the cells in a medium comprising at least about 8.5 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 9 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 9.5 ng / mL TGFp. In some aspects, the tranduction step comprises culturing the cells in a medium comprising at least about 10 ng / mL TGFp.II.B.1. Interleukins

[0132] As used herein, "IL-15" refers to native or recombinant IL- 15 or a variant thereof that acts as an agonist for one or more IL-15 receptor (IL-15R) subunits (e.g. mutants, muteins, analogues, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof). IL-15, like IL-2, is a known T-cell growth factor that can support proliferation of an IL-2- dependent cell line, CTLL-2. IL- 15 was first reported by Grabstein, et al. (Grabstein, et al. Science 1994. 264.5161: 965-969) as a 114-amino acid mature protein. The term "IL-15," as used herein, means native or recombinant IL-15 and muteins, analogs, subunits thereof, or complexes thereof (e.g. receptor complexes, e.g. sushi peptides, as described in WO 2007 / 046006), and each of which can stimulate proliferation of CTLL-2 cells. In the CTLL-2 proliferation assays, supernatants of cells transfected with recombinantly expressed precursor and in-frame fusions of mature forms of IL-15 can induce CTLL-2 cell proliferation.

[0133] Human IL- 15 can be obtained according to the procedures described by Grabstein, et al. or by conventional procedures such as polymerase chain reaction (PCR). A deposit of human IL-15 cDNA was made with the ATCC® on Feb. 19, 1993 and assigned accession number 69245.

[0134] The amino acid sequence of human IL-15 (Gene ID 3600) is found in Genbank under accession locator NP000576.1 GL 10835153 (isoform 1) and NP_751915.1 GI: 26787986 (isoform 2). The murine (Mus miisciihis) IL-15 amino acid sequence (Gene ID 16168) is found in Genbank under accession locator NP 001241676.1 GI: 363000984.

[0135] IL- 15 can also refer to IL- 15 derived from a variety of mammalian species, including, for example, human, simian, bovine, porcine, equine, and murine. An IL- 15 "mutein" or "variant", as referred to herein, is a polypeptide substantially homologous to a sequence of a native mammalian IL- 15 but that has an amino acid sequence different from a native mammalian IL-15 polypeptide because of an amino acid deletion, insertion or substitution. Variants can comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics.Examples of conservative substitutions include substitution of one aliphatic residue for another, such as He, Vai, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known. Naturally occurring IL- 15 variants are also encompassed by the disclosure. Examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the IL-15 protein, wherein the IL-15 binding property is retained. Alternate splicing of mRNA can yield a truncated but biologically active IL- 15 protein. Variations attributable to proteolysis include, for example, differences in the N- or C- termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the IL- 15 protein (generally from 1-10 amino acids). In some aspects, the terminus of the protein can be modified to alter its physical properties, for example, with a chemical group such as polyethylene glycol (Yang, etal. Cancer 1995. 76:687-694). In some aspects, the terminus or interior of the protein can be modified with additional amino acids (Clark-Lewis, et al. PNAS 1993. 90:3574-3577).

[0136] In some aspects, the methods defined herein include IL- 15 typically at a concentration of at least 0.1 ng / mL, such as at least 10 ng / mL (e.g. from 0.1 ng / mL to 10,000 ng / mL, from 1.0 ng / mL to 1,000 ng / mL, from 5 ng / mL to 800 ng / mL, from 10 ng / mL to 750 ng / mL, from 20 ng / mL to 500 ng / mL, from 50 ng / mL to 400 ng / mL, or from 100 ng / mL to 250 ng / mL, e.g. from 0.1 ng / mL to 1.0 ng / mL, from 1.0 ng / mL to 5.0 ng / mL, from 5.0 ng / mL to 10 ng / mL, from 10 ng / mL to 20 ng / mL, from 20 ng / mL to 100 ng / mL, from 20 ng / mL to 50 ng / mL, from 40 ng / mL to 70 ng / mL, from 50 ng / mL to 100 ng / mL, from 50 ng / mL to 60 ng / mL, from 100 ng / mL to 200 ng / mL, from 200 ng / mL to 500 ng / mL, or from 500 ng / mL to 1,000 ng / mL). In further aspects, the methods defined herein include IL- 15 typically at a concentration of less than 500 ng / mL, such as less than 250 ng / mL. In some aspects, the concentration of IL-15 is about 100 ng / mL. In some aspects defined herein, the IL-15 is included at a concentration from 5 ng / mL - 300 ng / mL (e.g., 5 ng / mL - 150 ng / mL) (e.g., 10 ng / mL - 150 ng / mL) (e.g., 10 ng / mL - 100 ng / mL). In some aspects, the IL-15 is included at a concentration from about 5 ng / mL - 250 ng / mL, about 5 ng / mL - 200 ng / mL, about 5 ng / mL - 150 ng / mL, about 10 ng / mL - 250 ng / mL, about 10 ng / mL - 200 ng / mL, about 10 ng / mL - 150 ng / mL, about 20 ng / mL - 250 ng / mL, about 20 ng / mL - 200 ng / mL, about 20 ng / mL - 150 ng / mL, about 30 ng / mL - 250 ng / mL, about 30 ng / mL - 200 ng / mL, about 30 ng / mL - 150 ng / mL, about 40 ng / mL - 250 ng / mL, about 40 ng / mL - 200 ng / mL, about 40 ng / mL -150 ng / mL, about 50 ng / mL - 250 ng / mL, about 50 ng / mL - 200 ng / mL, about 50 ng / mL - 150 ng / mL, about 10 ng / mL - 125 ng / mL, about 10 ng / mL - 100 ng / mL, or about 20 ng / mL - 100 ng / mL. In some aspects, the IL- 15 is included at a concentration from about 5 ng / mL to about 150 ng / mL. In some aspects, the IL- 15 is included at a concentration from about 5 ng / mL to about 125 ng / mL. In some aspects, the IL-15 is included at a concentration from about 5 ng / mL to about 100 ng / mL. In some aspects, the IL-15 is included at a concentration from about 10 ng / mL to about 150 ng / mL. In some aspects, the IL- 15 is included at a concentration from about 10 ng / mL to about 125 ng / mL. In some aspects, the IL-15 is included at a concentration from about 10 ng / mL to about 100 ng / mL. In some aspects, the IL- 15 is included at a concentration from about 15 ng / mL to about 150 ng / mL. In some aspects, the IL-15 is included at a concentration from about 5-300 (e.g., wherein the range is from 10-150). In some aspects, the IL-15 is included at a concentration from about 15 ng / mL to about 125 ng / mL. In some aspects, the IL- 15 is included at a concentration from about 15 ng / mL to about 100 ng / mL. In some aspects, the IL-15 is included at a concentration from about 20 ng / mL to about 150 ng / mL. In some aspects, the IL- 15 is included at a concentration from about 20 ng / mL to about 125 ng / mL. In some aspects, the IL-15 is included at a concentration from about 20 ng / mL to about 100 ng / mL. In some aspects, the IL-15 is included at a concentration from about 25 ng / mL to about 150 ng / mL. In some aspects, the IL- 15 is included at a concentration from about 25 ng / mL to about 125 ng / mL. In some aspects, the IL-15 is included at a concentration from about 25 ng / mL to about 100 ng / mL.

[0137] In some aspects, the IL- 15 is included at a concentration of about 5 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 6 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 7 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 8 ng / mL. In some aspects, the IL-15 is included at a concentration of about 9 ng / mL. In some aspects, the IL-15 is included at a concentration of about 10 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 11 ng / mL. In some aspects, the IL-15 is included at a concentration of about 12 ng / mL. In some aspects, the IL-15 is included at a concentration of about 13 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 14 ng / mL. In some aspects, the IL-15 is included at a concentration of about 15 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 16 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 17 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 18 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 19 ng / mL. In some aspects, the IL- 15 is included at aconcentration of about 20 ng / mL. In some aspects, the IL-15 is included at a concentration of about 21 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 22 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 23 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 24 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 25 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 26 ng / mL. In some aspects, the IL-15 is included at a concentration of about 27 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 28 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 29 ng / mL. In some aspects, the IL-15 is included at a concentration of about 30 ng / mL. In some aspects, the IL-15 is included at a concentration of about 35 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 40 ng / mL. In some aspects, the IL-15 is included at a concentration of about 45 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 50 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 60 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 70 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 80 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 90 ng / mL. In some aspects, the IL-15 is included at a concentration of about 100 ng / mL. In some aspects, the IL- 15 is included at a concentration of about 110 ng / mL. In some aspects, the IL-15 is included at a concentration of about 120 ng / mL. In some aspects, the IL-15 is included at a concentration of about 130 ng / mL. In some aspects, the IL-15 is included at a concentration of about 140 ng / mL. In some aspects, the IL-15 is included at a concentration of about 150 ng / mL.

[0138] In some aspects, the cells are contacted with IL-21. As used herein, "IL-21" refers to native or recombinant IL-21 or a variant thereof that acts as an agonist for one or more IL- 21 receptor (IL-21R) subunits (e.g. mutants, muteins, analogues, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof). Such agents can support proliferation of natural killer (NK) and cytotoxic (CD8+) T cells. Mature human IL-21 occurs as a 133 amino acid sequence (less the signal peptide, consisting of an additional 22 N-terminal amino acids). An IL-21 mutein is a polypeptide wherein specific substitutions to the Interleukin-21 protein have been made while retaining the ability to bind IL-21Ra, such as those described in US 9,388,241. The IL-21 muteins can be characterized by amino acid insertions, deletions, substitutions and modifications at one or more sites in or at the other residues of the native IL- 21 polypeptide chain. In accordance with this disclosure any such insertions, deletions, substitutions and modifications result in an IL-21 mutein that retains the IL-21R bindingactivity. Exemplary muteins can include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0139] Nucleic acid encoding human IL-21 can be obtained by conventional procedures such as polymerase chain reaction (PCR). The amino acid sequence of human IL-21 (Gene ID 59067) is found in Genbank under accession locator NC_000004.12. The murine (Mus musculus) IL-21 amino acid sequence (Gene ID 60505) is found in Genbank under accession locator NC_000069.6.

[0140] IL-21 can also refer to IL-21 derived from a variety of mammalian species, including, for example, human, simian, bovine, porcine, equine, and murine. Variants can comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as He, Vai, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known. Naturally occurring IL-21 variants are also encompassed by the disclosure. Examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the IL-21 protein, wherein the IL-21 binding property is retained. Alternate splicing of mRNA can yield a truncated but biologically active IL-21 protein. Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the IL-21 protein (generally from 1-10 amino acids). In some aspects, the terminus of the protein can be modified to alter its physical properties, for example, with a chemical group such as polyethylene glycol (Yang, et al. Cancer 1995. 76:687-694). In some aspects, the terminus or interior of the protein can be modified with additional amino acids (Clark-Lewis, et al. PNAS 1993. 90:3574-3577).

[0141] In further aspects, the methods defined herein include IL-21 typically at a concentration of at least 0.1 ng / mL, such as at least 1.0 ng / mL (e.g. from 0.1 ng / mL to 1,000 ng / mL, from 1.0 ng / mL to 100 ng / mL, from 1.0 ng / mL to 50 ng / mL, from 2 ng / mL to 50 ng / mL, from 3 ng / mL to 10 ng / mL, from 4 ng / mL to 8 ng / mL, from 5 ng / mL to 10 ng / mL, from 6 ng / mL to 8 ng / mL, e.g. from 0.1 ng / mL to 10 ng / mL, from 1.0 ng / mL to 5 ng / mL, from 1.0 ng / mL to 10 ng / mL, from 1.0 ng / mL to 20 ng / mL). In further aspects, the methods defined herein include IL-21 typically at a concentration of less than 100 ng / mL, such as less than 50ng / mL. In some aspects, the concentration of IL-21 is from 3 ng / mL to 40 ng / mL, from 4 ng / mL to 20 ng / mL, from 5 ng / mL to 15 ng / mL, or from 6 ng / mL to 10 ng / mL. In some aspects, the cells are not contacted with IL-21.

[0142] In some aspects, the IL-21 is included at a concentration of about 0.1 ng / mL. In some aspects, the IL-21 is included at a concentration of about 0.5 ng / mL. In some aspects, the IL-21 is included at a concentration of about 1 ng / mL. In some aspects, the IL-21 is included at a concentration of about 2 ng / mL. In some aspects, the IL-21 is included at a concentration of about 3 ng / mL. In some aspects, the IL-21 is included at a concentration of about 4 ng / mL. In some aspects, the IL-21 is included at a concentration of about 5 ng / mL. In some aspects, the IL-21 is included at a concentration of about 6 ng / mL. In some aspects, the IL-21 is included at a concentration of about 7 ng / mL. In some aspects, the IL-21 is included at a concentration of about 8 ng / mL. In some aspects, the IL-21 is included at a concentration of about 9 ng / mL. In some aspects, the IL-21 is included at a concentration of about 10 ng / mL. In some aspects, the IL-21 is included at a concentration of about 11 ng / mL. In some aspects, the IL-21 is included at a concentration of about 12 ng / mL. In some aspects, the IL-21 is included at a concentration of about 13 ng / mL. In some aspects, the IL-21 is included at a concentration of about 14 ng / mL. In some aspects, the IL-21 is included at a concentration of about 15 ng / mL. In some aspects, the IL-21 is included at a concentration of about 16 ng / mL. In some aspects, the IL-21 is included at a concentration of about 17 ng / mL. In some aspects, the IL-21 is included at a concentration of about 18 ng / mL. In some aspects, the IL-21 is included at a concentration of about 19 ng / mL. In some aspects, the IL-21 is included at a concentration of about 20 ng / mL. In some aspects, the IL-21 is included at a concentration of about 21 ng / mL. In some aspects, the IL-21 is included at a concentration of about 22 ng / mL. In some aspects, the IL-21 is included at a concentration of about 23 ng / mL. In some aspects, the IL-21 is included at a concentration of about 24 ng / mL. In some aspects, the IL-21 is included at a concentration of about 25 ng / mL. In some aspects, the IL-21 is included at a concentration of about 26 ng / mL. In some aspects, the IL-21 is included at a concentration of about 27 ng / mL. In some aspects, the IL-21 is included at a concentration of about 28 ng / mL. In some aspects, the IL-21 is included at a concentration of about 29 ng / mL. In some aspects, the IL-21 is included at a concentration of about 30 ng / mL. In some aspects, the IL-21 is included at a concentration of about 35 ng / mL. In some aspects, the IL-21 is included at a concentration of about 40 ng / mL. In some aspects, the IL-21 is included at a concentration of about 45 ng / mL. In some aspects, the IL-21 is included at a concentration of about 50 ng / mL. In some aspects,the IL-21 is included at a concentration of about 60 ng / mL. In some aspects, the IL-21 is included at a concentration of about 70 ng / mL. In some aspects, the IL-21 is included at a concentration of about 80 ng / mL. In some aspects, the IL-21 is included at a concentration of about 90 ng / mL. In some aspects, the IL-21 is included at a concentration of about 100 ng / mL. In some aspects, the IL-21 is included at a concentration of about 110 ng / mL. In some aspects, the IL-21 is included at a concentration of about 120 ng / mL. In some aspects, the IL-21 is included at a concentration of about 130 ng / mL. In some aspects, the IL-21 is included at a concentration of about 140 ng / mL. In some aspects, the IL-21 is included at a concentration of about 150 ng / mL.

[0143] In some aspects, the cells are contacted with IL-2. As used herein, "IL-2" refers to native or recombinant IL-2 or a variant thereof that acts as an agonist for one or more IL-2 receptor (IL-2R) subunits (e.g. mutants, muteins, analogues, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof). Such agents can support proliferation of an IL-2-dependent cell line, CTLL-2 (33; American Type Culture Collection (ATCC®) TIB 214). Mature human IL-2 occurs as a 133 amino acid sequence (less the signal peptide, consisting of an additional 20 N-terminal amino acids), as described in Fujita, et al. Cell 1986. 46.3:401- 407. An IL-2 mutein is a polypeptide wherein specific substitutions to the Interleukin-2 protein have been made while retaining the ability to bind IL-2RP, such as those described in US 2014 / 0046026. The IL-2 muteins can be characterized by amino acid insertions, deletions, substitutions and modifications at one or more sites in or at the other residues of the native IL- 2 polypeptide chain. In accordance with this disclosure any such insertions, deletions, substitutions and modifications result in an IL-2 mutein that retains the IL-2RP binding activity. Exemplary muteins can include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0144] Nucleic acid encoding human IL-2 can be obtained by conventional procedures such as polymerase chain reaction (PCR). The amino acid sequence of human IL-2 (Gene ID 3558) is found in Genbank under accession locator NP 000577.2 GI: 28178861. The murine (Mus musculus) IL-2 amino acid sequence (Gene ID 16183) is found in Genbank under accession locator NP_032392.1 GI: 7110653.

[0145] IL-2 can also refer to IL-2 derived from a variety of mammalian species, including, for example, human, simian, bovine, porcine, equine, and murine. Variants can comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as He, Vai, Leu, orAla for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known. Naturally occurring IL-2 variants are also encompassed by the disclosure. Examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the IL-2 protein, wherein the IL-2 binding property is retained. Alternate splicing of mRNA can yield a truncated but biologically active IL-2 protein. Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the IL-2 protein (generally from 1-10 amino acids). In some aspects, the terminus or interior of the protein can be modified to alter its physical properties, for example, with a chemical group such as polyethylene glycol (Yang, et al. Cancer 1995. 76: 687-694). In some aspects, the terminus or interior of the protein can be modified with additional amino acids (Clark-Lewis, el al. PNAS 1993. 90:3574-3577).

[0146] In some aspects, the methods defined herein include IL-2 typically at a concentration of at least 10 lU / mL, such as at least 100 lU / mL (e.g. from 10 lU / mL to 1,000 lU / mL, from 20 lU / mL to 800 lU / mL, from 25 lU / mL to 750 lU / mL, from 30 lU / mL to 700 lU / mL, from 40 lU / mL to 600 lU / mL, from 50 lU / mL to 500 lU / mL, from 75 lU / mL to 250 lU / mL, or from 100 lU / mL to 200 lU / mL, e.g. from 10 lU / mL to 20 lU / mL, from 20 lU / mL to 30 lU / mL, from 30 lU / mL to 40 lU / mL, from 40 lU / mL to 50 lU / mL, from 50 lU / mL to 75 lU / mL, from 75 lU / mL to 100 lU / mL, from 100 lU / mL to 150 lU / mL, from 150 lU / mL to 200 lU / mL, from 200 lU / mL to 500 lU / mL, or from 500 lU / mL to 1,000 lU / mL). In some aspects, the methods defined herein include IL-2 typically at a concentration of less than 1,000 lU / mL, such as less than 500 lU / mL. In some aspects, the concentration of IL-2 is about 100 lU / mL. In some aspects, the cells are not contacted with IL-2.

[0147] In some aspects, the cells are contacted with IL-ip. As used herein, "IL-ip" refers to native or recombinant IL-ip or a variant thereof that acts as an agonist for one or more IL-1 receptor (IL-1R) subunits (e.g. mutants, muteins, analogues, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof). IL-1 is a pro-inflammatory cytokine that plays a major role in a wide range of diseases, including inflammatory diseases. It consists of two molecular species, IL- la and IL-ip, which share only limited sequence identity but exert similar biological activities through binding to IL-1 receptor (type I and type II). Mature human IL-ip occurs as a 153 amino acid sequence after cleavage of 116 amino acids from the N-terminus in the precursor polypeptide by CASP1, as described in Andrei et al. (2004) PNAS 101(26): 9745-9750. An IL-ip mutein is a polypeptide wherein specific substitutions to the IL- ip protein have been made while retaining the ability to bind IL-1R. The IL-ip muteins can be characterized by amino acid insertions, deletions, substitutions and modifications at one or more sites in or at the other residues of the native IL-ip polypeptide chain. In accordance with this disclosure any such insertions, deletions, substitutions and modifications result in an IL- ip mutein that retains the IL-1R binding activity. Exemplary muteins can include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0148] Nucleic acid encoding human IL-ip can be obtained by conventional procedures such as polymerase chain reaction (PCR). The amino acid sequence of human IL-ip (Gene ID 3553) is found in Genbank under accession locator NP 000567 or in UniProt under accession number P01584. The murine (Mus musculus) IL-ip amino acid sequence (Gene ID 16176) is found in Genbank under accession locator NP 032387 or in UniProt under accession number P10749.

[0149] IL-ip can also refer to IL-ip derived from a variety of mammalian species, including, for example, human, simian, bovine, porcine, equine, and murine. Variants can comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as He, Vai, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known. Naturally occurring IL-ip variants are also encompassed by the disclosure. Examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the IL-ip protein, wherein the IL-ip binding property is retained.

[0150] In some aspects, the methods defined herein include IL-ip typically at a concentration of at least 100 lU / mL, such as at least 1,000 lU / mL (e.g. from 100 lU / mL to 8,000 lU / mL, from 250 lU / mL to 7,000 lU / mL, from 500 lU / mL to 6,000 lU / mL, or from 1,000 lU / mL to 5,000 lU / mL). In some aspects, the methods defined herein include IL-ip typically at a concentration of less than 8,000 lU / mL, such as less than 5,000 lU / mL. In some aspects, the concentration of IL-ip is about 4,500 lU / mL. In some aspects, the cells are not contacted with IL-ip.

[0151] In some aspects, the cells are contacted with IL-4. As used herein, "IL-4" refers to native or recombinant IL-4 or a variant thereof that acts as an agonist for one or more IL-4 receptor (IL-4R) subunits (e.g. mutants, muteins, analogues, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof). Such agents can support differentiation of naive helper T cells (ThO cells) to Th2 cells. Mature human IL-4 occurs as a 129 amino acid sequence (less the signal peptide, consisting of an additional 24 N-terminal amino acids). An IL-4 mutein is a polypeptide wherein specific substitutions to the Interleukin-4 protein have been made while retaining the ability to bind IL-4Ra, such as those described in US Patent No.6,313,272. The IL-4 muteins can be characterized by amino acid insertions, deletions, substitutions and modifications at one or more sites in or at the other residues of the native IL- 4 polypeptide chain. In accordance with this disclosure any such insertions, deletions, substitutions and modifications result in an IL-4 mutein that retains the IL-2Ra binding activity. Exemplary muteins can include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0152] Nucleic acid encoding human IL-4 can be obtained by conventional procedures such as polymerase chain reaction (PCR). The amino acid sequence of human IL-4 (Gene ID 3565) is found in Genbank under accession locator NG 023252. The murine (Mus musculus) IL-4 amino acid sequence (Gene ID 16189) is found in Genbank under accession locator NC_000077.6.

[0153] IL-4 can also refer to IL-4 derived from a variety of mammalian species, including, for example, human, simian, bovine, porcine, equine, and murine. Variants can comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as He, Vai, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known. Naturally occurring IL-4 variants are also encompassed by the disclosure. Examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the IL-4 protein, wherein the IL-4 binding property is retained. Alternate splicing of mRNA can yield a truncated but biologically active IL-4 protein. Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the IL-4 protein (generally from 1-10 amino acids).ILB.2. Serum and Serum Replacement

[0154] In some aspects, the first culture medium, the second culture medium, or both in are substantially free of serum (e.g. serum-free media or media containing a serum-replacement (SR)). In some aspects, the first culture medium is substantially free of serum. In some aspects, the first culture medium does not comprise serum. In some aspects, the first culture medium does not comprise human serum. In some aspects, the first culture medium does not comprise animal serum (e.g., bovine serum). In some aspects, the first culture medium comprises serum replacement.

[0155] In some aspects, the second culture medium is substantially free of serum. In some aspects, the second culture medium does not comprise serum. In some aspects, the second culture medium does not comprise human serum. In some aspects, the second culture medium does not comprise animal serum (e.g., bovine serum). In some aspects, the second culture medium comprises serum replacement.

[0156] In some aspects, the first culture medium and the second culture medium are substantially free of serum. In some aspects, the first culture medium and the second culture medium do not comprise serum. In some aspects, the first culture medium and the second culture medium do not comprise human serum. In some aspects, the first culture medium and the second culture medium do not comprise animal serum (e.g., bovine serum). In some aspects, the first culture medium and the second culture medium comprise serum replacement.

[0157] In some aspects, the first culture medium, the second culture medium, or both comprise serum. In some aspects, the first culture medium comprises serum. In some aspects, the first culture medium comprises human serum. In some aspects, the first culture medium comprises bovine serum.

[0158] In some aspects, the second culture medium, the second culture medium, or both in comprise serum. In some aspects, the second culture medium comprises serum. In some aspects, the second culture medium comprises human serum. In some aspects, the second culture medium comprises bovine serum.

[0159] In some aspects, the first culture medium and the second culture medium comprise serum. In some aspects, the first culture medium and the second culture medium comprise human serum. In some aspects, the first culture medium and the second culture medium comprise bovine serum.ILB.3. Culture Vessels

[0160] In some aspects, the methods described herein are performed in a vessel (e.g. an expansion vessel) comprising a gas permeable material. Such materials are permeable to gases such as oxygen, carbon dioxide, and / or nitrogen to allow gaseous exchange between the contents of the vessel and the surrounding atmosphere. It will be appreciated that references herein to "vessel" include culture dishes, culture plates, single-well dishes, multi-well dishes, multi-well plates, flasks, multi-layer flasks, bottles (such as roller bottles), bioreactors, bags, tubes, and the like. Such vessels have been used in methods involving expansion of nonadherent cells and other lymphocytes. However, vessels comprising a gas permeable material also surprisingly find utility in the isolation and expansion of y5 T cells which are considered as usually being adherent. The use of such vessels for culturing was found to greatly increase the yield of expanded y5 T cells. Such vessels were also found to preferentially support y5 T cells and other lymphocytes over fibroblasts and other stromal cells (e.g. epithelial cells), including adherent cell-types. See, e.g., Int'l Publication Nos. W02020095058 and W02020095059, each of which is incorporated by reference herein in its entirety. Thus, in some aspects, the vessels comprising a gas permeable material as defined herein preferentially support y5 T cells and other lymphocytes (e.g. aP T cells and / or NK cells). In some aspects, fibroblasts and / or other stromal cells (e.g. epithelial cells) are absent from cultures performed in vessels comprising a gas permeable material.

[0161] Such vessels comprising gas permeable materials can additionally comprise a gas permeable material that is non-porous. Thus, in some aspects, the gas permeable material in non-porous. In some aspects, the gas permeable material comprises a membrane film such as silicone, fluoroethylene polypropylene, polyolefin, or ethylene vinyl acetate copolymer. Furthermore, such vessels can comprise a portion of gas permeable material, gas permeable membrane film, or non-porous gas permeable material. Thus, according to some aspects, the vessel comprises a top, a bottom, and at least one sidewall, wherein at least part of the vessel bottom comprises a gas permeable material that is in a substantially horizontal plane when the top is above the bottom. In some aspects, the vessel includes a top, a bottom, and at least one sidewall, wherein at least a part of said bottom comprises the gas permeable material that is in a horizontal plane when said top is above said bottom. In some aspects, the vessel includes a top, a bottom, and at least one sidewall, wherein the at least one sidewall comprises a gas permeable material that is in a vertical plane when the top is above the bottom or in a horizontal plane when the top is not above the bottom. It will be appreciated that in some aspects, only aportion of the bottom or the side wall comprises a gas permeable material. Alternatively, the entire of the bottom or entire of the side wall can comprise a gas permeable material. In some aspects, the top of the vessel comprising a gas permeable material is sealed, for example by utilization of an O-ring. Such aspects will be appreciated to prevent spillage or reduce evaporation of the vessel contents. Thus, in some aspects, the vessel comprises a liquid sealed container comprising a gas permeable material to allow gas exchange. In some aspects, the top of the vessel comprising a gas permeable material is in the horizontal plane and above the bottom and is not sealed. Thus, in some aspects, the top is configured to allow gas exchange from the top of the vessel. In some aspects, the bottom of the gas permeable container is configured to allow gas exchange from the bottom of the vessel. In some aspects, the vessel comprising a gas permeable material is a liquid sealed container and further comprises inlet and outlet ports or tubes. Thus, in some aspects, the vessel comprising a gas permeable material includes a top, a bottom, and optionally at least one sidewall, wherein at least a part of the top and the bottom comprises a gas permeable material and, if present, at least part of the at least one sidewall comprises a gas permeable material. Example vessels are described in W02005035728 and US9255243, each of which is incorporated herein by reference in its entirety. These vessels are also commercially available, such as the G-REX® cell culture devices marketed by Wilson Wolf Manufacturing, such as the G-REX6 well-plate, G-REX24 well-plate and the G-REX10 vessel.II.C. Cell Modification

[0162] In some aspects, the population of cells is genetically modified. In some aspects, the population of cells is genetically modified prior to culture in the first culture medium. In some aspects, the population of cells is genetically modified prior to restimulation. In some aspects, the population of cells is genetically modified following restimulation. In some aspects, the population of cells is genetically modified at the start of the culture in the second culture medium. In some aspects, the population of cells is genetically modified during culture in the second culture medium. In some aspects, the population of cells is genetically modified at the end of the culture in the second culture medium.

[0163] In some aspects, the population of cells is transduced with an exogenous nucleic acid. In some aspects, the population of cells is transduced with an exogenous nucleic acid prior to culture in the first culture medium. In some aspects, the population of cells is transduced with an exogenous nucleic acid prior to restimulation. In some aspects, thepopulation of cells is transduced with an exogenous nucleic acid following restimulation. In some aspects, the population of cells is transduced with an exogenous nucleic acid at the start of the culture in the second culture medium. In some aspects, the population of cells is transduced with an exogenous nucleic acid during culture in the second culture medium. In some aspects, the population of cells is transduced with an exogenous nucleic acid at the end of the culture in the second culture medium.

[0164] In some aspects, the exogenous nucleic acid encodes a membrane-bound protein (e.g., a cell surface receptor, such as a chimeric antigen receptor (CAR), an aP TCR, a natural cytotoxicity receptor (e.g., NKp30, NKp44, or NKp46), a cytokine receptor (e.g., IL- 12 receptor), a chemokine receptor (e.g., CCR2 receptor), and / or a membrane-bound ligand or cytokine (e.g., membrane-bound IL- 15, membrane-bound IL-7, membrane-bound CD40L, membrane-bound 4- IBB, membrane-bound 4-1BBL, membrane bound CCL19), a soluble protein (e.g., soluble ligands or cytokines, e.g., soluble IL-15, soluble IL-7, soluble IL-12, soluble CD40L, soluble 4-1BBL, and / or soluble CCL19), a selectable marker (e.g., a reporter gene), or a suicide gene. In some instances, the disclosure provides a population of y5 T cells engineered to express IL-15RP, a CAR, and one or more additional transgene-encoded proteins (e.g., an armor protein).

[0165] Some aspects of the present disclosure are directed to a method for engineering y5 T cells, said method comprising the steps of: (i) preparing a composition enriched for y5 T cells according to a method disclosure herein; (ii) transducing the y5 T cells with an exogenous nucleic acid for expression in the y5 T cells; and (iii) culturing the transduced y5 T cells to expand the engineered y5 T cells. In some aspects, step (iii) comprises culturing the transduced composition in the absence of feeder cells. In some aspects, step (iii) comprises culturing the transduced composition in the presence of feeder cells.

[0166] The y5 T cells described herein can also be gene engineered for enhanced therapeutic properties. For example, the cells can be engineered to express exogenous nucleic acids which encode cell surface receptors and / or secretory proteins.

[0167] One example of cell engineering includes chimeric antigen receptor T cell (CAR- T) therapy. This can involve the generation of engineered receptors (such as chimeric antigen receptors or modified T cell receptors) to re-program the T cell with a new specificity, e.g. the specificity of a monoclonal antibody. The engineered receptor can make the T cells specific for malignant cells and therefore useful for cancer immunotherapy. For example, the T cells can recognize cancer cells expressing a tumor antigen, such as a tumor specific antigen that isnot expressed by normal somatic cells from the subject tissue, a tumor associated antigen which is preferentially overexpressed on cancer cells compared to healthy somatic cells or antigens expressed in the context of stress events such as oxidative stress, DNA damage, UV radiation, EGF receptor stimulation; or other means for identifying cancerous versus noncancerous cells. Thus, the CAR-modified T cells can be used for adoptive T cell therapy of, for example, cancer patients.

[0168] According to some aspects of the disclosure, there is provided a method for engineering y5 T cells, said method comprising the steps of: (i) preparing a composition enriched for y5 T cells using a method as described herein; (ii) transducing the composition with an exogenous nucleic acid for expression in the y5 T cells; and (iii) culturing the transduced composition to expand the engineered y5 T cells.

[0169] It will be understood that transducing the composition with an exogenous nucleic acid for expression in the y5 T cells according to step (ii) thereby produces engineered y5 T cells expressing the exogenous nucleic acid. In some aspects, the exogenous nucleic acid encodes a surface receptor. In some aspects, the surface receptor is a CAR recognizing a tumor antigen. In some aspects, the method comprises: (i) preparing a composition enriched for y5 T cells according to the methods as described herein; (ii) transducing the composition to express a chimeric antigen receptor (CAR); and (iii) culturing the transduced composition to expand the engineered y5 T cells.

[0170] In some aspects, step (ii) is performed using between 1 xlO6and 1 xlO8TU / ml, such as about 1 xlO6, about 5 xlO6, about 1 xlO7, about 5 x 107or about 1 xlO8TU / ml of viral vector. In some aspects, step (ii) is performed using 1 x 107TU / ml of viral vector. In other aspects, step (ii) is performed using an MOI of viral vector between 0.5 and 50, such as an MOI of about 0.5, about 1, about 1.5, about 2.5, about 5, about 10, about 25, about 40 or about 50. In some aspects, step (ii) is performed using an MOI of viral vector of 2.5. In another aspect, step (ii) is performed using an MOI of viral vector of 5. In some aspects, step (ii) is performed using an MOI of viral vector of 10.

[0171] In some aspects, the CAR comprises an antigen-binding domain that specifically binds a tumor antigen. In some aspects, the tumor antigen is a tumor specific antigen that is not expressed by normal somatic cells from the subject tissue. In some aspects, the tumor antigen is a tumor associated antigen which is preferentially overexpressed on cancer cells compared to healthy somatic cells. In some aspects, the tumor antigen is an antigen expressed by a cell experiencing oxidative stress, DNA damage, UV radiation, EGF receptor stimulation, or anycombination thereof. In some aspects, the tumor antigen is an antigen expressed by a solid tumor.

[0172] In some aspects, the tumor associated antigen is an antigen associated with a solid tumor. Thus, in some aspects the tumor and / or cancer is a solid tumor. Constitutive expression of CD70, a member of the tumor necrosis family, has been described in both hematological and solid cancers where it increases the survival of tumor cells and regulatory T cells within the tumor microenvironment by signaling through its receptor, CD27. Thus, in some aspects the solid tumor is a CD70+tumor. It will be appreciated that CD70 can be used to target engineered y5 T cells to said tumors. Therefore, in a yet further aspect the tumor associated antigen is CD70.

[0173] In an alternative aspect, the tumor associated antigen is mesothelin (also referred to "MSLN" herein). Mesothelin is a 40 kDa protein that is expressed in mesothelial cells and is overexpressed in several tumors, including mesothelioma, ovarian cancer, pancreatic adenocarcinoma, lung adenocarcinoma and cholangiocarcinoma. It has therefore been proposed as a tumor marker or tumor associated antigen which can be targeted in immunotherapy (Hassan et al. Clin. Cancer Res., 2004, 10(12):3937-3942). The expression of mesothelin in these tumors can contribute to the implantation and peritoneal spread of tumors by cell adhesion (Rump et al., Biological Chemistry, 2004, 279(10):9190-9198).

[0174] In some aspects, the CAR targets CD19, TRAC, TCRP, BCMA, CLL-1, CS1, CD38, CD 19, TSHR, CD 123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPC AM, B7H3, KIT, IL- 13Ra2, mesothelin, IL-1 IRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, S SEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF -I receptor, CAIX, LMP2, gplOO, bcr- abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT- 2, Fos- related antigen 1, p53, p53 mutant, prostein, surviving, telomerase, PCTA- 1 / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, humantelomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3s, CD4, CD5, CD7, the extracellular portion of the APRIL protein, or any combinations thereof. In some aspects, the TCR targets AFP, CD 19, TRAC, TCRP, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, R0R1, R0R2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL- 13Ra2, mesothelin, IL-1 IRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, S SEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl- GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CX0RF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD- CT- 2, Fos-related antigen 1, p53, p53 mutant, prostein, surviving, telomerase, PCTA- 1 / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML- IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE- 1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70- 2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3s, CD4, CD5, CD7, the extracellular portion of the APRIL protein, or any combinations thereof. In some aspects, the CAR comprises an antigenbinding domain that specifically binds CD 19. In some aspects, the CAR comprises an antigenbinding domain that specifically binds mesothelin.

[0175] In some aspects, the population of cells is transduced with a viral vector comprising the exogenous nucleic acid molecule (e.g., encoding a CAR). In some aspects, the viral vector is a retroviral vector. In some aspects, the viral vector is a lentiviral vector. In some aspects, the viral vector comprises a gammaretroviral vector or a lentiviral vector. In some aspects, the gammaretroviral vector is a murine stem cell virus (MSCV) or Moloney Murine Leukaemia Virus (MLV). In some aspects, the viral vector is pseudotyped with an envelope other than vesicular stomatitis virus-G (VSV-G). In some aspects, the envelope comprises a betaretroviral envelope. In some aspects, the envelope comprises a baboon endogenous virus (BaEV) or RD114. In some such aspects, the cell stably expresses a transgene encoded by the exogenousnucleic acid molecule. In some aspects, the cell transiently expresses a transgene encoded by the exogenous nucleic acid molecule.

[0176] In some aspects, the disclosure features a cell population (e.g., an isolated cell population) of engineered y5 T cells (e.g., atleast 10, 102, 103, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, or 1013cells), wherein at least 3% (e.g., at least 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or substantially all) of the cell population are of engineered y5 T cells expressing a transgene (e.g., IL-15RP and / or a CAR and / or one or more additional proteins).

[0177] Some aspects of the present disclosure are directed to a population of y5 T cells (e.g., a population of Vdl+ y5 cells derived from blood or skin) comprising y5 T cells comprising a heterologous nucleic acid encoding a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds human mesothelin. In some aspects, the CAR comprises an antigen-binding domain that specifically binds human mesothelin (e.g., wherein the antigen-binding domain comprises a sequence of any of SEQ ID NOs: 1 - 8). In some aspects, the cells can further comprise a 4- IBB and CD3 zeta domain. In still some aspects, the cells are directed to a population of y5 T cells (e.g., a population of Vdl+ y5 cells derived from blood or skin) comprising y5 T cells comprising a heterologous nucleic acid encoding a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds human mesothelin (e.g., wherein the antigen-binding domain comprises a sequence of any of SEQ ID NOs: 1 - 8), wherein the y5 T cells further comprise a CD8 transmembrane domain. In yet some aspects, the cells are directed to a population of y5 T cells (e.g., a population of Vdl+ y5 cells derived from blood or skin) comprising y5 T cells comprising a heterologous nucleic acid encoding a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds human mesothelin (e.g., wherein the antigen-binding domain comprises a sequence of any of SEQ ID NOs: 1 - 8), wherein the y5 T cells further comprise (a) IL-15R-beta or (b)(i) IL-15R-alpha tethered with an IL- 15 or variant thereof and (ii) IL-15R-beta. In some aspects, the y5 T cells described herein comprising a heterologous nucleic acid encoding a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds human mesothelin (e.g., wherein the antigen-binding domain comprises a sequence of any of SEQ ID NOs: 1 - 8), can further comprise a 4- IBB and CD3 zeta domain and / or a CD8 domain and (a) IL-15R-beta or (b)(i) IL-15R-alpha tethered with an IL- 15 or variant thereof and (ii) IL-15R- beta.ILE. Methods of Treatment

[0178] The expanded / engineered y5 T cell population obtained by the methods described herein can be used as a medicament, for example for adoptive T cell therapy. As such, some aspects of the present disclosure are directed to methods of treating a subject in need thereof, comprising administering to the subject a population of cells obtained by a method disclosed herein. In some aspects, the method comprises transfer of an expanded / engineered population obtained by the methods into a patient. The therapy can be autologous, i.e. the y5 T cells can be transferred back into the same patient from which they were obtained, or the therapy can be allogeneic, i.e. the y5 T cells from one person can be transferred into a different patient. In instances involving allogeneic transfer, the expanded / engineered population can be substantially free of aP T cells. For example, aP T cells can be depleted from the expanded / engineered population, e.g. after engineering, using any suitable means known in the art (e.g. by negative selection, e.g. using magnetic beads). A method of treatment can include: providing a sample obtained from a donor individual; expanding and / or engineering the y5 T cells as described herein to produce an expanded / engineered population; and administering the expanded / engineered population of y5 T cells to a recipient individual.

[0179] In some aspects, the expanded / engineered y5 T cell population obtained by methods described herein has increased persistence of in vivo cytotoxicity as compared to similarly engineered cells obtained by standard methods. In some aspects, the expanded / engineered y5 T cell population obtained by methods described herein are capable of in vivo cytotoxicity for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, or at least about 21 days. In some aspects, the expanded / engineered y5 T cell population obtained by methods described herein are capable of in vivo cytotoxicity for at least about 7 days. In some aspects, the expanded / engineered y5 T cell population obtained by methods described herein are capable of in vivo cytotoxicity for at least about 8 days. In some aspects, the expanded / engineered y5 T cell population obtained by methods described herein are capable of in vivo cytotoxicity for at least about 9 days. In some aspects, the expanded / engineered y5 T cell population obtained by methods described herein are capable of in vivo cytotoxicity for at least about 10 days. In some aspects, the expanded / engineered y5 T cell population obtained by methods described herein are capable of in vivo cytotoxicity for at least about 11 days. In some aspects, the expanded / engineered y5T cell population obtained by methods described herein are capable of in vivo cytotoxicity for at least about 12 days. In some aspects, the expanded / engineered y5 T cell population obtained by methods described herein are capable of in vivo cytotoxicity for at least about 13 days. In some aspects, the expanded / engineered y5 T cell population obtained by methods described herein are capable of in vivo cytotoxicity for at least about 14 days. In some aspects, the expanded / engineered y5 T cell population obtained by methods described herein are capable of in vivo cytotoxicity for at least about 15 days. In some aspects, the expanded / engineered y5 T cell population obtained by methods described herein are capable of in vivo cytotoxicity for at least about 16 days. In some aspects, the expanded / engineered y5 T cell population obtained by methods described herein are capable of in vivo cytotoxicity for at least about 17 days. In some aspects, the expanded / engineered y5 T cell population obtained by methods described herein are capable of in vivo cytotoxicity for at least about 18 days. In some aspects, the expanded / engineered y5 T cell population obtained by methods described herein are capable of in vivo cytotoxicity for at least about 19 days. In some aspects, the expanded / engineered y5 T cell population obtained by methods described herein are capable of in vivo cytotoxicity for at least about 20 days. In some aspects, the expanded / engineered y5 T cell population obtained by methods described herein are capable of in vivo cytotoxicity for at least about 21 days.

[0180] In some aspects, the subject is afflicted with a cancer. "Cancer," as used herein, refers to the abnormal growth or division of cells. Generally, the growth and / or life span of a cancer cell exceeds, and is not coordinated with, that of the normal cells and tissues around it. Cancers can be benign, pre-malignant or malignant. Cancer occurs in a variety of cells and tissues, including the oral cavity (e.g. mouth, tongue, pharynx, etc.), digestive system (e.g. oesophagus, stomach, small intestine, colon, rectum, liver, bile duct, gall bladder, pancreas, etc.), respiratory system (e.g. larynx, lung, bronchus, etc.), bones, joints, skin (e.g. basal cell, squamous cell, meningioma, etc.), breast, genital system, (e.g. uterus, ovary, prostate, testis, etc.), urinary system (e.g. bladder, kidney, ureter, etc.), eye, nervous system (e.g. brain, etc.), endocrine system (e.g. thyroid, etc.), and haematopoietic system (e.g. lymphoma, myeloma, leukaemia, acute lymphocytic leukaemia, chronic lymphocytic leukaemia, acute myeloid leukaemia, chronic myeloid leukaemia, etc.).

[0181] In some aspects, the cancer is a solid malignancy (also referred to herein as a solid tumor). Solid tumors can occur in several places, for example in the tissues, bones, muscles and / or organs. It will be understood that a solid tumor does not include cancers of the blood (i.e. haematological cancers). In an alternative aspect, the cancer is a haematological cancer.

[0182] In some aspects, the cancer comprises bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)), uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia (ALL), chronic myelogenous leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, or any combination thereof. In some aspects, the cancer is locally advanced. In some aspects, the cancer is metastatic. In some aspects, the cancer is refractory. In some aspects, the cancer is relapsed. In some aspects, the cancer is refractory or relapsed following one or more prior anti-cancer therapy. In some aspects, the one or more prior anti-cancer therapies comprise a standard of care therapy.

[0183] The patient or subject to be treated is preferably a human cancer patient (e.g. a human cancer patient being treated for a solid tumor) or a virus-infected patient (e.g. a CMV- infected or HIV infected patient). In some instances, the patient has and / or is being treated for a solid tumor. Because they are normally resident in non-hematopoietic tissues, tissue-resident V51 T cells are also more likely to home to and be retained within tumor masses than their systemic blood-resident counterparts and adoptive transfer of these cells is likely to be more effective at targeting solid tumors and potentially other non-hematopoietic tissue-associated immunopathologies.

[0184] As y5 T cells are non-MHC restricted, they do not recognize a host into which they are transferred as foreign, which means that they are less likely to cause graft-versus-host disease. This means that they can be used "off the shelf and transferred into any recipient, e.g. for allogeneic adoptive T cell therapy.

[0185] y5 T cells obtained by methods described herein express NKG2D and respond to a NKG2D ligand (e.g. MICA), which is strongly associated with malignancy. They also express a cytotoxic profile in the absence of any activation and are therefore likely to be effective at killing tumor cells. For example, the expanded / engineered y5 T cells obtained as described herein can express one or more, preferably all of IFN-y, TNF-a, GM-CSF, CCL4, IL-13, Granulysin, Granzyme A and B, and Perforin in the absence of any activation. IL-17A can not be expressed.

[0186] The expanded / engineered y5 T cells obtained by the methods described herein can be suitable as an "off-the-shelf immunotherapeutic reagent. These cells possess innate-like killing, have no MHC restriction and display improved homing to and / or retention within tumors than do other T cells.

[0187] In some aspects, a method of treatment of an individual with a solid tumor in a non- hematopoietic tissue can include: expanding / engineering y5 T cells from a sample from the individual as described herein to produce an expanded / engineered population; and administering the expanded / engineered population of y5 T cells to the individual. In alternative aspects, the method of treatment comprises expanding / engineering y5 T cells from a sample from a different individual as described herein to produce an expanded / engineered population; and administering the expanded / engineered population of y5 T cells to the individual with a solid tumor. In some aspects, the amount of expanded / engineered y5 T cells administered to the individual is a therapeutically effective amount.

[0188] In further aspects, the method of treatment and / or the therapeutically effective amount comprises those disclosed in W02020095058 or W02020095059, the contents of each of which are incorporated by reference herein in their entirety.III. Compositions of the DisclosureIII.A. Cells

[0189] In some aspects, the population of cells cultured according to the methods disclosed herein is obtained from a biological sample obtained from a human subject. In some aspects, the biological sample is a haematopoietic sample or a fraction thereof. As used herein, a "haematopoietic sample" refers to a biological sample comprising or consisting of peripheral blood, umbilical cord blood, lymphoid tissue, thymus, bone marrow, spleen, node tissue or fractions thereof.

[0190] In some aspects, the biological sample is selected from peripheral blood, umbilical cord blood, lymphoid tissue, thymus, bone marrow, spleen, lymph node tissue, or fractions thereof. In some aspects, the biological sample comprises peripheral blood or a fraction thereof. In some aspects, the biological sample comprises PBMCs. In some aspects, the biological sample consists of PBMCs. In some aspects, the biological sample comprises low density mononuclear cells (LDMCs). In some aspects, the biological sample consists of LDMCs.

[0191] In some aspects, the biological sample is a non-haematopoietic tissue. As used herein, a "non-haematopoietic sample" refers to a biological sample comprising obtained from a subject that is other than peripheral blood, umbilical cord blood, lymphoid tissue, thymus, bone marrow, spleen, node tissue or fractions thereof. In some aspects, the non-haematopoietic sample comprises a skin (e.g. human skin) sample. In some aspects, the non-haematopoietic sample comprises a sample obtained from the gastrointestinal tract (e.g. human gut or human colon). In some aspects, the non-haematopoietic sample comprises a gut (e.g. human gut) sample. In some aspects, the non-haematopoietic sample comprises a colon (e.g. human colon) sample. In some aspects, the non-haematopoietic sample comprises a mammary gland (e.g. human mammary gland) sample. In some aspects, the non-haematopoietic sample comprises a lung (e.g. human lung) sample. In some aspects, the non-haematopoietic sample comprises a prostate (e.g. human prostate) sample. In some aspects, the non-haematopoietic sample comprises a liver (e.g. human liver) sample. In some aspects, the non-haematopoietic sample comprises a spleen (e.g. human spleen) sample. In some aspects, the non-haematopoietic sample comprises a pancreas (e.g. human pancreas) sample. In some aspects, the non- haematopoietic sample comprises a uterus (e.g. human gut) sample. In some aspects, the non- haematopoietic sample comprises a vagina (e.g. human vagina) sample. In some aspects, the non-haematopoietic sample comprises a sample obtained from a cutaneous, mucosal or serous membranes (e.g. in a human gut). As used herein, a non-haematopoietic sample does not need to be free from any residual blood cells, i.e., in some aspects, a non-haematopoietic sample comprises some blood.

[0192] In some aspects, the biological sample comprises a cancer tissue sample, e.g., from a tumor of the breast or prostate, in particular a human cancer tissue sample. In other aspects, the biological sample is not obtained from cancer tissue (e.g. a tissue without a substantial number of tumor cells). For example, the sample can be from a region of skin (e.g. healthy skin) separate from a nearby or adjacent cancer tissue. Thus, in some aspects, the y5 T cells are not obtained from human cancer tissue.

[0193] In some aspects the biological sample has been obtained from a human. In an alternative aspect, the biological sample has been obtained from a non-human animal subject. In some aspects, the composition of y5 T cells is derived from a single donor. In some aspects, the composition is derived from multiple donors, i.e., the composition is a "pooled" composition.

[0194] In some aspects, the single or multiple donors comprise a subject that is to be treated with the cell populations or compositions of the disclosure. In some aspects, the single or multiple donors do not comprise a subject that is to be treated with the cell populations or compositions of the disclosure.

[0195] Any methods can be used to obtain the non-haematopoietic samples. Examples of such methods include scalpel explant or punch biopsy and can vary in size according to the method. In some aspects, the non-haematopoietic tissue sample is obtained by punch biopsy.

[0196] In some aspects, the y5 T cells expanded by the methods defined herein comprise a population of V51 T cells. In some aspects, the methods disclosed herein result in depletion of aP T cells, i.e. the composition enriched for y5 T cells comprises few or no aP T cells. In some aspects, the methods disclosed herein comprise one or more aP T cells depletion steps. In some aspects, there is one aP T cells depletion step during the expansion method. In still further aspects, there are more than one (e.g., two) depletion steps during the expansion method. In yet a further aspect, the methods disclosed herein comprise one more depletion steps wherein a second depletion step is performed anywhere from Day 2 - Day 21 (e.g., on Day 2, Day 3, Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, Day 11, Day 12, Day 13, Day 14, Day 15, Day 16, Day 17, Day 18, Day 19, Day 20 or Day 21). In a further aspect, the methods disclosed herein comprise two aP T cells depletion steps during the expansion process.

[0197] y5 T cells represent a subset of T cells that express on their surface a distinct, defining y5 T-cell receptor (TCR). This TCR is made up of one gamma (y) and one delta (5) chain. Human y5 T cells can be broadly classified as one or two types — peripheral bloodresident y5 T cells and non-haematopoietic tissue-resident y5 T cells. Most blood-resident y5 T cells express a V52 TCR, whereas this is less common among tissue-resident y5 T cells, which more frequently use V51 and / or other V5 chains.

[0198] In some aspects, y5 T cells cultured according to the methods disclosed herein include V51 cells, V52 cells, V53 cells, V55 cells, and V58 cells. In some aspects, the y5 T cells are derived from a population of V51 cells or V52 cells. In some aspects, the y5 T cellsare derived from a population of non-V51 / V52 T cells. In some aspects, the y5 T cells are derived from a mixed population of V51 cells and V52 cells.

[0199] The y5 T cells described herein (e.g., endogenous y5 T cells or primed y5 T cells) can lack a vesicular stomatis virus G glycoprotein (VSV-G) entry receptor (e.g., LDL). The y5 T cell (e.g., endogenous y5 T cells or primed y5 T cells) can express ASCT-1 and / or ASCT-2. The expression of ASCT-1 and / or ASCT-2 can permit transduction with a betaretroviral pseudotyped vector (e.g., BaEV and RD114). The lack of expression of VSV-G can prevent transduction with a VSV-G pseudotyped vector.

[0200] In some aspects, the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, or at least about 21 days. In some aspects, the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 7 days. In some aspects, the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 8 days. In some aspects, the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 9 days. In some aspects, the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 10 days. In some aspects, the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 11 days. In some aspects, the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 12 days. In some aspects, the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 13 days. In some aspects, the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 14 days. In some aspects, the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 15 days. In some aspects, the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 16 days. In some aspects, the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 17 days. In some aspects, the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 18 days. In some aspects, the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 19 days. In some aspects, the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 20 days. In some aspects, the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 21 days.

[0201] In some aspects, the population of expanded y5 T cells comprises NK cells. The presence of NK cells in the composition can be advantageous as these cells are also effective cytotoxic cells. NK cells (also known as large granular lymphocytes (LGL)) are cytotoxiclymphocytes of the innate immune system. They provide rapid responses to e.g. virus-infected cells and tumor cells independently of MHC expression on the surface of the target cell. Therefore, similarly to y5 T cells, the recognition of target cells by NK cells is not MHC restricted and they are not allo-HLA reactive, meaning HLA matching of patients is not required for NK cell-based therapies.

[0202] In some aspects, at least 80%, such as 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, of the cells present in the cell population comprise NK cells and y5 T cells (e.g., V51 T cells). In some aspects, at least 80%, such as 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, of the cells present in the cell population obtained by the methods described herein comprise NK cells and y5 T cells. In some aspects, at least 85% of the cells present in the cell population comprise NK and y5 T cells. In some aspects, at least 90% of the cells present in the cell population comprise NK cells and y5 T cells. In a yet further aspect, at least 95% of the cells present in the cell population comprise NK cells and y5 T cells.

[0203] In some aspects, at least 50% of the cells present in the composition are y5 T cells.In some aspects, the composition comprises at least about 50% y5 T cells, such as at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% y5 T cells. In some aspects, the composition comprises at least about 60% y5 T cells, such as greater than about 70% y5 T cells. In other aspects, at least 80%, such as at least 90%, of cells present in the composition are y5 T cells. In some aspects, the composition comprises at least about 82% y5 T cells.

[0204] In some aspects, the composition comprises at least about 30% V51 T cells, such as at least about 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50% V51 T cells. In some aspects, the composition comprises at least about 40% V51 T cells, such as at least about 50% V51 T cells. In some aspects, the composition comprises from about 50% to about 70% V51 T cells. In some aspects, the composition comprises about 60% V51 T cells.

[0205] In some aspects, the composition comprises less than about 1% aP T cells. In some aspects, the composition comprises less than about 1% aP T cells, less than about 0.9% aP T cells, less than about 0.8% aP T cells, less than about 0.7% aP T cells, less than about 0.6% aP T cells, less than about 0.5% aP T cells, less than about 0.4% aP T cells, less than about 0.3% aP T cells, less than about 0.2% aP T cells, less than about 0.1% aP T cells, less than about 0.09% aP T cells, less than about 0.08% aP T cells, less than about 0.07% aP T cells, less than about 0.06% aP T cells, less than about 0.05% aP T cells, less than about 0.04% aP T cells, less than about 0.05% aP T cells, less than about 0.04% aP T cells, less than about 0.03% aPT cells, less than about 0.02% aP T cells, less than about 0.01% aP T cells, less than about 0.009% aP T cells, less than about 0.008% aP T cells, less than about 0.007% aP T cells, less than about 0.006% aP T cells, less than about 0.005% aP T cells. In some aspects, the composition comprises less than about 0.9% aP T cells. In some aspects, the composition comprises less than about 0.8% aP T cells. In some aspects, the composition comprises less than about 0.7% aP T cells. In some aspects, the composition comprises less than about 0.6% aP T cells. In some aspects, the composition comprises less than about 0.5% aP T cells. In some aspects, the composition comprises less than about 0.4% aP T cells. In some aspects, the composition comprises less than about 0.3% aP T cells. In some aspects, the composition comprises less than about 0.2% aP T cells. In some aspects, the composition comprises less than about 0.1% aP T cells. In some aspects, the composition comprises less than about 0.09% aP T cells. In some aspects, the composition comprises less than about 0.08% aP T cells. In some aspects, the composition comprises less than about 0.07% aP T cells. In some aspects, the composition comprises less than about 0.06% aP T cells. In some aspects, the composition comprises less than about 0.05% aP T cells. In some aspects, the composition comprises less than about 0.04% aP T cells. In some aspects, the composition comprises less than about 0.03% aP T cells. In some aspects, the composition comprises less than about 0.02% aP T cells. In some aspects, the composition comprises less than about 0.01% aP T cells. In some aspects, the composition comprises a concentration of aP T cells that is below detectable limits.

[0206] In some aspects, at least about 50% of cells isolated prior to the expansion methods disclosed herein are aP T cells, and less than about 1% of the cells are aP T cells in the composition following expansion according to the methods disclosed herein. In some aspects, at least about 45% of cells isolated prior to the expansion methods disclosed herein are aP T cells, and less than about 1% of the cells are aP T cells in the composition following expansion according to the methods disclosed herein. In some aspects, at least about 40% of cells isolated prior to the expansion methods disclosed herein are aP T cells, and less than about 1% of the cells are aP T cells in the composition following expansion according to the methods disclosed herein. In some aspects, at least about 35% of cells isolated prior to the expansion methods disclosed herein are aP T cells, and less than about 1% of the cells are aP T cells in the composition following expansion according to the methods disclosed herein. In some aspects, at least about 30% of cells isolated prior to the expansion methods disclosed herein are aP T cells, and less than about 1% of the cells are aP T cells in the composition following expansion according to the methods disclosed herein. In some aspects, at least about 25% of cells isolatedprior to the expansion methods disclosed herein are aP T cells, and less than about 1% of the cells are aP T cells in the composition following expansion according to the methods disclosed herein. In some aspects, at least about 20% of cells isolated prior to the expansion methods disclosed herein are aP T cells, and less than about 1% of the cells are aP T cells in the composition following expansion according to the methods disclosed herein. In some aspects, at least about 15% of cells isolated prior to the expansion methods disclosed herein are aP T cells, and less than about 1% of the cells are aP T cells in the composition following expansion according to the methods disclosed herein. In some aspects, at least about 10% of cells isolated prior to the expansion methods disclosed herein are aP T cells, and less than about 1% of the cells are aP T cells in the composition following expansion according to the methods disclosed herein. In some aspects, at least about 5% of cells isolated prior to the expansion methods disclosed herein are aP T cells, and less than about 1% of the cells are aP T cells in the composition following expansion according to the methods disclosed herein.

[0207] In some aspects, at least 50% of the expanded y5 T cells present in the cell population express CD56. In some aspects, at least about 60%, such as at least 65%, 70%, 75% or 80% of the expanded y5 T cells present in the cell population express CD56. In some aspects, at least 80% of the expanded y5 T cells present in the cell population express CD56, for example after a period of about 14 days (such as 14 days) of culture.

[0208] Some aspects of the present disclosure are directed to a population of expanded y5 T cells prepared according to a method disclosed herein. In some aspects, at least 10% of the y5 T cells present in the population of expanded y5 T cells express CD56. In some aspect, at least 20% of the y5 T cells present in the population of expanded y5 T cells express CD56. In some aspect, at least 30% of the y5 T cells present in the population of expanded y5 T cells express CD56. In some aspect, at least 40% of the y5 T cells present in the population of expanded y5 T cells express CD56. In some aspect, at least 50% of the y5 T cells present in the population of expanded y5 T cells express CD56. In some aspect, at least 60% of the y5 T cells present in the population of expanded y5 T cells express CD56. In some aspect, at least 70% of the y5 T cells present in the population of expanded y5 T cells express CD56. In some aspect, at least 80% of the y5 T cells present in the population of expanded y5 T cells express CD56. In some aspect, at least 90% of the y5 T cells present in the population of expanded y5 T cells express CD56.

[0209] CD56, also known as Neural Cell Adhesion Molecule (NCAM), is an adhesion molecule of the immunoglobulin (Ig) superfamily that correlates with high cytotoxicity in NKcells, aP T cells and y5 T cells. It has been shown to participate in cis and trans binding to itself which contributes to lymphocyte activation. It has also been shown to be fundamental for immunological synapse formation between lymphocytes, lymphocytes and antigen presenting cells (APCs), as well as lymphocytes and target cells (Nussbaumer and Thumher (2020) Cells 9(3): 772). Cell phenotype can also be defined by the cell-surface density of CD56. There are therefore recognized sub-types known in the art of CD56bnghtand CDSb^111cells. The increased expression and / or intensity of CD56 on y5 T cells have been shown to have a correlation with enhanced killing. CD56 surface expression can be determined using methods known in the art, such as through analysis of the staining intensity via flow cytometry. Dividing cells into CD56brigbtand CD56dimis understood in the art, for example as described in Van Acker et al. (2017) Front. Immunol. 8: 892. Such methods compare the sample population to a reference population of known CD56 expression levels, such as a cell population containing NK cells. NK cells have distinct CD56 expression levels that can be identified as bright and dim, so by establishing gating strategies using this reference population, the sample population can also be sorted into CD56bngbtand CD56dimfractions.

[0210] In some aspects, the population of cells following the culture in the second culture medium comprises y5 T cells that express NKp30, CD57, GITR, TIGIT, CCR6, CCR2, CCR5 and / or CXCR6. In some aspects, at least about 10% of the cells in the population of expanded y5 T cells express NKp30, CD57, GITR, TIGIT, CCR6, CCR2, CCR5 and / or CXCR6. In some aspects, at least about 20% of the cells in the population of expanded y5 T cells express NKp30, CD57, GITR, TIGIT, CCR6, CCR2, CCR5 and / or CXCR6. In some aspects, at least about 30% of the cells in the population of expanded y5 T cells express NKp30, CD57, GITR, TIGIT, CCR6, CCR2, CCR5 and / or CXCR6. In some aspects, at least about 40% of the cells in the population of expanded y5 T cells express NKp30, CD57, GITR, TIGIT, CCR6, CCR2, CCR5 and / or CXCR6. In some aspects, at least about 50% of the cells in the population of expanded y5 T cells express NKp30, CD57, GITR, TIGIT, CCR6, CCR2, CCR5 and / or CXCR6. In some aspects, at least about 60% of the cells in the population of expanded y5 T cells express NKp30, CD57, GITR, TIGIT, CCR6, CCR2, CCR5 and / or CXCR6. In some aspects, at least about 70% of the cells in the population of expanded y5 T cells express NKp30, CD57, GITR, TIGIT, CCR6, CCR2, CCR5 and / or CXCR6. In some aspects, at least about 80% of the cells in the population of expanded y5 T cells express NKp30, CD57, GITR, TIGIT, CCR6, CCR2, CCR5 and / or CXCR6. In some aspects, at least about 90% of the cells in the population of expanded y5 T cells express NKp30, CD57, GITR, TIGIT, CCR6, CCR2, CCR5 and / or CXCR6. In someaspects, at least about 95% of the cells in the population of expanded y5 T cells express NKp30, CD57, GITR, TIGIT, CCR6, CCR2, CCR5 and / or CXCR6.

[0211] In some aspects, the cell population comprises y5 T cells that express CD56, NKp30, CD57, GITR, TIGIT, CCR6, CCR2, CCR5 and / or CXCR6, such as CD56, NKp30, CD57, GITR and / or TIGIT. In some aspects, the cell population comprises y5 T cells that express CD56, NKp30, CD57, GITR and TIGIT. In some aspects, the cell population comprises y5 T cells that express NKp30. In some aspects, the cell population comprises y5 T cells that express NKG2D. In some aspects, the cell population comprises y5 T cells that express DNAM-1. In some aspects, the cell population comprises y5 T cells that express CD57. In some aspects, the cell population comprises y5 T cells that express GITR. In some aspects, the cell population comprises y5 T cells that express TIGIT. In some aspects, the cell population comprises y5 T cells that express CXCR3. In some aspects, the cell population comprises y5 T cells that express CXCR4. In some aspects, the cell population comprises y5 T cells that express CCR4. In some aspects, the cell population comprises y5 T cells that express CCR6. In some aspects, the cell population comprises y5 T cells that express CCR2. In some aspects, the cell population comprises y5 T cells that express CCR5. In some aspects, the cell population comprises y5 T cells that express CXCR6.

[0212] In some aspects, the cell population comprises y5 T cells that secrete IFN5. In some aspects, the cell population comprises y5 T cells that secrete IFN5 at higher levels than cells expanded using standard techniques. In some aspects, the cell population comprises y5 T cells that secrete GM-CSF. In some aspects, the cell population comprises y5 T cells that secrete GM-CSF at higher levels than cells expanded using standard techniques. In some aspects, the cell population comprises y5 T cells that secrete CXCL9. In some aspects, the cell population comprises y5 T cells that secrete CXCL9 at higher levels than cells expanded using standard techniques. In some aspects, the cell population comprises y5 T cells that secrete CXCL10. In some aspects, the cell population comprises y5 T cells that secrete CXCL10 at higher levels than cells expanded using standard techniques. In some aspects, the cell population comprises y5 T cells that secrete CCL3. In some aspects, the cell population comprises y5 T cells that secrete CCL3 at higher levels than cells expanded using standard techniques. In some aspects, the cell population comprises y5 T cells that secrete CCL4. In some aspects, the cell population comprises y5 T cells that secrete CCL4 at higher levels than cells expanded using standard techniques. In some aspects, the cell population comprises y5 T cells that secrete CCL7. In some aspects, the cell population comprises y5 T cells that secrete CCL7 at higher levels thancells expanded using standard techniques. In some aspects, the cell population comprises y5 T cells that secrete CCL20. In some aspects, the cell population comprises y5 T cells that secrete CCL20 at higher levels than cells expanded using standard techniques.

[0213] Methods of expansion as provided herein, in some aspects, yield an expanded cell population of y5 T cells (in particular, V51 T cells) having a high expression of particular markers, relative to a reference population (e.g. the isolated population of y5 T cells prior to the expansion step). Such markers can include, for example, CD56, NKp30, CD57, GITR and / or TIGIT. In some aspects, the expanded cell population of y5 T cells (in particular, V51 T cells) can have a frequency of CD56+ cells of more than about 20%, such as more than about 40%, more than about 50%, or more than about 60%. In some aspects, more than about 60% of the cell population comprises y5 T cells (in particular, V51 T cells) that express CD56. In some aspects, the expanded cell population of y5 T cells (in particular, V51 T cells) can have a frequency of NKp30+ cells of more than about 20%, such as more than about 25%, more than about 30%, or more than about 35%. In some aspects, more than about 35% of the cell population comprises y5 T cells that express NKp30. In some aspects, the expanded cell population of y5 T cells (in particular, V51 T cells) can have a frequency of CD57+ cells of more than about 20%, such as more than about 30%, more than about 40%, or more than about 50%. In some aspects, more than about 50% of the cell population comprises y5 T cells that express CD57. In some aspects, the expanded cell population of y5 T cells (in particular, V51 T cells) can have a frequency of GITR+ cells of more than about 20%, such as more than about 30%, more than about 40%, or more than about 50%. In some aspects, more than about 50% of the cell population comprises y5 T cells that express GITR. In some aspects, the expanded cell population of y5 T cells (in particular, V51 T cells) can have a frequency of TIGIT+ cells of more than about 25%, such as more than about 30%, more than about 35%, more than about 40%, or more than about 45%. In some aspects, more than about 45% of the cell population comprises y5 T cells that express GITR.

[0214] In some aspects, the cell population comprises y5 T cells with a low (or undetectable) expression of LAG-3, PD-1 and CTLA-4. In some aspects, less than about 15% of the cell population comprises y5 T cells that express LAG-3, PD-1 or CTLA-4 (i.e. >15% y5 T cells are LAG-3+, PD-1+ or CTLA-4+).

[0215] Methods of expansion as provided herein, in some aspects, yield an expanded cell population of y5 T cells (in particular, V51 T cells) having a low expression of particular markers, relative to a reference population (e.g. the isolated population of y5 T cells prior tothe expansion step). Such markers can include, for example, CD27. In some aspects, the expanded cell population of y5 T cells (in particular, V51 T cells) can have a frequency of CD27+ cells of less than about 90%, such as less than about 85%, less than about 80%, or less than about 75%. In some aspects, the expanded cell population of y5 T cells (in particular, V51 T cells) has a frequency of CD27+ cells of less than 75%. In some aspects, less than about 75% of the cell population comprises y5 T cells that express CD27.

[0216] In some aspects, the cell population comprises y5 T cells with a low (or undetectable) expression of LAG-3, PD-1 and CTLA-4. In some aspects, less than about 15% of the cell population comprises y5 T cells that express LAG-3, PD-1 or CTLA-4 (i.e. has a frequency of LAG-3+, PD-1+ or CTLA-4+ cells of less than 15%).

[0217] In some aspects, the cell population comprises y5 T cells with a low (or undetectable) expression of CD62L and CCR7. In some aspects, less than about 15% of the cell population comprises y5 T cells that express CD62L or CCR7 (i.e. has a frequency of CD62L+ or CCR7+ cells of less than 15%).

[0218] In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein has increased persistence of in vivo cytotoxicity as compared to similarly engineered cells obtained by standard methods. In some aspects, the y5 T cell population obtained by methods described herein are capable of in vivo cytotoxicity for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, or at least about 21 days. In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least about 7 days. In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least about 8 days. In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least about 9 days. In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least about 10 days. In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least about 11 days. In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least about 12 days. In some aspects, the y5 T cellpopulation (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least about 13 days. In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least about 14 days. In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least about 15 days. In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least about 16 days. In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least about 17 days. In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least about 18 days. In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least about 19 days. In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least about 20 days. In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least about 21 days.

[0219] In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity, despite having little proliferation. Though the efficacy of aP T cell therapies are dependent on T cell proliferation, the methods disclosed herein generate y5 T cell capable of highly effective cytotoxicity despite having very minimal proliferation. In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) has a proliferation rate of less than about 15-fold, less than about 14-fold, less than about 13-fold, less than about 12-fold, less than about 11-fold, less than about 10-fold, less than about 9-fold, less than about 8-fold, less than about 7-fold, less than about 6-fold, less than about 5-fold, less than about 4-fold, less than about 3-fold, or less than about 2-fold. In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least 7 days, wherein the y5 T cell population (e.g., V51 y5 T cell population) has a proliferation rate of less than a 15-fold increase over the at least 7 days. In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least 10 days, wherein the y5 T cell population (e.g., V51 y5 T cell population) has a proliferation rate of less than a 15-fold increase over the at least 10 days. In some aspects, the y5 T cell population (e.g.,V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least 14 days, wherein the y5 T cell population (e.g, V51 y5 T cell population) has a proliferation rate of less than a 15-fold increase over the at least 14 days. In some aspects, the y5 T cell population (e.g, V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least 18 days, wherein the y5 T cell population (e.g., V51 y5 T cell population) has a proliferation rate of less than a 15-fold increase over the at least 18 days. In some aspects, the y5 T cell population (e.g., V51 y5 T cell population) obtained by methods described herein are capable of in vivo cytotoxicity for at least 21 days, wherein the y5 T cell population (e.g., V51 y5 T cell population) has a proliferation rate of less than a 15-fold increase over the at least 21 days.

[0220] In some aspects, the in vivo cytotoxicity is against a cancer cell. In some aspects, the cancer cell is of a solid tumor. In some aspects, the cancer comprises bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)), uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia (AML) (e.g., relapsed or refractory AML), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia (ALL), chronic myelogenous leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, or any combination thereof. In some aspects, the cancer is locally advanced. In some aspects, the cancer is metastatic. In some aspects, the cancer is refractory. In some aspects, the cancer is relapsed. In some aspects, the cancer is refractory or relapsed following one or more prior anti-cancer therapy. In some aspects, the one or more prior anticancer therapies comprise a standard of care therapy.III.B. Pharmaceutical Compositions

[0221] Some aspects of the present disclosure are directed to a pharmaceutical composition comprising a population of expanded cells disclosed herein. Pharmaceutical compositions can include expanded and / or engineered y5 T cells as described herein in combination with one or more pharmaceutically or physiologically acceptable carrier, diluents, or excipients. Such compositions can include buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g. aluminum hydroxide); and preservatives. Cryopreservation solutions which can be used in the pharmaceutical compositions of the disclosure include, for example, DMSO. Compositions can be formulated, e.g. for intravenous administration.

[0222] Thus, according to another aspect of the disclosure, there is provided a pharmaceutical composition comprising the expanded y5 T cell population or the engineered y5 T cell population as described herein.

[0223] In some aspects, the pharmaceutical composition is substantially free of (e.g. there are no) detectable levels of a contaminant, e.g. endotoxin or mycoplasma.

[0224] According to a yet further aspect of the disclosure, there is provided the expanded y5 T cell population, the engineered y5 T cell population or the pharmaceutical composition as described herein for use as a medicament. In another aspect, there is provided the expanded y5 T cell population, the engineered y5 T cell population or the pharmaceutical composition as described herein for use in the treatment of cancer. In some aspects, the cancer is a solid tumor.

[0225] It will be understood that all aspects described herein can be applied to all aspects of the disclosure.

[0226] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized CellsAnd Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986)); Crooke, Antisense drug Technology: Principles, Strategies and Applications, 2nd Ed. CRC Press (2007) and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0227] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.

[0228] Some aspects and aspects of the disclosure will now be illustrated by way of the following examples and with reference to the figures described above.EXAMPLES EXAMPLE 1. Materials and Methods

[0229] V51+ y5 T cells were generated according to the methods described below and as summarized in FIG. 1.

[0230] Method 1: V51+ y5 T cells were generated from aP-TCR+ depleted peripheral blood mononuclear cells using serum-free culture medium. Between days 0 and 4, the isolated cells were seeded and activated in the presence of 186 U / mL recombinant IL-4 (Miltenyi), 630 U / mL IL-ip (Miltenyi), 3.75 ng / mL IL-21 (CellGenix), 420 U / mL IFNy (Bio-Techne) and 42 ng / mL of soluble anti-CD3 (clone OKT3) antibody (Biolegend). After setup, cultures were incubated at 37°C and 5% CO2 in a humidified incubator. An optional transduction step was performed on day 4 as detailed below. Cells were then expanded by introduction of culture media supplemented with fresh IL-21 (CellGenix) and IL- 15 (Bio-Techne) on day 7 and IL- 15 (Bio-Techne) on day 11. Cells were harvested after 14 days of culture and cryopreserved as a mixed population in Cryostor5 (STEMCELL Technologies).

[0231] Method 2: V51+ y5 T cells were generated from aP-TCR+ depleted peripheral blood mononuclear cells (PBMC) using serum-free culture medium. Between days 0 and 4, the isolated cells were seeded and activated in the presence of 50 ng / mL IL- 18 (MBL Life Science),20 ng / ml of IL-21 (Peprotech), 300 ng / ml of soluble anti-CD30 (clone MAB2291) antibody (R&D Systems) and 3 ug / ml of plate-bound anti-CD3 (clone OKT3) antibody (Biolegend). After setup, cultures were incubated at 37°C and 5% CO2 in a humidified incubator. An optional transduction step was performed on day 4 as detailed below. Cells were then expanded by introduction of culture media supplemented on day 5 with IL- 15 (Peprotech), IL-21 (Peprotech), IL-2 (Peprotech), and anti-CD30 (R&D Systems) antibody. On day 11 cells were supplemented with the same reagents added on Day 5. Cells were harvested after 14 days of culture and cryopreserved as a mixed population in Cryostor5 (STEMCELL Technologies).

[0232] Method 3: V51+ y5 T cells were generated from aP-TCR+ depleted peripheral blood mononuclear cells using serum-free culture medium. Between days 0 and 4, the isolated cells were seeded and activated in the presence of 186 U / mL recombinant IL-4 (Miltenyi), 630 U / mL IL-ip (Miltenyi), 3.75 ng / mL IL-21 (CellGenix), 420 U / mL IFNy (Bio-Techne) and 42 ng / mL of soluble anti-CD3 (clone OKT3) antibody (Biolegend). After setup, cultures were incubated at 37°C and 5% CO2 in a humidified incubator. An optional transduction step was performed on day 4 as detailed below. Cells were then expanded by introduction of culture media supplemented with fresh IL-21 (CellGenix) and IL- 15 (Bio-Techne) on day 7 and IL- 15 (Bio-Techne) on day 11. Cells were harvested after 14 days of culture and cryopreserved as a mixed population in Cryostor5 (STEMCELL Technologies).

[0233] Transduction: On day 4 or 5, cells were expanded, transduced, and optionally restimulated with T Cell TransAct™, ImmunoCult™ T Cell Activator, or Dynabeads™ magnetic beads using concentrations recommended by the vendors. Transduction was achieved by seeding and spinning expanded (activated ) ybT cells with an immobilized y-retroviral vector encoding for GCC targeting chimeric antigen receptor (CAR) in cell culture wells coated with RetroNectin (20 pg / mL). After spinning, cells were incubated at 37°C for 24 hrs. Transduction efficiency was determined by flow cytometry at the end of the expansion process.

[0234] T Cell TransAct™ (a polymeric nanomatrix structure comprising a CD3 agonist and a CD28 agonist), ImmunoCult™ (a medium comprising a CD3 agonist, a CD28 agonist, and a CD2 agonist), and Dynabeads (a composition comprising 4.5 pm beads linked to an anti- CD3 antibody and an anti-CD28 antibody) restimulate cells by contacting the population of cells with a matrix of CD3 and CD28 agonists. T Cell TransAct™ includes a CD3 / CD28 agonist polymeric nanomatrix structure. ImmunoCult™ T Cell Activator includes a CD3 / CD28 / CD2 soluble antibody complex. Dynabeads™ magnetic beads include anti-CD3 and anti-CD28 agonistic monoclonal antibodies.

[0235] Flow cytometry assessment of fold expansion, cell purity and surface phenotype.

[0236] Purity and Composition Characterization: Immunophenotyping was performed using a BD FACSymphony A5™ flow cytometry instrument. The same protocol was applied to characterize both cell products at harvest and post-cryopreservation. Dead cells were excluded using LIVE / DEAD™ Fixable Aqua Dead Cell Stain kit (Invitrogen). Cells were analyzed for the expression of surface markers using APC-pan-ybT, APCvio770-V51, BUV396-CD27, BUV805-CD45RA, BV785-abTCR, BUV563-CD56, PE Dazzle 594-NKp46, PE-NKp44, BUV737-NKp30, BV605-NKG2D, BUV496-DNAM1, R718-KLRG1, BV421- CD30, BV711-CXCR3, PE Cy7-CXCR6, BUV563-CD56, V605-CD38, BV711-CD39, PerCP Cy5.5-CD28, BUV563-CD69, BUV496-OX40, BV785-4-1BB, PE Cy7-PD-1, BV421-LAG3, BUV737-TIM3, PE-TIGIT, PE Dazzle 594-CD86, R718-PD-L1 antibodies, available from Miltenyi, BioLegend and BD Biosciences.

[0237] T-cell Subset Characterization: Immunophenotyping was performed using a BD FACSymphony A5flow cytometry instrument. The same protocol was applied to characterize both cell products at harvest and post-cryopreservation. Dead cells were excluded using LIVE / DEAD™ Fixable Aqua Dead Cell Stain kit (Invitrogen).

[0238] Thawing Cryopreserved Cell Products

[0239] Frozen cryovials were thawed in a 37°C water bath and thawed cells were added to pre-warmed OpTmizer + 2.5% allogeneic plasma. A 50 uL sample was transferred to an Eppendorf tube and counted with an NC200 to evaluate cell density and viability. Cells were then spun at 300g for 5 minutes, supernatants discarded and the cells were then resuspended at 2xl06cells per mL for phenotyping and downstream assays.

[0240] Cytotoxicity Assays

[0241] Expanded cells were co-cultured with luciferase expressing tumour targets (e.g.OVCAR3, LS1034, A549, HT29, GSU), at various effector to target cell ratios. Target cells in the absence of effectors served as a control (Ctrl). After 20 hours, quantification of target viability was assessed using ONE-Glo™ Luciferase Assay System kit (Promega) or CellTiter- Glo® Luminescent Cell Viability Assay (Promega). Luminescence was measured on Biotek Synergy H4 plate reader.

[0242] Repeated Antigen Stimulation (RAS) Assay

[0243] Cryopreserved V51+ y5 T cell products were thawed and co-cultured in presence of cytokines with adherent tumour targets expressing GFP or NLR (e.g., OVCAR3, LS1034, A549, HT55) at various effector to target cell ratios. Cells were incubated at 37°C and 5% CO2in an Incucyte® Live-Cell Analysis System (Sartorius). y5 T cell products were challenged by adding fresh tumor targets to the culture at regular intervals. % reporters (GFP or Nuclight red (NLR)) expressed by cell lines was used as a measure of residual tumor targets at each time point across the assay. To calculate V51+ y5 fold expansion, cells were counted at each time point and flow cytometry was performed to determine V51+ y5 enrichment.EXAMPLE 2. Development of Method for V51+ y5 T cells Expansion

[0244] aP-TCR+ depleted peripheral blood mononuclear cells were expanded using Method 1 described in Example 1 and as summarized in FIG. 1. The expanded cell product was then analyzed.

[0245] The generation of V51+ enriched y5 T cells using Method 1 demonstrated that CD28 is significantly upregulated at day 4 of culture (FIGs. 2A-2B), suggesting that restimulation may further enhance the expansion of V51+ y5 T cells while maintaining or improving proliferative potential and cytotoxicity. In order to improve upon Method 1, restimulation in the context of costimulation and cytokine conditions were further explored to improve cell expansion and differentiation.

[0246] The addition of supplemental cytokine support and / or restimulation was further explored using Method 1 as a baseline control. Specifically, V51+ enriched y5 T cells were generated using Method 1, Method 1 with transactivation using TransAct™, Method 2 with IL-18 supplementation, and Method 3 (i.e., Method 2 with IL-18 supplementation and TransAct™).

[0247] The fold expansion and expansion normalized to Method 1 was first assessed (FIGs.3A-3B). The addition of TxA led to a two-fold increase in y5 T cell expansion for both Method 1 and Method 2 with IL- 18 supplementation (Method 3). Further, the purity of the generated product demonstrates that the composition (e.g., aP T-cells, Natural Killer (NK) cells, V51+ y5 T cells, Non-V51+ y5 T cells, triple negative cells) did not markedly change with or without TransAct™ restimulation (FIG. 4). This finding suggests that transactivation leads to marked increase in cell number, but does not impact the purity of the expanded cells.

[0248] The effector and memory subsets of the resulting V51+ y5 T cells generated using the above methods were then assessed (FIGs. 5A-5B). Method 3 (Method 2 with IL-18 and TransAct™) resulted in an increase in the T-cell effector memory cell population compared to Method 1, Method 1 + TransAct™ and Method 2 with IL-18, respectively (FIG. 5A). Similarly, the V51+ y5 T-cell population generated using Method 3 had the largest sub-population of Teffector memory cells relative to Method 1, Method 1 with TxA, and Method 2 with IL- 18 supplementation. Method 3 yielded a subset distribution most similar to the populations found in skin. Method 1 + TransAct™ led to a similar cell subset distribution as Method 1 with most cells showing a Tcm-like differentiation.EXAMPLE 3: Analysis of IL- 15 Concentration

[0249] y5 T cells were generated from aP-TCR+ depleted peripheral blood mononuclear cells as described in the above examples. The post-thaw viability of the different expansion processes was then assessed and compared to the other methods (FIG. 6). Importantly, there was no significant difference between each of these groups. The post-thawed cells were then cultured with or without IL- 15 (0 and 10 ng / mL) over 7 days (FIGs. 7A-7D). IL- 15 supplementation after thaw was found to be necessary in order to maintain cell viability (FIG.7A, FIG. 7C) and total cell numbers over 7 days of culture (FIG. 7B, FIG. 7D). Notably, cells produced by Method 1 + TransAct™, Method 2 with IL- 18 and Method 3 in the context of supplementation with 10 ng / mL IL- 15 exhibited enhanced total live cell fold expansion and retained high cell viability at day 7 of culture (FIG. 7C, FIG. 7D). In contrast, absence of IL- 15 supplementation after thaw resulted in a significant drop in cell viability and total live cells by day 7 post-thaw (FIG. 7A, FIG. 7B). This demonstrates the increased responsiveness of y8T cells to cytokine stimulus (IL-15) with the new expansion Methods (Method 1 + TransAct™, Method 2, and Method 3). Similar results were seen when comparing only Method 1 and Method 3 in an independent experiment demonstrating the increased responsiveness to cytokine-induced proliferation conferred by TransAct™ restimulation (FIG. 8).

[0250] The impact of costimulation on expanded y5 T-cells was assessed. Briefly, y5 T- cells were expanded using Method 1 or Method 1 + TransAct™, subsequently frozen and thawed, labeled with CellTrace, and left unstimulated or stimulated with plate-bound OKT3 antibody in the presence or absence of 3 pg / mL of plate-bound ligand-Fc (e.g., 4-lBBL-Fc, CD80-Fc, CD27-Fc) for 7 days. y5 T-cells expanded with Method 1 + TransAct™ and stimulated with OKT3 antibody were found to be more responsive to costimulation by 4-1BBL, CD80, and CD27 than cells expanded with Method 1 as evidenced by increased cell proliferation (FIG. 9).

[0251] These results demonstrates that Method 1 + TransAct™ expanded y5 T-cells have higher proliferative potential and response to costimulation relative to Method 1 expanded y5 T-cells, while retaining comparable post thaw viability to Method 1.EXAMPLE 4: Comparison of Restimulation Agents and Effect on Expansion, Viability, and Differentiation

[0252] The effect of restimulation agent (e.g., TransAct™, ImmunoCult™, Dynabeads™) and cytokine combinations (i.e., IL-15 and IL-21; IL-15, IL-21, and IL-2; IL-15, IL-21, IL-2, and IL-18) on the subsequent expansion, viability, and differentiation was further investigated to optimize Method 3.

[0253] y5 T cells were generated from aP-TCR+ depleted peripheral blood mononuclear cells as described in the above examples. Cells were cultured using CTS Optimizer complete media with IFN-y, IL-ip, IL-21, and OKT3 from Day 0 to Day 4 followed by restimulation on day 4 using TransAct™, ImmunoCult™ or Dynabeads ™ at concentrations recommended by the vendors. From day 4 to day 14, cells were cultured in CTS Optimizer complete media supplemented with the cytokine combination shown in FIGs. 10A-10D. The fold expansion (FIG. 10 A), total yield (FIG. 10B), and viability (FIG. 10C) were then assessed at Day 14 using a NucleoCounter® NC-200.

[0254] Transactivation using TransAct™ or ImmunoCult™ resulted in superior fold expansion and total yield compared to Dynabeads™ (FIGs. 10A-10B) while retaining high cell viability after the expansion protocol for all cytokine combinations (FIG. 10C). Similarly, the post-thaw viability of cells transactivated with TransAct™ or ImmunoCult™ resulted in higher viability relative to Dynabeads™ (FIG. 10D). This finding suggests that transactivation using TransAct™ or ImmunoCult™, in contrast to Dynabeads™, leads to marked increase in cell number, but does not impact the viability of the expanded cells.

[0255] The effector and memory subsets of the resulting V51+ y5 T cells generated using the above methods were then assessed (FIG. 11). The naive, effector memory, central memory, and effector populations were assessed using CD45RA and CD27 staining as quantified by flow cytometry. Transactivation using TransAct™ supplemented with IL- 15, IL-21, IL-2, and IL-18 or ImmunoCult™ supplemented with IL-15, IL-21, and IL-2 with or without IL-18 resulted in a substantial increase in T-cell effector memory cells compared to all other groups (FIG. 11). Transactivation with TransAct™ supplemented with IL-15 and IL-21 led to very similar T cell subset distribution as the control (Method 1) group.EXAMPLE 5: Expression of Natural Cytotoxicity Receptors and Phenotypic Profile of V51 y8 T- Cells

[0256] The expression of natural cytotoxicity receptors (NCRs) and the phenotypic profile of the V51 y8 T-cells generated by the above methods was also investigated. The NCR inhibitory receptor expression, and chemokines production was assessed in V51 y8 T-cells produced by Method 1, Method 1 with TransAct™, Method 2 with IL- 18, and Method 3 (FIGs.12A-12B). The V51 y8 T-cells produced with TransAct™ restimulation maintained similar expression of these markers and cytokines as the V51 y8 T-cells produced by Method 1.EXAMPLE 6: Killing Capacity of Expanded Cells

[0257] The functionality of the engineered cell products was also investigated in an overnight cytotoxicity assay (FIGs. 13A-13E) and repeated tumor challenging experiments (FIGs. 14A-14C) in four different models using OVCAR3, A549, GSU, and LS1034 tumor cell lines. In the context of an overnight cytotoxicity assay, Method 1 + TransAct™, Method 2 and Method 3 resulted in higher cytolysis relative to cells generated by Method 1, observed particularly at the higher effectortarget cell ratios.

[0258] In the context of a 21 -day Repeated Antigen Stimulation (RAS) assay, where the cell products are challenged with A549 cells every 3 to 4 days, Method 1 + TransAct™ and Method 3 resulted in significantly higher tumor control compared to Method 1 and Method 2 without TransAct™ restimulation. This result was consistent across three independent studies (FIGs. 14A-14C).

[0259] The proliferation of y8 T-cells after RAS assay against target A549 or OVCAR3 was then assessed. y8 T-cells produced by Method 1 + TransAct™ or Method 3 resulted in similar or higher post RAS proliferation relative to the other methods tested when the target was A549 cells (FIG. 15 A) or OVCAR3 cells (FIG. 15B).

[0260] These results demonstrate that cells expanded by Method 1 + TransAct™ and Method 3 resulted in superior tumor control over 21 days. Additionally, the superior tumor control exhibited by the products expanded by Method 1 + TransAct™ and Method 3 was possible with similar or higher expansion during the assay when compared to cell products grown by the other methods. This is indicative that cell products restimulated with TransAct™ are more capable of sustaining functionality and expansion over multiple rounds of stimulation with target cells. .EXAMPLE 7. Incorporation of New Expansion Process with Viral Transduction

[0261] Expanding cells were transduced with y-retroviral vector before or after transactivation with TransAct™ (TxA). The timing of the expansion process relative to viral transduction was evaluated including (1) transduction control without restimulation (Mock), (2) restimulation 24 hr prior to transduction (TxA 24 hr pre-TD) or restimulation 48 hr prior to transduction (TxA 48 hr pre-TD), and (3) transduction followed by restimulation (TxA 24 hr post-TD) or transduction followed by restimulation 48 hr later (TxA 48 hr post-TD). For the transduction step, cells were either transduced with a y-retroviral vector encoding for an anti- GCC CAR or mock transduced with no virus (Mock). Cell numbers were then normalized relative to Method 1 (i.e., number of cells used for transduction, number of cells used for expansion).

[0262] The resulting fold expansion post-transduction was then assessed (FIGs. 16A-16B).The fold expansion that took place after the transduction step when cells were placed in a Grex® for the expansion phase was quantified on day 14 (FIG. 16 A). Restimulation with TransAct™ (TxA) at 24 hr pre-TD resulted in significantly higher fold expansion compared to restimulation with TransAct™ at 48 hr pre-TD. A similar trend is seen when restimulation was performed after transduction, where restimulation with TransAct™ at 24 hr post-TD resulted in higher fold expansion than restimulation with TransAct™ at 48 hr post-TD. For all conditions, the anti-GCC CAR condition had higher fold expansion than the mock condition. Similar insights were demonstrated when calculating the theoretical fold expansion (FIG. 16B), where the theoretical fold expansion considers hypothetical expansion if all cells were used after restimulation for viral transduction and subsequent expansion.

[0263] For all tested anti-GCC CAR transduced groups, the vector copy number (VCN) was determined by ddPCR at day 14 (FIG. 17). There was no significant difference between the groups, demonstrating that groups with superior fold expansion maintained a similar VCN.

[0264] The effect of the restimulation timing relative to transduction with mock or anti- GCC CAR on cell viability and cell product composition was then considered (FIGs. 18A- 18B). For all groups, the cell viability was high (FIG. 18A), and the composition of aP T-cells (abT), V51 y5 T-cells (Vdl gdT), Non-V51 y5 T-cells (Non-Vdl gdT), Natural Killer cells (NK), and triple negative cells (abT-gdT-NK-) did not significantly change from condition to condition (FIG. 18B).

[0265] The effect of the restimulation timing relative to transduction with mock or anti- GCC CAR on the expression of functional, chemokine, inhibitory, and activation markers onthe cell products’ V51 y5 T-cell population was further investigated (FIGs. 19A-19B). Importantly, the expression of anti-GCC CAR led to an increase in the expression of activation / differentiation markers (CD38, CD39, CD69) and inhibitory markers (LAG3, TIGIT, TIMIT).EXAMPLE 8: Further optimization of restimulation and transduction timing

[0266] The timing of restimulation with TransAct™ (TxA) relative to transduction was further explored, specifically Method 1 + TxA with either (1) TxA restimulation 24 hr prior to TD (TxA 24 h pre-TD) or (2) TxA restimulation after TD (TxA post-TD) using the same methodology as Example 6. Cells were transduced with anti-GCC CAR using the variations of Method 1 + TxA timing and compared to anti-GCC CAR transduction using Method 1 without TxA restimulation.

[0267] The resulting cell products demonstrate that Method 1 + TxA with mock or anti- GCC CAR transduction and either TxA 24 h pre-TD or TxA post-TD achieved a 2-fold increase in expansion (FIG. 20A) and total cell yield (FIG. 20B) when compared to samples expanded with Method 1. Similar post-thaw viability and CAR expression was observed between Method 1 + TxA groups and Method 1 (FIG. 20C-20D).

[0268] Therefore, restimulation using Method 1 + TxA enhances the fold expansion and yield of y5 T-cells while preserving their viability and CAR expression with similar outcomes whether TransAct™ restimulation takes place 24 hrs before TD or after the TD step.EXAMPLE 9: Cell product functionality after transduction.

[0269] The impact of Method 1 + TxA on the expression of relevant cell markers on V51 y5 T-cells (FIGs. 21 A-21B) and resulting innate killing capacity was then assessed. Similar to our previous results, the expression of anti-GCC CAR led to an increase in the expression of activation / differentiation markers (CD38, CD39, CD69) and inhibitory markers (LAG3, TIGIT, TIMIT) relative to mock transduction for both Method 1 and Method 1 + TxA (FIG.2 IB). The innate killing capacity of these cell products were assessed against GCC+ cell lines (LSI 034, GSU) and GCC- cell lines (HT-29, A549) over a range of effectortarget cell ratios (10:1, 3:1, 1:1, 1:3). Pronounced cell killing was observed against GCC+ cell lines by cell products expanded by methods that included GCC transduction (FIG. 22) and largely comparable between Method 1 and Method 1 + TxA expanded cells in this short-term killing experiment.

[0270] The functionality of the engineered cell products was also investigated in a repeated tumor challenge experiment using two different models using GCC111LS1034 and GCC10HT55 tumor cells followed by an assessment of post RAS assay fold expansion (FIGs. 23 A-23B and 24A-24B). In the context of a 14-day Repeated Antigen Stimulation (RAS) assay, where the cell products are challenged with LSI 034 cells every 3 to 4 days at an effectortarget ratio of 1:1, mock transduced cells expanded with Method 1 + TxA resulted in higher tumor control compared to mock transduced cells from Method 1 (FIG. 23 A) representing innate activity. CAR-mediated killing for both Methods was comparable in this anti-GCC CAR sensitive tumor model. Similar results were seen for a 14-day RAS assay where the cell products are instead challenged with HT55 cells every 3 to 4 days at an effectortarget ratio of 1:1 (FIG.24A). In this more resistant tumor model, anti-GCC transduced cells from Method 1 + TxA showed slightly improved tumor control relative to Method 1 expanded cells. The post-RAS proliferation of the cell product was enhanced in the Method 1 + TxA groups transduced with anti-GCC CAR when HT55 was the target, but similar expansion was seen in Method 1 and Method 1 + TxA groups when LSI 034 was the target (FIGs. 24B and 23B, respectively).

[0271] These results demonstrate that Method 1+ TxA preserves the functionality of y5 T- cells while improving the innate responses in a target-specific manner.EXAMPLE 10: Incorporation of Expansion Methods with Bioreactor Expansion

[0272] Method 1 and Method 1 + TxA were further explored by incorporating these methods with bioreactor expansion. Cells were transferred to an Ambr® bioreactor either after transduction (TxA 24 hr pre-TD) or two days after TxA restimulation (TxA post-TD), and cells were maintained at a concentration of 0.15 x io6cells / mL. Cells were transduced with anti- GCC CAR using the TxA variations of Method 1 + TxA and compared to anti-GCC CAR transduction using Method 1 without TxA restimulation.

[0273] The fold expansion of the cell products were significantly higher using Method 1 + TxA with either TxA 24 hr pre-TD or TxA post-TD relative to Method 1 without TxA (FIG.25A). Similarly, the cell yield was the highest for the Method 1 + TxA groups, specifically with TxA 24 h pre-TD providing the highest yield followed by TxA post-TD (FIG. 25D). The cellular viability and (%) CAR+ cells were similar across all groups (FIGs. 25B-25C).

[0274] The T-cell subset composition and cellular phenotype was then assessed for Method 1 and Method 1 + TxA using an Ambr® bioreactor or GRex® bioreactor. The cells were transduced either with tEGFR (control) or anti-GCC CAR as detailed above, and anti-GCCtransduced groups were either restimulated with TxA 24 hr pre-TD or TxA post-TD as detailed above. The T-cell subset composition (FIG. 26A) and phenotype (FIG. 26B) were largely similar when comparing Ambr® bioreactor and GRex® groups although more Tern memorylike cells were observed with Ambr® expansion.

[0275] These findings demonstrate that these methods can be successfully incorporated into bioreactor processes, and that Method 1 with TransAct™ restimulation resulted in significantly higher fold proliferation and cell yield without sacrificing cellular viability and (%) CAR+ y5 T-cells. Furthermore, these expansion methods can be applied to multiple different bioreactor setups, demonstrating the ability to scale up this process while retaining cell composition and phenotype.EXAMPLE 11 : Use of TGFp to Increase Cell Yield (Prophetic)

[0276] Donor variability can lead to poor cell yield in some instances. To increase cell yield, recombinant human TGFP will be added to culture medium in one or more of the activation medium, the transduction medium, or the expansion medium, according to any of the methods described above. Where TGFp is included during activation, the medium will be supplemented with about 0.5 ng / mL to about 10 ng / mL TGFp. Where TGFP is included during transduction, the medium will be supplemented with about 0.5 ng / mL to about 10 ng / mL TGFp. Where TGFP is included during expansion, the medium will be supplemented with about 0.5 ng / mL to about 10 ng / mL TGFp. V51+ y5 T cell yield and phenotype will be monitored.***

[0277] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary aspects of the present disclosure as contemplated by the inventor(s), and thus, are not intended to limit the present disclosure and the appended claims in any way.

[0278] The foregoing description of the specific aspects will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and / or adapt for various applications such specific aspects, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects, based on the teaching and guidance presented herein.It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0279] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.

[0280] The contents of all cited references (including literature references, U.S. or foreign patents or patent applications, and websites) that are cited throughout this application are hereby expressly incorporated by reference as if written herein in their entireties for any purpose, as are the references cited therein. Where any inconsistencies arise, material literally disclosed herein controls.

[0281] While various specific aspects have been illustrated and described, the above specification is not restrictive. It will be appreciated that various changes can be made without departing from the spirit and scope of the disclosure(s). Many variations will become apparent to those skilled in the art upon review of this specification.

Claims

CLAIMS1. A method for expanding y5 T cells, wherein said method comprises the steps of:(1) culturing a population of cells comprising one or more y5 T cells in a first culture medium, wherein the first culture medium comprises a T cell mitogen and interleukin-4 (IL-4), wherein the first culture medium does not comprise interleukin- 15 (IL- 15); and(2) culturing the population of cells from (1) in a second culture medium, wherein the second culture medium comprises IL- 15, and wherein the second culture medium does not comprise IL-4;wherein the population of cells is restimulated before (2), concurrently with (2), or after (2).

2. The method of claim 1, wherein the population of cells is cultured in the first culture medium for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.

3. The method of claim 1 or 2, wherein the population of cells is cultured in the first culture medium for about 4 days.

4. The method of any one of claims 1 to 3, wherein the population of cells is cultured in the second culture medium for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days or about 17 days.

5. The method of any one of claims 1 to 4, wherein the population of cells is cultured in the second medium for about 6 days.

6. The method of any one of claims 1 to 4, wherein the population of cells in cultured in the second medium for about 9 days.

7. The method of any one of claims 1 to 6, wherein the population of cells is restimulated on day 1 of the culture in the second culture medium.

8. The method of any one of claims 1 to 7, wherein the population of cells is restimulated by contacting the population of cells with a T cell mitogen, a CD28 agonist, or both.

9. The method of claim 8, wherein the population of cells is restimulated by contacting the population of cells with a T cell mitogen and a CD28 agonist.

10. The method of claim 8 or 9, wherein the T cell mitogen and the CD28 agonist are soluble.

11. The method of any one of claims 8 to 10, wherein the T cell mitogen and the CD28 agonist are associated with each other.

12. The method of claim 11, wherein the T cell mitogen is linked to the CD28 agonist by a covalent bond.

13. The method of claim 11 or 12, wherein the T cell mitogen is linked to the CD28 agonist by a peptide bond.

14. The method of claim 8 or 9, wherein the population of cells is restimulated by contacting the population of cells with a matrix, wherein the T cell mitogen, the CD28 agonist, or both, are associated with the matrix.

15. The method of claim 14, wherein both the T cell mitogen and the CD28 agonist are associated with the matrix.

16. The method of any one of claims 1 to 15, wherein the first culture medium further comprises IL-21, interferon gamma (IFNy), IL1-P, or any combination thereof.

17. The method of claim 16, wherein the first culture medium further comprises IL-21, interferon gamma (IFNy), and IL-ip.

18. The method of any one of claims 1 to 17, wherein the second culture medium comprises a CD30 agonist, IL-2, IL-18, or any combination thereof.

19. The method of claim 18, wherein the second culture medium comprises a CD30 agonist, IL-2, and IL- 18.

20. The method of any one of claims 1 to 19, wherein after the cells are cultured in the second culture medium for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days following restimulation, the population of cells is contacted with fresh second culture medium.

21. The method of claim 20, wherein fresh second culture medium is added to the population of cells about 6 days after restimulation.

22. The method of any one of claims 1 to 21, wherein the population of cells is obtained from a biological sample obtained from a human subject.

23. The method of claim 22, wherein the biological sample is a haematopoietic sample or a fraction thereof.

24. The method of claim 22 or 23, wherein the biological sample is selected from peripheral blood, umbilical cord blood, lymphoid tissue, thymus, bone marrow, spleen, lymph node tissue or fractions thereof.

25. The method of any one of claims 22 to 24, wherein the biological sample comprises peripheral blood or a fraction thereof.

26. The method of any one of claims 22 to 25, wherein the biological sample consists of peripheral blood mononuclear cells (PBMCs) or low density mononuclear cells (LDMCs).

27. The method of claim 22, wherein the biological sample is a non-haematopoietic tissue.

28. The method of any one of claims 1 to 27, wherein the method comprises culturing the population of cells in the first culture medium and the second culture medium for between 7 and 21 days.

29. The method of claim 28, wherein the method comprises culturing the composition in the first culture medium and the second culture medium for about 10, about 11, about 12, about 13, or about 14 days.

30. The method of any one of claims 1 to 29, wherein the T cell mitogen comprises a CD3 agonist, an antibody or an antigen-binding portion thereof that specifically binds TCRyS, or an antibody or antigen-binding portion thereof that specifically binds TCRV51.

31. The method of claim 30, wherein the CD3 agonist comprises an antibody or antigenbinding portion thereof the specifically binds CD3.

32. The method of claim 31, wherein the antibody or antigen-binding portion thereof that specifically binds CD3 is OKT3.

33. The method of any one of claims 1 to 32, wherein the number of y5 T cells in the population of cells following the culture in the second culture medium is expanded by at least 5-fold, at least 10-fold, or at least 20-fold relative to the number of y5 T cells in the population of cells at the start of the culture.

34. The method of any one of claims 1 to 33, wherein the population of cells following the culture in the second culture medium comprises y5 T cells that express NKG2D,DNAM1, NKp30, NKp80, NKp46, CD57, GITR, TIGIT, CCR6, CCR2, CCR5, CXCR3 and / or CXCR6.

35. The method of any one of claims 1 to 34, wherein the y5 T cells are derived from a single donor.

36. The method of any one of claims 1 to 34, wherein the y5 T cells are derived from multiple donors.

37. The method of any one of claims 1 to 36, wherein the method further comprises freezing the population of cells comprising the y5 T cells following culture in the second culture medium.

38. A method for engineering y5 T cells, said method comprising the steps of:(i) preparing a composition enriched for y5 T cells using the method of any one of claims 1 to 38;(ii) transducing the y5 T cells with an exogenous nucleic acid for expression in the y5 T cells; and(iii) culturing the transduced y5 T cells to expand the engineered y5 T cells.

39. The method of claim 38, wherein the exogenous nucleic acid encodes a chimeric antigen receptor (CAR), a chimeric costimulatory receptor (CCR), a chimeric switch receptor (CSR), a chimeric TCR or other chimeric recombinant protein recognizing a tumour antigen, a stromal antigen, a costimulatory receptor, an inhibitory receptor, a cytokine or other cell surface antigen.

40. The method of claim 39, wherein the tumour antigen is a tumour specific antigen that is not expressed by normal somatic cells from the subject tissue.

41. The method of claim 39 or claim 40, wherein the tumour antigen is a tumour associated antigen which is preferentially overexpressed on cancer cells compared to healthy somatic cells.

42. The method of any one of claims 39 to 41, wherein the tumour antigen is an antigen expressed by a cell experiencing oxidative stress, DNA damage, UV radiation, EGF receptor stimulation, or any combination thereof.

43. The method of any one of claims 39 to 42, wherein the tumour antigen is an antigen expressed by a solid tumour.

44. The method of any one of claims 38 to 43, wherein the composition is transduced using a viral vector or electroporation.

45. The method of claim 44, wherein the viral vector comprises a retroviral vector.

46. The method of claim 44 or 45, wherein the viral vector comprises a gammaretroviral vector or a lentiviral vector.

47. The method of claim 46, wherein the gammaretroviral vector is a murine stem cell virus (MSCV) or Moloney Murine Leukaemia Virus (MLV).

48. The method of any one of claims 44 to 47, wherein the viral vector is pseudotyped with an envelope other than vesicular stomatitis virus-G (VSV-G).

49. The method of claim 48, wherein the envelope comprises a betaretroviral envelope.

50. The method of claim 48 or 49, wherein the envelope comprises a baboon endogenous virus (BaEV) or RD 114.

51. The method of any one of claims 38 to 50, wherein the y5 T cells are restimulated before, concurrently with, or after transduction.

52. The method of any one of claims 38 to 51, wherein the y5 T cells are restimulated by contacting the y5 T cells with a T cell mitogen, a CD28 agonist, or both.

53. The method of claim 52, wherein the y5 T cells are restimulated by contacting the y5 T cells with a T cell mitogen and a CD28 agonist.

54. The method of claim 52 or 53, wherein the T cell mitogen and the CD28 agonist are soluble.

55. The method of claim 52 or 53, wherein the T cell mitogen and the CD28 agonist are associated with each other.

56. The method of claim 55, wherein the T cell mitogen is linked to the CD28 agonist by a covalent bond.

57. The method of claim 55 or 56, wherein the T cell mitogen is linked to the CD28 agonist by a peptide bond.

58. The method of claim 51 or 57, wherein the y5 T cells are restimulated by contacting the y5 T cells with a matrix, and wherein the T cell mitogen, the CD28 agonist, or both, are associated with the matrix.

59. The method of claim 58, wherein both the T cell mitogen and the CD28 agonist are associated with the matrix.

60. The method of any one of claims 38 to 59, wherein step (iii) comprises culturing the transduced composition in the absence of feeder cells.

61. The method of any one of claims 38 to 60, wherein step (iii) comprises culturing the transduced composition in the presence of feeder cells.

62. An expanded y5 T cell population obtained by the method of any one of claims 1 to 61.

63. A pharmaceutical composition comprising the expanded y5 T cell population of claim 62.

64. The expanded y5 T cell population of claim 62 or the pharmaceutical composition of claim 53 for use as a medicament.

65. The expanded y5 T cell population of claim 62 or the pharmaceutical composition of claim 48 for use in the treatment of cancer.

66. The expanded y5 T cell population or the pharmaceutical composition for use of claim 65, wherein the cancer is a solid tumour.

67. The expanded y5 T cell population of claim 62 or the pharmaceutical composition of claim 63, wherein the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, or at least about 21 days.

68. The expanded y5 T cell population of claim 62 or the pharmaceutical composition of claim 63, wherein the expanded y5 T cells are capable of in vivo cytotoxicity for at least about 14 days.

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