Enhanced gamma delta t cells for immunotherapy

Abstract

Aspects of the present disclosure relate to methods and compositions relating to the selection and expansion of immune cells, including T cells expressing CD16Hi in combination with a V [delta] 2 T cell receptor. The T cells generated by the methods disclosed herein are suitable for allogeneic cell therapy as they do not induce graft versus host disease (GvHD) and resist host immune allogeneic rejection. Thus, such cells are suitable for ready-to-use in clinical therapy for diseases such as cancer, infectious diseases, autoimmune diseases, heart diseases and neuronal diseases.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 432,814, filed Dec. 15, 2022, U.S. Provisional Patent Application No. 63 / 479,278, filed Jan. 10, 2023, and U.S. Provisional Patent Application No. 63 / 502,881, filed May 17, 2023, all of which are commonly owned and co-pending, and all entitled "ENHANCED GAMMA DELTA T CELLS FOR IMMUNOTHERAPY", the disclosures of all of which are incorporated herein by reference. FIELD OF THE DISCLOSURE

[0003] Embodiments of the present disclosure relate at least to the fields of immunology, cell biology, molecular biology, and medicine. BACKGROUND OF THE DISCLOSURE

[0004] Gamma delta (γδ) T cells are a small subset of T lymphocytes with the ability to bridge innate and adaptive immunity. Most γδ T cells in adult blood exhibit the Vγ9Vδ2 T cell receptor and respond to small phosphorylated non-peptide antigens called phosphoantigens (pAgs), which are typically produced by malignant cells (see, e.g., Yang et al., Immunity 50, 1043-1053.e5 (2019)). Unlike conventional αβ T cells, γδ T cells do not recognize polymorphic classical major histocompatibility complex (MHC) molecules and thus have no risk of graft-versus-host disease (GvHD) when adoptively transferred into allogeneic hosts. In addition, γδ T cells have several other unique features that make them an ideal cellular vehicle for the development of off-the-shelf cell therapies for cancer. These features include: 1) γδ T cells play a role in cancer immunosurveillance; 2) γδ T cells have a remarkable ability to target tumors independent of tumor antigens and major histocompatibility complex (MHC) restriction; 3) γδ T cells can employ multiple mechanisms to attack tumor cells through direct killing and adjuvant effects; and 4) γδ T cells can express the surface receptor FcγRIII (CD16) involved in antibody-dependent cell cytotoxicity (ADCC) and can potentially be combined with monoclonal antibodies for cancer therapy.

[0005] However, unfortunately, the development of allogeneic off-the-shelf γδ T cell products has been greatly hindered by their availability - these cells are present in low numbers and have high variability in humans (approximately 1 - 5% of T cells in human blood), making it difficult to generate therapeutic numbers of γδ T cells using blood cells from allogeneic human donors. In addition, γδ T cells are a heterogeneous population of cells with distinct transcriptional programs, phenotypes, and functions, and there is donor-to-donor variability in γδ T cell profiles. Certain subsets and donor-specific properties of γδ T cells may be ideal for cancer adoptive cell immunotherapy (ACT), and thus it is important to identify biomarkers indicative of greater potency.

[0006] There is a need in the art for means to generate large numbers of γδ T cells with potent effector capabilities. Specifically, new methods and materials that can reliably generate large numbers of γδ T cells are crucial for the development of off-the-shelf γδ T cell therapies that can be used to treat a variety of pathological conditions. SUMMARY OF THE INVENTION

[0007] Cancer remains the leading cause of death worldwide as well as in the United States. Chimeric alpha beta (αβ) antigen receptor-T (CAR-T) cells have transformed the treatment of hematological malignancies but have not had the same success against solid tumors. In addition, all FDA-approved CAR-T cell therapies are autologous, which is a key bottleneck in their scalability, affordability, and accessibility and can lead to serious adverse events. To generate allogeneic "off-the-shelf" cell therapies that are safe and effective for treating both liquid and solid tumor types, we have focused on gamma delta (γδ) T cells as a cell source for genetic engineering and cancer treatment, and in particular, the Vδ2 subset of γδ T cells (a population of T cells that does not cause graft-versus-host disease and that also has intrinsic cancer-killing capabilities). Unfortunately, however, their clinical application in cancer immunotherapy is limited by their scarcity and persistence as well as tumor immunosuppressive mechanisms. In this context, the ability to manufacture therapeutic γδ T cell populations or cell populations that can be used to generate "off-the-shelf" therapeutic γδ T cell populations increases the availability and usefulness of new cell therapies.

[0008] As disclosed herein, we have invented a unique γδ T cell platform designed to address the need for new methods and materials for cell therapy, and more particularly, to address the need for cell therapies that are not hindered by the challenges presented in personalized therapies using autologous cells. As described below, using selected CD16 high (CD16 Hi)Vδ2 T cell screening and expansion methods, where we have overcome many limitations associated with conventional reagents and methods used in T lymphocyte growth and expansion methodologies. As disclosed herein, we screen donors for CD16 expression on Vδ2 T cells in order to focus on CD16 as a biomarker for Vδ2 T cell donor selection. Our studies have found that CD16 Hi Vδ2 T cells exhibit more robust cytotoxic activity than CD16 low (CD16 Lo ) Vδ2 T cells and perform well, for example, in antibody-dependent cellular cytotoxicity assays. Additionally, CD16 Hi Vδ2 T cells were found to exhibit a gene profile associated with reduced Th17 function. Based on our findings, we have developed a Vδ2 T lymphocyte expansion method and associated culture medium materials that utilize a combination of γδ T cell stimulants (such as the bisphosphonate zoledronic acid), cytokines, and GSK-3β inhibitors to achieve an expansion rate of more than 10,000-fold for CD16 Hi Vδ2 T cells, which is approximately 10 to 20 times the expansion rate observed in conventional methods. Vδ2 T cells prepared by embodiments of the present invention can be expanded for more than a month and restimulated while maintaining a very desirable lower differentiated memory state. Additionally, in long-term stress assays, the resulting memory-like Vδ2 T cells are superior to Vδ2 T cells expanded by conventional methods. These findings and related advancements in Vδ2 T cell technology can be used to generate potent, high-quality Vδ2 cells on a scale suitable for a wide range of clinical applications.

[0009] The present invention disclosed herein has multiple embodiments. Embodiments of the present invention include methods of culturing mammalian cells (typically human T lymphocytes), the methods comprising: obtaining a population of lymphocytes (e.g., obtaining a population of lymphocytes from the peripheral blood of one or more donors); identifying donors based on Vδ2 T cell CD16 expression; selecting and / or purifying CD16-expressing cells within the population of T lymphocytes HiDonor cells in combination with Vδ2; then expand the selected and / or purified T lymphocytes expressing the combination of CD16 and Vδ2. In certain embodiments of the present invention, the cells are expanded 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000-fold. In some embodiments, the expansion phase lasts more than 3, 4, 5, or 6 weeks. In some embodiments, the cells are restimulated during the expansion phase. Generally, in such methods, the selected and / or purified T lymphocytes expressing the combination of CD16 and Vδ2 are expanded by placing the T lymphocytes in a culture medium that contains a combination of a γδ T cell stimulant (e.g., the bisphosphonate zoledronic acid), cytokines (e.g., IL-2, IL-15, IL-12, IL-18, IL-7, IL-21, TGF-β), a Wnt activator (e.g., Wnt3a), and / or a GSK-3β inhibitor (e.g., TWS119). In certain embodiments of the present invention, the method includes testing / examining CD16 in a cellular cytotoxicity assay such as an antibody-dependent cellular cytotoxicity assay Hi cells within the Vδ2 lymphocyte population.

[0010] Embodiments of the present invention include a cell culture medium for expanding T lymphocytes expressing the combination of CD16 and Vδ2, the medium containing: a γδ T cell stimulant (e.g., a bisphosphonate), a combination of cytokines, a Wnt activator, and / or a GSK-3β inhibitor. In certain embodiments of the present invention, this medium further contains a population of T cells that has been selected for T lymphocytes expressing the combination of CD16 and Vδ2. Optionally, the T cell population has been selected or enriched by: using an antibody that specifically binds CD16, an antibody that specifically binds Vδ2; magnetic bead sorting; and / or fluorescence-activated cell sorting (FACS). Related embodiments of the present invention also include a method for preparing a cell culture medium for expanding T lymphocytes expressing the combination of CD16 and Vδ2, the method including combining a γδ T cell stimulant (e.g., a bisphosphonate), a combination of cytokines, a Wnt activator, and / or a GSK-3β inhibitor to form the cell culture medium. In some embodiments of the present invention, a tyrosine kinase inhibitor is included in the cell culture medium. Certain embodiments of the present invention include further placing the selected T cells expressing the combination of CD16 and Vδ2 in the cell culture medium. In certain embodiments of the present invention, the population of T cells has been modified to regulate the expression of one or more endogenous genes (e.g., human leukocyte antigen genes, etc.). In some embodiments of the present invention, the cell population is engineered to express an exogenous transgene (e.g., a chimeric alpha beta (αβ) antigen receptor transgene).

[0011] Embodiments of the invention also include methods of treating a patient with the CD16 Hi Vδ2 T cells or cell populations disclosed herein. Such embodiments of the invention include methods of treating a subject in need of γδ T cells (e.g., to combat diseases such as autoimmune diseases or cancer or infections such as viral, bacterial, or parasitic infections), which comprise administering to the subject functional CD16 Hi Vδ2 γδ T cells selected to target the appropriate pathology.

[0012] Other objects, features, and advantages of the invention will become apparent to those skilled in the art from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating some embodiments of the invention, are given by way of illustration and not limitation. Many changes and modifications may be made within the scope of the invention without departing from the spirit of the invention, and the invention includes all such modifications. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : CD16 serves as a biomarker for screening peripheral blood mononuclear cell (PBMC) donors for high-performance Vδ2 T cells. a Experimental design for generating PBMC-derived Vγ9Vδ2T (referred to as Vδ2T) cells. Zoledronic acid (ZOL) and IL-2 are used to activate and expand Vδ2T cells. b Pie chart showing the proportion of CD16 high (CD16 Hi ) and CD16 low (CD16 Lo ) Vδ2T cell donors. Note: A total of 30 healthy donors were screened. The cut-off value between CD16 Hi and CD16 Lo donors is 35% of CD16 + cells in total Vδ2T cells. c FACS quantification of % CD16 + cells in total Vδ2T cells before and after activation and expansion. PBMC from 30 different donors were used. Note: Vδ2T cells were expanded for 10 - 14 days. d Representative FACS plot of (c). e Expansion of CD16 Hi and CD16 Lo Vδ2T cells over 14 days (n = 5; n represents different donors). f - h Study of CD16 Hi and CD16 LoIn vitro anti-tumor efficacy of Vδ2 T cells. Two human ovarian cancer cell lines were studied as tumor targets: OVCAR3-FG and SKOV3-FG; both cell lines were engineered to express the dual reporter genes of firefly luciferase and green fluorescent protein (FG). f Experimental design. g Tumor cell killing data at 24 h (n = 3, from 3 different donors). h At 24 h, ELISA measurement of IFN-γ production (left), and FACS measurement of intracellular perforin (middle) and granzyme B (right) production (E:T ratio = 1:1; n = 3 from 3 different donors). i-k Study of CD16 overexpression in CD16 LO Vδ2 T cells. CD16 Lo Vδ2 T cells were transduced with a lentiviral vector encoding the human CD16 gene (Lenti / CD16). i Schematic design of the overexpression experiment. j FACS detection of CD16 overexpression on CD16 Lo Vδ2 T cells transduced with titrated Lenti-CD16. k At 24 h after co-culture, in the presence of ZOL, CD16 Lo Vδ2 T cell in vitro killing of OVCAR3-FG cells (n = 3). Representative of >10 experiments (a-d), 5 experiments (e), 3 experiments (f-h), and 1 experiment (i-k). Data are represented as mean ± SEM. By Student's t-test (e, g) or one-way ANOVA (h): ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0014] Figure 2 : CD16 Hi Vδ2 T cells show an enhanced cytotoxic gene signature. RNA-Seq analysis was performed on Vδ2 T cells amplified from 13 PBMC donors. a Dot plot showing CD16 mRNA expression in Vδ2 T cells (CPM: counts per million mapped reads). Samples were classified into CD16 Hi (n = 3) and CD16 Lo (n = 10) Vδ2 T cells using the k-means clustering algorithm (k = 2). b Principal component analysis (PCA) plot showing the coordination of CD16 Hi and CD16 Lo Vδ2 T cells. c Volcano plot showing differential gene expression between CD16 Hi and CD16 Lo Vδ2 T cells. FC, fold change. NS, not significant. d Gene set enrichment analysis (GSEA) plot showing CD16 HiSignificant enrichment of the gene signature shown in Vδ2 T cells. NES, normalized enrichment score. e shows the Cnet plot of the enriched GO pathways. f shows the correlation plot of the relationship between CD16 expression and immune cell type scores in Vδ2 T cell samples. Circle size and color intensity are proportional to the correlation; red and blue represent positive and negative correlations, respectively. Representative of 1 experiment.

[0015] Figure 3 : CD16 Hi Vδ2 T cells can be engineered with CAR / IL-15 while retaining the expansion capacity and memory state. a Generation of MCAR / IL-15-engineered CD16 Hi Experimental design of Vδ2 T (MCAR15-Vδ2 T) cells. Note: ZOL and IL-2 were used to activate and expand Vδ2 T cells. CAR, chimeric antigen receptor. b Schematic diagram of the indicated lentiviral vectors. Lenti / MCAR, lentiviral vector encoding mesothelin (MSLN)-targeted CAR (MCAR); Lenti / MCAR15: lentiviral vector encoding the same MCAR and secreted form of human IL-15. scFv: single-chain variable fragment; V H : variable heavy chain; V L : variable light chain; H: CD8 hinge; TM: CD28 transmembrane domain; CD28: CD28 intracellular domain; CD3ζ: CD3ζ intracellular domain; IL-15: interleukin 15. c CD16 transduced with mock (represented as untransduced Vδ2 T cells; NT-Vδ2 T cells), or with Lenti / MCAR (represented as mCAR-Vδ2 T cells), or with Lenti / MCAR15 (represented as mCAR15-Vδ2 T cells) Hi FACS analysis of CAR expression on Vδ2 T cells. d Quantification of c (n = 10; n represents different donors). e Expansion of the indicated Vδ2 T cells in 14-day culture (n = 5; n represents different donors). f FACS analysis of the cell composition of expanded MCAR15-Vδ2 T cells. g Quantification of f (n = 10; n represents different donors), NK cells were gated as CD45 + CD56 + CD3 -cells. FACS detection of IL-15 signaling events in hMCAR15-Vδ2 T cells. Phosphorylation of STAT5 (pSTAT5) and upregulation of the anti-apoptotic transcription factors Bcl-xL and Bcl-2 were studied. Cells were cultured in cytokine-free medium for 48 h and then subjected to intracellular staining. i IL-15 production ELISA measurement (n = 3) from MCAR15-Vδ2 T cells for 48 h in the presence or absence of the human ovarian cancer cell line OVCAR3-FG. j-l Study of the memory phenotype of MCAR15-Vδ2 T cells. j Schematic diagram defining memory T cell subsets by cell surface expression of the CD27 and CD45RA biomarkers. Stem cell-like memory (T SCM ): CD27 + CD45RA + ; Central memory (T CM ): CD27 + CD45RA - ; Effector memory (T EM ): CD27 - CD45RA - ; Terminally differentiated effector memory (T EMRA ): CD27 - CD45RA + . k FACS detection of CD27 and CD45RA expression on CAR15 + and CAR15 - MCAR15-Vδ2 T cells. l Quantification of k (n = 5; n represents different donors). Representative of 1 experiment (b), 3 experiments (h, i), 5 experiments (j-l), and >10 experiments (c-g). Data are represented as mean ± SEM. By Student's t-test (d, l) or one-way ANOVA (e, i): ns, not significant; *p < 0.05; ****p < 0.0001.

[0016] Figure 4 : MCAR15-Vδ2 T cells can effectively kill tumor cells via a CAR / TCR dual-targeting mechanism. Three human ovarian cancer cell lines (OVCAR3-FG, OVCAR8-FG, and SKOV3-FG) and four effector cells (Vδ2T, MCAR-Vδ2T, MCAR15-Vδ2T, and MCAR-T) were used in the study. MCAR-T represents conventional αβ T cells engineered to express the same MCAR (as a benchmark control). The same CD16 HiDonor PBMCs were used to generate all 4 types of effector cells. ae In vitro 24-hour tumor cell killing assay. a Experimental design. Tumor cells and effector cells were co-cultured for 24 hours and then analyzed. b FACS plots showing the expression of mesothelin (MSLN) tumor antigen on the indicated ovarian cancer cell lines. c Tumor cell killing data collected at 24 hours (n = 3). d FACS detection of perforin and granzyme B production by MCAR15-Vδ2 T cells at 24 hours (E:T ratio = 1:1). e ELISA measurement of IFN-γ production at 24 hours (E:T ratio = 1:1; n = 3). fi In vitro repeated tumor cell attack assay. f Experimental design. Effector cells were mixed with tumor cells and attacked again every 3 days, and tumor cell killing data were collected 24 hours after each tumor cell dosing (E:T ratio = 2:1; n = 3). g Tumor cell killing data collected over time. h MSLN knockout OVCAR3-FG ( KO Generation and FACS validation of OVCAR3-FG cell lines. i Collected after the third tumor cell re-challenge KO OVCAR3-FG tumor cell killing data. Representative of 3 (a, cg, i) and 1 (b, h) experiments. Data are expressed as mean ± SEM. By Student's t test (c, e) or one-way ANOVA (i): ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0017] Figure 5 :MCAR15-Vδ2T cells can also effectively target tumor cells via antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. ag in vitro ADCC assay. Three human ovarian cancer cell lines (OVCAR3-FG, OVCAR8-FG and SKOV3-FG) and four effector cells (Vδ2T, MCAR-Vδ2T, MCAR15-Vδ2T and MCAR-T as a baseline control) were included in the study. The same CD16 HiDonor PBMCs were used to generate all 4 types of effector cells. a Experimental design. Data were collected 24 hours after co-culture. Anti-HER2 Ab: monoclonal antibody trastuzumab. b FACS detection of HER2 expression on the indicated tumor cell lines. c Killing of OVCAR3-FG tumor cells by Vδ2T cells in the presence of titrated isotype control or anti-HER2 Ab (n = 3). d-f Tumor cell killing data of (d) OVCAR3-FG, (e) OVCAR8-FG, and (f) SKOV3-FG in the presence or absence of anti-HER2 Ab (anti-HER2 Ab concentration = 0.1 μg / mL; E:T ratio = 0.5:1; n = 3). g ELISA measurement of IFN-γ production at 24 hours in the presence or absence of anti-HER2 Ab (anti-HER2 Ab concentration = 0.1 μg / mL; E:T ratio = 1:1; n = 3). h,i In vitro repeated tumor cell attack assay to study ADCC. h Experimental design. Effector cells were mixed with tumor cells and re-attacked every 3 days. Tumor cell killing data measured 24 hours after co-culture with or without anti-HER2 antibody (anti-HER2 Ab concentration = 0.1 μg / mL; E:T ratio = 2:1; n = 3). i KO OVCAR3-FG tumor cell killing data. Representative of 3 experiments. Data are presented as mean ± SEM. By Student's t test (c) or one-way ANOVA (d-g): ns, not significant; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0018] Figure 6 : In vivo anti-tumor efficacy and safety of MCAR15-Vδ2T cells in an intraperitoneal tumor model. OVCAR3-FG human ovarian cancer cells were injected i.p. into NSG mice on day 0, and then effector cells were administered i.p. on day 14 (n = 5 mice per group). Four experimental groups were included: vehicle (mice not receiving effector cells), MCAR-T (mice receiving MCAR-T cells), MCAR-Vδ2T (mice receiving MCAR-Vδ2T cells), and MCAR15-Vδ2T (mice receiving MCAR15-Vδ2T cells). Note: The same CD16 HiDonor PBMCs were used to generate all three types of effector cells. a Experimental design. b BLI images showing the tumor burden of experimental mice over time. c Quantification of b. TBL, total body luminescence. d Tumor burden on day 55. e Kaplan–Meier survival curves of experimental mice over time. Representative of three experiments. Data are expressed as mean ± SEM. By one-way ANOVA (d), or by log-rank (Mantel–Cox) test with adjustment for multiple comparisons (e): ns, not significant; *p < 0.05; **p < 0.01; ****p < 0.0001.

[0019] Figure 7 : In vivo anti-tumor efficacy and safety of MCAR15-Vδ2 T cells in a subcutaneous tumor model. OVCAR8 human ovarian cancer cells were injected s.c. into NSG mice on day 0, and then effector cells were administered intravenously (i.v.) on day 7. Four experimental groups were included: vehicle (mice not receiving effector cells), MCAR-T (mice receiving MCAR-T cells), MCAR-Vδ2T (mice receiving MCAR-Vδ2T cells), and MCAR15-Vδ2T (mice receiving MCAR15-Vδ2T cells). Note: The same CD16 Hi Donor PBMCs generated all three types of effector cells. a Experimental design. b Tumor growth over time. c Tumor size measurements collected on day 57 (n = 5). d FACS quantification of MCAR-Vδ2T and MCAR15-Vδ2T cells in the tissues of experimental mice collected on day 57 (n = 5). e H&E-stained tissue sections collected on day 57. Scale bar, 100 μm. Representative of three experiments. Data are expressed as mean ± SEM. By one-way ANOVA (c): ns, not significant; ****p < 0.0001.

[0020] Figure 8 : From CD16 Hi or CD16 Lo Characterization of CD16 + / - Vδ2 T cells generated from PBMC donors. According to the experimental design shown in Figure 1 a, Vδ2 T cells were generated from CD16 Hi or CD16 Lo donor PBMCs, and then FACS analysis was performed for the a, b gating strategy to select CD16 Hi or CD16 Lo from donors, and CD16 + and CD16 -Vδ2 T cells, surface expression markers (n = 5), and intracellular perforin and granzyme B production 24 hours after co-culture with e-h tumors (E:T ratio = 1:1; n = 3 from 3 different donors). CD16 was gated based on cell surface CD16 expression. Representative of >10 experiments (a,b), 2 experiments (c,d), and 3 experiments (e-h). Data are presented as mean ± SEM. By Student's t-test (c,d) or one-way ANOVA (e-h): ns, not significant; *p < 0.05; **p < 0.01; ****p < 0.0001. + and CD16 - Vδ2 T cells were gated. Representative of >10 experiments (a,b), 2 experiments (c,d), and 3 experiments (e-h). Data are presented as mean ± SEM. By Student's t-test (c,d) or one-way ANOVA (e-h): ns, not significant; *p < 0.05; **p < 0.01; ****p < 0.0001.

[0021] Figure 9 : Transcriptomic characterization of Vδ2 T cells associated with CD16 expression. a shows a heatmap of the expression levels of selected genes in Vδ2 T cells generated from three CD16 Hi PBMC donors and ten CD16 Lo PBMC donors. b Genome enrichment analysis, which shows the relationship between CD16 expression and biological process pathways in Vδ2 T cells generated from all 13 PBMC donors (including 3 CD16 Hi donors and 10 CD16 Lo donors). The top 30 most significantly activated or inhibited biological process pathways are shown. Representative of 1 experiment.

[0022] Figure 10 : Comparison of the in vitro anti-tumor efficacy of CD16 Hi and CD16 Lo MCAR15-Vδ2 T cells. Vδ2 T cells were cultured from CD16 Hi or CD16 Lo donor PBMCs and engineered to express MCAR and IL-15. The resulting cell products are designated as 16H MCAR15-Vδ2 T or 16HMCAR15-Vδ2 T cells. a, b In vitro tumor cell killing assay. The OVCAR3-FG human ovarian cancer cell line was used. a Experimental design. b Tumor cell killing data collected 24 h after co-culture (n = 3; n represents different donors). c, d In vitro antibody-dependent cell-mediated cytotoxicity (ADCC) assay. The SKOV3-FG human ovarian cancer cell line was used. c Experimental design. d Tumor cell killing data collected 24 h after co-culture (anti-HER2 Ab concentration = 0.1 μg / mL; n = 3; n represents different donors). Representative of 3 experiments. Data are presented as mean ± SEM. By Student's t test (b, d): ns, not significant; *p < 0.05; ***p < 0.001; ****p < 0.0001.

[0023] Figure 11 : FMCAR15-Vδ2 T cells can target M2-polarized human macrophages. a Experimental design for generating unpolarized (M0) or M2-polarized human monocyte-derived macrophages (MDMs). M-CSF, macrophage colony-stimulating factor; macrophages. b FACS detection of CD11b and CD14 expression on M2-polarized macrophages. c FACS detection of M2 macrophage markers (i.e., CD163 and CD206) on M2-polarized macrophages. d, e Investigation of the in vitro killing of M2-polarized macrophages by MCAR15-Vδ2 T effector cells. d Experimental design. e Data collected 24 h after co-culture Data are presented as mean ± SEM. By one-way ANOVA (e): ns, not significant; *p < 0.05; ***p < 0.001; ****p < 0.0001.

[0024] Figure 12 : Comparison of CD16 Hi and CD16 Lo The in vitro anti-tumor efficacy of MCAR15-Vδ2 T cells. Vδ2 T cells were cultured from CD16 Hi or CD16 Lo donor PBMCs and engineered to express MCAR and IL-15. The resulting cell products were designated as 16H MCAR15-Vδ2 T or 16H MCAR15-Vδ2 T cells. a Experimental design. Three experimental groups were included: vehicle (mice not receiving effector cells; n = 5), 16L MCAR15-Vδ2 T (mice receiving 16H MCAR15-Vδ2 T cells; n = 9 from 3 different donors) and 16L MCAR15-Vδ2 T (mice receiving 16LMice with MCAR15-Vδ2 T cells; n = 9 from 3 different donors). b Tumor growth over time. c Tumor size measured on day 57. Representative of 2 experiments. Data are presented as mean ± SEM. By one-way ANOVA (c): **p < 0.01; ****p < 0.0001. Detailed implementation

[0025] In the description of the embodiments, reference may be made to the accompanying drawings that form a part of this specification and in which specific embodiments in which the invention may be practiced are illustrated by way of example. It should be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the invention.

[0026] Of particular note is that any embodiment discussed herein in the context of a particular cell or cell population embodiment may be employed with respect to any other cell or cell population embodiment. Additionally, any embodiment employed in the context of a particular method may be implemented in the context of any other method described herein. Further, aspects of the different methods described herein may be combined to achieve other methods, as well as to generate any cell or cell population or described use. It is specifically contemplated that aspects of one or more embodiments may be combined with aspects of one or more other embodiments described herein. Additionally, any method described herein may be expressed as reciting one or more uses of the cells or cell populations described herein. For example, the use of γδ T cells or a population of γδ T cells may be recited by any method described herein.

[0027] As noted above, there is a need in the art for methods and materials capable of reliably generating large quantities of potent γδ T cells. These technologies are critical for the development of off-the-shelf T cell therapies. Such methods and materials can, for example, provide γδ T cells that can be used in allogeneic or autologous recipient subjects for the treatment of a variety of pathological conditions, including, for example, viral infections, bacterial infections, fungal infections, protozoal infections, and cancer.

[0028] Embodiments of the invention include an expansion medium and methods of using the same, as well as functional CD16 produced by the methods disclosed herein HiVδ2 γδ T cells and cell populations. Generally, these populations consist essentially of functional γδ T cells (e.g., excluding conventional αβ T cells). Specific embodiments of the invention include a cell culture medium for amplifying T lymphocytes expressing the combination of CD16 and Vδ2, the medium comprising: a γδ T cell stimulant (e.g., bisphosphonate), a combination of cytokines, and a combination of small molecules (e.g., a Wnt activator, a GSK-3β inhibitor). The medium can be a serum-containing or serum-free medium, or a xeno-free medium. From the viewpoint of preventing contamination caused by heterogeneous animal-derived components, the serum can be derived from the same animal as the stem cells. A serum-free medium refers to a medium that does not contain unprocessed or unpurified serum, and thus can include a medium having purified blood-derived components or animal tissue-derived components (such as growth factors).

[0029] Embodiments of the invention include a method of culturing mammalian (e.g., human) cells, the method comprising: obtaining a population of lymphocytes (e.g., obtained from the peripheral blood of one or more donors); identifying and / or selecting a population of cells from one or more donors based on their Vδ2 T cell CD16 expression profile; and then amplifying the identified, selected, and / or purified T lymphocytes expressing the combination of CD16 and Vδ2. In this context, aspects of the human γδ T cell subset and the Vδ2 TCR stimulation mechanism are discussed in Lee et al., Cancers 2022, 14, 3005, the content of which is incorporated by reference. In some embodiments of the invention, the population of lymphocytes is selected from one or more donors identified as having at least 35% CD16-positive cells within the population of Vδ2 T lymphocytes obtained from an individual (referred to herein as "CD16" Hi "). In particular, as used herein, "CD16 high" means a cut-off value of 35% or more CD16-positive cells in the total Vδ2 (e.g., Vg9Vd2) T cells from primary donor PBMCs measured by flow cytometry. In other embodiments of the invention, the population of lymphocytes is selected from one or more individuals identified as having less than 35% CD16-positive cells within the population of Vδ2 T lymphocytes obtained from an individual (referred to herein as "CD16" Lo ") (e.g., as observed in flow cytometry analysis).

[0030] In certain embodiments of the invention, the cells are expanded 10, 100, 500, 1000, 2,000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000-fold. In some embodiments, the expansion phase lasts at least 3, 4, 5 or 6 weeks. In some embodiments, the cells are restimulated one or more times in the culture. Typically in such methods, the identified, selected and / or purified T lymphocytes expressing the combination of CD16 and Vδ2 are expanded by placing the T lymphocytes in a medium comprising a combination of a γδ T cell stimulant (e.g., an antibody specific for the γδ receptor on the cells, bisphosphonates such as zoledonic acid, etc.), cytokines (e.g., IL-2, IL-15, IL-12, IL-18, IL-7, IL-21, TGF-β) and a Wnt activator (e.g., Wnt3a) and / or a GSK-3β inhibitor (e.g., TWS119). For example, in certain embodiments, the medium comprises a combination of IL-7 and IL-21. In some embodiments of the invention, the medium comprises a bisphosphonate such as zoledonic acid at a concentration of about 0.1 uM to about 100 uM (e.g., 5-10 uM). In some embodiments of the invention, the medium comprises a phosphoantigen at a concentration of about 1 nM to about 1 uM, such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP). In certain embodiments of the invention, the medium comprises one or more cytokines at a concentration of about 1 ng / mL to about 500 ng / mL. In certain embodiments of the invention, the medium comprises a Wnt activator (e.g., Wnt3a) at a concentration of about 1 ng / mL to about 1 ug / mL. In certain embodiments of the invention, the medium comprises a GSK-3β inhibitor (e.g., TWS119) at a concentration of about 0.1 uM to about 50 uM.

[0031] In some embodiments, CD16 Hi The Vδ2 T cells are cultured and / or expanded in a medium containing 1, 2, 3, 4, 5, 6, 7 or 8 of the following Wnt activators and / or GSK-3β inhibitors: Wnt3A, CHIR99021, AR-A014418, TWS119, LY2090314, 9-ING-41, lithium chloride (LiCl) or BIO (6-bromoindirubin-3-oxime, 6-bromoindirubin-3'-oxime). In additional embodiments, CD16 HiVδ2 T cells are cultured and / or expanded in a medium containing 1, 2, 3, or 4 of the following tyrosine kinase inhibitors: dasatinib, ibrutinib, acalabrutinib, or zanubrutinib.

[0032] In general embodiments, CD16 Hi Vδ2 T cells are cultured and / or expanded in a serum-free medium. In certain embodiments, the serum-free medium further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all 16 (or any range derivable therefrom) of the following externally added components: interleukin 7 (IL-7), stromal cell-derived factor 1α (SDF-1α), IL-2, IL-4, IL-6, IL-12, IL-15, IL-18, IL-21, IL-23, TNF-α, TGF-β, interferon-γ, interferon-λ, TSLP, thymopentin, pleotrophin, or midkine. In additional embodiments, the serum-free medium comprises one or more vitamins. In some cases, the serum-free medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any range derivable therefrom) of the following vitamins: biotin, DLα-tocopheryl acetate, DLα-tocopherol, vitamin A, vitamin C, choline chloride, calcium pantothenate, pantothenic acid, folic acid, nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or a salt thereof. In certain embodiments, the medium comprises or at least comprises biotin, DLα-tocopheryl acetate, DLα-tocopherol, vitamin A, vitamin C, or a combination or salt thereof. In additional embodiments, the serum-free medium comprises one or more proteins. In some embodiments, the serum-free medium comprises 1, 2, 3, 4, 5, 6, or more (or any range derivable therefrom) of the following proteins: albumin or bovine serum albumin (BSA), fractions of BSA, catalase, insulin, transferrin, superoxide dismutase, or a combination thereof. In other embodiments, the serum-free medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the following compounds: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-L-thyronine, or a combination thereof. In additional embodiments, the serum-free medium comprises supplements, xeno-free Supplements, GS21TM supplements, or combinations thereof. In additional embodiments, the serum-free medium contains or further contains amino acids, monosaccharides, and / or inorganic ions. In some aspects, the serum-free medium contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the following amino acids: arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In other aspects, the serum-free medium contains 1, 2, 3, 4, 5, or 6 of the following inorganic ions: sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In additional aspects, the serum-free medium contains 1, 2, 3, 4, 5, 6, or 7 of the following elements: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain embodiments, the serum-free medium further contains externally added ascorbic acid. In specific embodiments, the method involves adding ascorbic acid to the medium.

[0033] The medium in certain embodiments of the present invention can be prepared using a medium for culturing animal cells as its basal medium, such as any one of AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640, and Fischer's media, and any combination thereof. However, the medium is not particularly limited thereto as long as it can be used to culture animal cells. In particular, the medium can be xeno-free or chemically defined.

[0034] The medium can contain or not contain any serum replacements. Serum replacements can include materials that appropriately contain albumin (such as lipid-rich albumin, bovine albumin, albumin substitutes such as recombinant albumin or humanized albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transport proteins), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiol glycerol, or equivalents thereof. Serum replacements can be prepared by the methods disclosed in, for example, International Publication No. 98 / 30679, the entire content of which is incorporated herein. Alternatively, for greater convenience, any commercially available materials can be used. Commercially available materials include knockout serum replacement (KSR), chemically defined lipid concentrate (Gibco), and Glutamax (Gibco).

[0035] In general embodiments of the present invention, the expansion medium is a serum-free medium suitable for cell development. For example, the medium can comprise supplements, xeno-free supplements (available at the World Wide Web address thermofisher.com / us / en / home / technical-resources / media-formulation.250.html), NS21 supplement (Chen et al., J Neurosci Methods, June 30, 2008; 171(2):239–247, which is incorporated herein in its entirety), GS21 TM supplement (available at the World Wide Web address amsbio.com / B-27.aspx) or a combination thereof, at a concentration effective for generating T cells from 3D cell aggregates.

[0036] As discussed in the examples below, in some embodiments, the CD16 Hi Vδ2 T cells of the present invention are placed in selected medium conditions during growth and differentiation. The cells produced by the preparation method can be frozen. The produced cells can be in a solution containing dextran, one or more electrolytes, albumin, dextran, and DMSO. The solution can be sterile, non-pyrogenic, and isotonic. In some embodiments, the γδ T cells have been previously frozen, and the previously frozen cells are stable at room temperature for at least one hour. In some embodiments, the CD16 Hi Vδ2 T cells have been previously frozen, and the previously frozen cells are stable at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 24, 30, or 48 hours (or any derivable range therein). In certain embodiments, the CD16 Hi Vδ2 T cells or a population of CD16 Hi Vδ2 T cells are in a solution containing dextran, one or more electrolytes, albumin, dextran, and / or DMSO.

[0037] As described below, in certain embodiments of the present invention, PBMCs are cultured to generate memory-like Vδ2 T cells (the accompanying drawings show cell markers associated with memory-like Vδ2 T cells, such as CD27 and CD45RA surface markers). Vδ2 T cells can be stimulated and expanded by supplementing the cell culture with Vδ2 stimulating reagents, including but not limited to TCR cognate antigens, phosphoantigens, small molecules, and / or antibodies. Examples of such reagents include isopentenylpyrophosphate, zoledronate, pamidronate, risedronate, alendronate, ibandronate, tiludronate, etidronate, anti-γδ TCR antibodies, non-specific TCR stimulating reagents (anti-CD3 / anti-CD28 antibodies or antibody-coated beads, Concanavalin A, PMA / ionomycin, and artificial APCs), anti-CD16 antibodies, etc. The memory-like characteristics of the cultured Vδ2 T cells can be achieved by supplementing the cell culture with memory-promoting reagents, including but not limited to serum albumin, L-ascorbic acid, 2-mercaptoethanol, IL-2, IL-4, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, TNFα, SDF-1α, TGF-β, and Wnt activators or glycogen synthase kinase-3 (GSK-3) inhibitors such as Wnt3A, CHIR99021, AR-A014418, TWS119, LY2090314, 9-ING-41, lithium chloride (LiCl), BIO (6-bromoindirubin-3-oxime, 6-bromoindirubin-3'-oxime). The memory medium can also include tyrosine kinase inhibitors, such as dasatinib, ibrutinib, acalabrutinib, zanubrutinib, etc. The memory medium can be used to culture and expand all types of Vδ2 T cells, including CD16 Hi Vδ2 T cells. In some embodiments, CD16 Lo Vδ2 T and / or CD16 Lo Vδ2 T and CD16 Hi Vδ2 T cell mixtures are cultured / expanded using the aforementioned medium and method.

[0038] In embodiments involving multiple cells, a CD16 Hi Vδ2 T cell population can contain, at least contain, or at most contain about 10 2 、10 3 、10 4 ,、10 5 、10 6 、10 7 、10 8 、10 9 、10 10 、1011 、 10 12 、 10 13 、 10 14 、 10 15 or more cells (or any range derivable therefrom), where in some embodiments are CD16 Hi Vδ2 T cells. In some cases, the cell population comprises at least about 10 6 - 10 12 CD16 Hi Vδ2 T cells. It is contemplated that in some embodiments, cell populations having these numbers are produced from a single batch of cells rather than being the result of combining multiple batches of separately produced cells.

[0039] Embodiments of the invention also include methods of treating a patient with the CD16Hi Vδ2 T cells or cell populations disclosed herein. Such embodiments of the invention include methods of treating a subject in need of γδ T cells (e.g., to combat a disease such as an autoimmune disease or cancer or an infection such as COVID-19), which include administering to the subject the functional CD16 Hi Vδ2 T cells disclosed herein. In this way, such T cells can be used to treat patients with a variety of pathological conditions. In certain treatment methods of the invention, the patient has been diagnosed with cancer. In some embodiments of the invention, the patient has a disease or disorder involving inflammation, which in some embodiments does not include cancer. In certain embodiments, the patient has an autoimmune disease or disorder. In a particular aspect of the invention, the cells or cell population are allogeneic to the patient. In certain embodiments, the patient does not exhibit signs of rejection or depletion of the cells or cell population. Some treatment methods also include administering to the patient an antibody or stimulatory molecule that activates γδ T cells (e.g., alone or loaded onto an APC) and / or a compound that initiates a suicide gene product.

[0040] Treating a cancer patient with CD16 Hi Vδ2 T cells may result in killing of the tumor cells of the cancer patient after administering the CD16 Hi Vδ2 T cells or cell population to the patient. Treatment of an inflammatory disease or disorder may result in a reduction in inflammation. In other embodiments, a patient with an autoimmune disease or disorder may experience improvement in the symptoms of the disease or disorder or may experience other therapeutic benefits from the CD16 Hi Vδ2 T cell population disclosed herein. A combination treatment of CD16 Hi Vδ2 T cells and a standard treatment regimen or another immunotherapy regimen can be employed.

[0041] As described below, in some embodiments, CD16 HiVδ2 T cells can be engineered to regulate endogenous genes and / or express exogenous transgenes. Embodiments of the invention include a composition of matter comprising a CD16 as disclosed herein Hi Vδ2 T cells or a population of T cells, such as those comprising a gene expression profile characterized as: HLA-I negative; HLA-II negative; HLA-E positive; expressing a suicide gene; and expressing one or more exogenous nucleic acids such as those encoding a T cell receptor gamma chain polypeptide and an exogenous T cell receptor delta chain polypeptide. Optionally, the CD16 Hi Vδ2 T cells further comprise an exogenous nucleic acid molecule encoding another polypeptide such as a T cell receptor alpha chain polypeptide and / or a T cell receptor beta chain polypeptide and / or an iNKT receptor polypeptide; and / or a cytokine; and / or comprise an inhibited endogenous TCR.

[0042] In certain embodiments, the CD16 Hi Vδ2 T cells lack or have reduced surface expression of at least one HLA-I or HLA-II molecule. In some embodiments, the lack of surface expression of HLA-I and / or HLA-II molecules is achieved by disrupting the gene encoding a single HLA-I / II molecule, or by disrupting the gene encoding B2M (beta-2 microglobulin), which is a common component of all HLA-I complex molecules, or by disrupting the gene encoding CIITA (class II major histocompatibility complex transactivator), which is a key transcription factor controlling the expression of all HLA-II genes. In a specific embodiment, the cell lacks surface expression of one or more HLA-I and / or HLA-II molecules, or expresses such molecules at a reduced level (or at least reduced) by 50%, 60%, 70%, 80%, 90%, 100% (or any range derivable therein). In some embodiments, due to genetic editing of the gamma-delta T cells, HLA-I or HLA-II is not expressed in the cell. In some embodiments, the genetic editing involved is CRISPR-Cas9. In addition to Cas9, CasX or CasY may be involved. Zinc finger nucleases (ZFNs) and TALENs are other gene editing techniques, as well as Cpf1, all of which may be employed. In other embodiments, the gamma-delta T cells comprise one or more different siRNA or miRNA molecules targeted to reduce the expression of HLA-I / II molecules, B2M, and / or CIITA.

[0043] In a specific embodiment, there is a CD16 expressing an exogenous nucleic acid such as an exogenous nucleic acid encoding a suicide gene product HiVδ2 T cells. Methods for the use of suicide genes in the art can be employed, such as those in U.S. Patent No. 8,628,767, U.S. Patent Application Publication 2014 / 0369979, U.S. Patent Application Publication 2014 / 0242033, and U.S. Patent Application Publication 2004 / 0014191, all of which are incorporated by reference in their entirety. In further embodiments, the TK gene is a viral TK gene, i.e., a TK gene from a virus. In certain embodiments, the TK gene is the herpes simplex virus TK gene. In some embodiments, the suicide gene product is activated by a substrate. Thymidine kinase is a suicide gene product activated by ganciclovir, penciclovir, or a derivative thereof. In certain embodiments, the substrate that activates the suicide gene product is labeled for detection. In some cases, the substrate can be labeled for imaging. In some embodiments, the suicide gene product can be encoded by the same or different nucleic acid molecules encoding one or both of TCR-γ or TCR-δ. In certain embodiments, the suicide gene is sr39TK or inducible caspase 9. In alternative embodiments, the cells do not express an exogenous suicide gene.

[0044] In a specific embodiment, there is a population of T cells comprising: clonal CD16 Hi Vδ2 T cells that comprise one or more exogenous nucleic acids encoding molecules such as a γδ T cell receptor and a thymidine kinase suicide gene product, wherein the clonal γδ T cells have been engineered not to express functional β-2-microglobulin (B2M) and / or class II major histocompatibility complex or class II transactivator (CIITA), and wherein the population of cells is at least about 10 6 -10 12 total cells and comprises at least about 10 2 -10 6 γδ T cells. In certain cases, the cells are frozen in solution.

[0045] In some embodiments, the CD16 Hi Vδ2 T cells of the present invention comprise a recombinant vector or a nucleic acid sequence from a recombinant vector introduced into the γδ cells. In certain embodiments, the recombinant vector is or was a viral vector. In further embodiments, the viral vector is or was a lentivirus, retrovirus, adeno-associated virus (AAV), herpesvirus, or adenovirus. It should be understood that the nucleic acid of certain viral vectors integrates into the host genomic sequence.

[0046] In exemplary embodiments, the exogenous nucleic acid can be transduced into CD16 HiVδ2 T cells, such as those comprising a nucleotide sequence encoding a γ-TCR and / or a δ-TCR. In certain embodiments, one nucleic acid encodes both the γ-chain and the δ-chain of the TCR. In some embodiments, additional nucleic acids may comprise nucleic acid sequences encoding an α-TCR and / or a β-TCR polypeptide and / or one or more iNKT TCR polypeptides. In further embodiments, the nucleic acid further comprises a nucleic acid sequence encoding a suicide gene product. In some embodiments, the nucleic acid molecule introduced into the selected cells encodes a TCR and a suicide gene product. In other embodiments, the method further involves introducing a nucleic acid encoding a suicide gene product into the selected cells, in which case a nucleic acid molecule different from the nucleic acid encoding at least one TCR gene encodes the suicide gene product.

[0047] As described above, in some embodiments, CD16 Hi Vδ2 T cells do not express HLA-I and / or HLA-II molecules on the cell surface, which can be achieved by disrupting the expression of genes encoding β-2-microglobulin (B2M), class II transactivator (CIITA), or HLA-I and HLA-II molecules. In certain embodiments, the method involves eliminating the surface expression of one or more HLA-I / II molecules in isolated human cells. In specific embodiments, the elimination of expression can be accomplished by gene editing of the cellular genomic DNA. Some methods include introducing CRISPR and one or more guide RNAs (gRNAs) corresponding to B2M or CIITA into the cells. In specific embodiments, CRISPR or one or more gRNAs are transfected into the cells by electroporation or lipid-mediated transfection. Thus, the method may involve introducing CRISPR and one or more gRNAs into the cells by transfecting the cells with a nucleic acid encoding CRISPR and one or more gRNAs. In some embodiments, different gene editing techniques may be employed. Similarly, in some embodiments, one or more nucleic acids encoding a TCR receptor are introduced into the cells. This can be accomplished by transfecting or infecting the cells with a recombinant vector, which may or may not be a viral vector as discussed herein. In some embodiments, the exogenous nucleic acid may be incorporated into the genome of the cells.

[0048] In some embodiments, the method includes introducing one or more additional nucleic acids into a cell population, which may or may not have been previously frozen and thawed. Such use provides for the production of a finished product CD16 HiOne of the advantages of Vδ2 T cells. In certain embodiments, one or more additional nucleic acids encode one or more therapeutic gene products. Examples of therapeutic gene products include at least the following: 1. Antigen recognition molecules such as CAR (chimeric antigen receptor) and / or TCR (T cell receptor); 2. Costimulatory molecules such as CD28, 4-1BB, 4-1BBL, CD40, CD40L, ICOS; and / or 3. Cytokines such as IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-9, IL-15, IL-12, IL-17, IL-21, IL-23, IFN-γ, TNF-α, TGF-β, G-CSF, GM-CSF; 4. Transcription factors such as T-bet, GATA-3, RORγt, FOXP3 and Bcl-6. Include therapeutic antibodies, as well as chimeric antigen receptors, single-chain antibodies, monomeric antibodies, humanized antibodies, bispecific antibodies, single-chain FV antibodies or combinations thereof.

[0049] Aspects of the present disclosure relate to human cells comprising: i) an exogenous expression or activity inhibitor of one or more of β2-microglobulin (B2M), CIITA, TRAC, TRBC1, or TRBC2; or ii) a genomic mutation. In some embodiments, the cell comprises a genomic mutation. In some embodiments, the genomic mutation comprises a mutation of one or more endogenous genes in the cell genome, wherein the one or more endogenous genes comprise the B2M, CIITA, TRAC, TRBC1, or TRBC2 gene. In some embodiments, the mutation comprises a loss-of-function mutation. In some embodiments, the inhibitor is an expression inhibitor. In some embodiments, the inhibitor comprises an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid comprises one or more of siRNA, shRNA, miRNA, or an antisense molecule. In some embodiments, the cell comprises an activity inhibitor. In some embodiments, after modification, the cell lacks any detectable expression of one or more of the B2M, CIITA, TRAC, TRBC1, or TRBC2 proteins. In some embodiments, the cell comprises a B2M inhibitor or a B2M genomic mutation. In some embodiments, the cell comprises a CIITA inhibitor or a CIITA genomic mutation. In some embodiments, the cell comprises a TRAC inhibitor or a TRAC genomic mutation. In some embodiments, the cell comprises a TRBC1 inhibitor or a TRBC1 genomic mutation. In some embodiments, the cell comprises a TRBC2 inhibitor or a TRBC2 genomic mutation. In some embodiments, at least 90% of the genomic DNA encoding B2M, CIITA, TRAC, TRBC1, and / or TRBC2 is deleted. In some embodiments, at least or at most 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% (or any range derivable therein) of the genomic DNA encoding B2M, CIITA, TRAC, TRBC1, and / or TRBC2 is deleted. In other embodiments, deletions, insertions, and / or substitutions are made in the genomic DNA. In some embodiments, the cell is a descendant of a human stem cell or progenitor cell.

[0050] In which CD16 HiVδ2 T cells contain one or more suicide genes for subsequent ablation when needed, and the suicide gene can be of any suitable type. For example, the γδ T cells of the present disclosure can express a suicide gene product that may be enzyme-based. Examples of suicide gene products include herpes simplex virus thymidine kinase (HSV-TK), purine nucleoside phosphorylase (PNP), cytosine deaminase (CD), carboxypeptidase G2, cytochrome P450, linamarase, β-lactamase, nitroreductase (NTR), carboxypeptidase A, or inducible caspase 9. Thus, in certain cases, the suicide gene can encode thymidine kinase (TK). In certain cases, the TK gene is a viral TK gene, such as the herpes simplex virus TK gene. In certain embodiments, the suicide gene product is activated by a substrate such as ganciclovir, penciclovir, or a derivative thereof.

[0051] In some embodiments, CD16 Hi Vδ2 T cells can be imaged or otherwise detected. In certain cases, the cells contain exogenous nucleic acid encoding a polypeptide having a substrate that can be labeled for imaging, and the imaging can be fluorescent, radioactive, colorimetric, and the like. In certain cases, the cells are detected by positron emission tomography. In at least some cases, the cells express the sr39TK gene, which is a positron emission tomography (PET) reporter gene / thymidine kinase gene that allows tracking of these genetically modified cells by PET imaging and elimination of these cells by the sr39TK suicide gene function.

[0052] The present disclosure encompasses CD16 Hi Vδ2 T cell populations. In certain aspects, CD16 + Vδ2 T clonal cells contain exogenous nucleic acid, such as exogenous nucleic acid encoding a γδ T cell receptor, and lack surface expression of one or more HLA-I or HLA-II molecules. The γδ T cells can contain exogenous nucleic acid encoding a suicide gene, which includes an enzyme-based suicide gene such as thymidine kinase (TK). The TK gene can be a viral TK gene, such as the herpes simplex virus TK gene. In the cells of the population, the suicide gene can be activated by a substrate, for example, by a substrate such as ganciclovir, penciclovir, or a derivative thereof. The cells can contain exogenous nucleic acid encoding a polypeptide having a substrate that can be labeled for imaging, and in some cases, the suicide gene product is the polypeptide having a substrate that can be labeled for imaging. In a specific aspect, the suicide gene is sr39TK. In certain cases of the γδ T cell population, the γδ T cells contain nucleic acid sequences from a recombinant vector introduced into the cells, such as a viral vector (including at least lentivirus, retrovirus, adeno-associated virus (AAV), herpesvirus, or adenovirus).

[0053] In certain embodiments, CD16Hi Cells of the Vδ2 T cell population may have been exposed to, or may not have been exposed to, or have been exposed to one or more specific conditions. In some cases, for example, the cells of the population are not exposed to or have not been exposed to a medium containing animal serum. The cells of the population can be frozen or not frozen. In some cases, the cells of the population are in a solution containing dextran, one or more electrolytes, albumin, dextran and / or DMSO. The solution can contain dextran, one or more electrolytes, albumin, dextran and DMSO. The cells can be in a sterile, non-purulent and isotonic solution. In specific cases, γδ T cells have been activated, such as with ZOL. In a specific aspect, the cell population contains at least about 10 2 -10 6 clonal cells. In some cases, the cell population can contain at least about 10 6 -10 12 total cells.

[0054] In a specific embodiment, there is a CD16 Hi Vδ2 T cell population: clonal CD16 Hi Vδ2 T cells that contain one or more exogenous nucleic acids encoding a γδ T cell receptor and thymidine kinase suicide, wherein the clonal γδ T cells have been engineered to not express functional β-2-microglobulin (B2M), class II major histocompatibility complex transactivator (CIITA), and / or HLA-I and HLA-II molecules, and wherein the cell population is at least about 10 6 -10 12 total cells and contains at least about 10 2 -10 6 clonal cells. In some cases, the cells are frozen in the solution.

[0055] As described above, genetic modifications can also be introduced into certain components to generate antigen-specific T cells and mimic positive and negative selection. Examples of such modifications include transducing HSCs with a lentiviral vector encoding an antigen-specific T cell receptor (TCR) or chimeric antigen receptor (CAR) to generate antigen-specific, allelically excluded naïve T cells; transducing HSCs with one or more genes to direct lineage commitment to specialized lymphoid cells.

[0056] Embodiment

[0057] Example 1: Use of allogeneic Vδ2 T cells for ovarian cancer therapy by CD16 biomarker selection and CAR / IL-15 engineering

[0058] Recently, the success of chimeric antigen receptor (CAR)-T cell therapy in treating hematological malignancies has highlighted the transformative potential of gene-engineered cell therapies 1-3 A CAR is a fusion protein that links a targeting moiety (usually the single-chain variable fragment of an antibody) to a T cell-stimulating domain, which allows CAR-engineered cells to target and kill cancer cells 4 Conventional αβ T cells are the basis for six FDA-approved CAR-engineered cell products, targeting CD19 for treating B cell malignancies and B cell maturation antigen (BCMA) for treating multiple myeloma. CAR-T therapy has also been actively tested in the solid tumor setting, but although CAR-T cells targeting GD2 and Claudin18.2 have shown encouraging results in small patient cohorts 6,7 , the therapeutic benefits remain limited 5 Solid tumors pose several challenges to CAR-T cells, including infiltration barriers, antigen heterogeneity, and an immunosuppressive tumor microenvironment (TME) 8 In addition to concerns about solid tumor efficacy, CAR-T cells can cause severe adverse events, such as cytokine release syndrome (CRS), and the autologous nature of CAR-T cell therapy limits its accessibility 9 Complex manufacturing and patient-derived starting materials further contribute to exorbitant costs, time constraints, and variability of the final product

[0059] Allogeneic cell therapies hold promise for addressing the limitations of today's CAR-T cell paradigm 10 Extensive research on αβ T cells and their clinical validation in autologous products have motivated active research into the development of allogeneic αβ CAR-T cells. To avoid the risk of graft-versus-host disease (GvHD) due to the recognition of mismatched MHC molecules by endogenous αβ TCRs, gene editing is usually required. CAR-engineered antigen-specific αβ T cells, such as CMV-specific T cells, are another approach to avoid GvHD and generate off-the-shelf αβ CAR-T cells. Other alternative strategies focus on innate and innate-like immune cell populations that are inherently safer for allogeneic transfer, such as macrophages, natural killer (NK) cells, invariant natural killer T (iNKT) cells, and γδ T cells 11,12

[0060] T cells expressing a T cell receptor (TCR) composed of γ and δ chains, i.e., γδ T cells, account for only 1-10% of circulating T cells but have conserved antimicrobial and antitumor functions 13 γδ T cells display characteristics of both the innate and adaptive immune systems, mediate cytotoxicity through both TCR and NK receptor signaling, and express a range of environmentally dependent immunomodulatory cytokines 14,15 ​。Gentles et al. have highlighted the anti-cancer potential of γδ T cells, where tumor-infiltrating γδ T cells are the strongest favorable prognostic factor among all immune cell subsets in several hematological and solid cancers. 16 。As non-peptide-MHC-dependent responders, γδ T cells are not expected to cause GvHD and have been shown to be safe in the clinical allogeneic setting. 17 。

[0061] More than 90% of peripheral γδ T cells have Vγ9Vδ2 TCR, which senses elevated phosphorylated non-peptide metabolites or phosphoantigens (pAg). 18 。Specifically, dysregulation of the mevalonate metabolism leads to intracellular pAg accumulation, which results in conformational changes of the B7-related membrane protein butyrophilin (BTN) 3A1, allowing BTN3A1 to interact with BTN2A1, and this complex is recognized by the Vγ9Vδ2 TCR. 19,20 。Cellular energy dysregulation is an emerging hallmark of cancer. 21 Thus, the pAg / BTN pathway enables Vγ9Vδ2 T cells to TCR-dependently kill a variety of liquid and solid tumors. 22 。Cell stimulation can also be achieved using bisphosphonates, a class of drugs that prevent or slow bone loss. Examples of such drugs include zoledronate (ZOL) and pamidronate (PAM). These small molecule compounds inhibit farnesyl pyrophosphate synthase in the mevalonate metabolic pathway. As a result, pAg accumulates in the treated cells, leading to the activation of the Vγ9Vδ2 TCR.

[0062] Although the development of Vγ9Vδ2 (Vδ2) T cells as cell carriers for off-the-shelf CAR therapies with favorable properties is encouraged, the literature on CAR-Vδ2 T cells is limited, especially compared to the literature on conventional αβ T cells. In addition, the engineering of therapeutic cells with transgenic IL-15, which has been shown to enhance the preclinical efficacy of CAR-engineered NK 23 、iNK 24 、NKT 25 and Vδ1T 26 cells, has not been reported for CAR-Vδ2 T cells.

[0063] CD16 (FcγRIII), as an IgG receptor that mediates antibody-dependent cell-mediated cytotoxicity (ADCC), is well established. Previous studies have explored the presence and function of CD16 on Vδ2 T cells. 27-30 。In this work, we report a simple method to generate Vδ2 T cells with enhanced anti-tumor activity by screening donors with CD16 expression on Vδ2 T cells. Derived from CD16 HiVδ2 T cells from donors exhibited phenotypic characteristics favorable for cancer treatment, including increased expression of effector molecules and ADCC activity. In addition, we engineered mesothelin-targeting CAR and IL-15 to further enhance CD16 Hi antitumor potential of Vδ2 T cells. Our results highlighted the feasibility, therapeutic potential, and high safety profile of engineered CD16 Hi Vδ2 T cells in the context of cancer treatment.

[0064] Results

[0065] CD16 served as a biomarker for screening PBMC donors for high-performance Vδ2 T cells. Expansion of Vγ9Vδ2 (Vδ2) T cells from a large cohort of peripheral blood mononuclear cell (PBMC) donors for the development of γδT cell-based cancer therapies revealed significant differences in CD16 expression ( Figure 1 a-d; Figure 8 a, b). Initial CD16 expression (before stimulation) on PBMC-derived Vδ2 T cells ranged from almost absent CD16 expression to over 35%. CD16 high (CD16 Hi ) Vδ2 T cells and CD16 low (CD16 Lo ) Vδ2 T cells were defined as ≥35% CD16 expression and ≤20% CD16 expression, respectively. A total of 30 healthy donors were screened in this experiment, and 7 (23.3%) were classified as CD16 Hi donors ( Figure 1 b). In particular, CD16 Hi expression on Vδ2 T cells not only persisted but also increased after activating Vδ2 T cells with ZOL and expanding for 14 days ( Figure 1 c, d). Using the ZOL and IL-2 expansion method, we achieved comparable Vδ2 T cell expansion regardless of CD16 expression ( Figure 1 e).

[0066] Previous studies have shown that Vδ2 T cells exert potent cytotoxicity against various types of tumors. We compared the killing of human ovarian tumor cells by Vδ2 T cells expanded from CD16 Hi and CD16 Lo donors. The high-grade serous ovarian cancer cell lines OVCAR3 and SKOV3 were engineered with a dual reporter gene of firefly luciferase and green fluorescent protein (FG) and co-cultured with various ratios of effector cells (effector to tumor, E:T ratio) in the presence or absence of ZOL ( Figure 1 f, g). Twenty-four hours after co-culture, tumor cell killing was measured by bioluminescence; three CD16 Hi and three CD16Lo Vδ2 T cell donors. In the presence of ZOL, for both cancer cell lines, CD16 Hi Vδ2 T cells showed significantly enhanced cytotoxicity at almost all tested E:T ratios. After co-culturing cancer cells and effector cells at an E:T ratio of 1:1 for 24 hours (h) in the presence or absence of ZOL, from CD16 Hi The improved cytotoxicity of Vδ2 T cells expanded from donors was associated with increased IFN-γ secretion (as measured by ELISA) and production of perforin and granzyme B (as measured by intracellular staining) ( Figure 1 h). Depending on the cancer cell type used and the assay, Vδ2 T cells were able to perform tumor killing and cytokine, perforin, and granzyme B production in the absence of ZOL; in some cases, ZOL or other preconditioning was used to exert effective cancer killing 31,32 . However, for the ovarian cancer cells we tested, Vδ2 T cells showed a dependence on ZOL with respect to cytotoxicity and effector molecule production during in vitro co-culture ( Figure 1 h). Despite the observed differences in cytotoxic potential, the expression of chemokine receptors CXCR3, CCR4, and CCR5 was comparable within and between donors, while CD56 was upregulated on CD16 + cells within donors, and was expressed at a higher overall level on Vδ2 T cells from CD16 Hi donors. CCR2 was upregulated on CD16 - cells within donors, and was expressed at an overall higher level on Vδ2 T cells from CD16 Lo donors ( Figure 8 c, d). Importantly, the expression of granzyme B and perforin was similar between CD16 + and CD16 - Vδ2 T cells within donors, and both types of cells showed higher expression levels in Vδ2 T cells expanded from CD16 Hi donors than in Vδ2 T cells expanded from CD16 Lo donors ( Figure 8 e-h). These results suggest that Vδ2 T cells from CD16 Hi donors may be a favorable cell type for the development of Vδ2 T cell-based cancer therapies.

[0067] Then, we created a lentiviral vector encoding CD16a 33 (Lenti / CD16) for engineering CD16 Lo Vδ2 T cells to express the transgenic CD16a (Figure 1 i-j). Importantly, titration of CD16 expression did not affect cytotoxicity during in vitro co-culture with OVCAR3-FG cells ( Figure 1 k). This provides evidence that CD16 can be used as a biomarker to select donors with highly potent Vδ2 T cells, rather than functioning as an activating receptor to enhance tumor killing, and that genetic introduction of CD16 into Lo Vδ2 T cells may not recapitulate the enhanced activity of Vδ2 T cells expanded from CD16 Hi donors.

[0068] CD16 Hi Vδ2 T cells display an enhanced cytotoxic gene signature. To further evaluate the differences between Vδ2 T cells expanded from different donors, we performed bulk RNA-Seq on Vδ2 T cells expanded from 13 PBMC donors, three of which were classified as CD16 Hi . CD16 mRNA expression was evaluated ( Figure 2 a), and samples were grouped into CD16 Hi and CD16 Lo Vδ2 T cells using the k-means clustering algorithm (k = 2). Three Vδ2 T cell samples were identified as CD16 Hi , and the rest as CD16 Lo , confirming our flow cytometry results. Principal component analysis (PCA) showed clustering of CD16 Hi T cells, potentially indicating transcriptomic differences related to CD16 expression among Vδ2 T cells ( Figure 2 b). Differential expression analysis based on CD16 expression revealed upregulation of genes related to effector functions such as GNLY, CD86, and CX3CR1 in the high CD16-expressing subset ( Figure 2 c). Various genes related to anti-tumor effector functions were also evaluated, such as genes encoding transcription factors, activation / homing markers, and cytotoxic molecules ( Figure 9 a). CD16 expression was positively correlated with the expression of granzyme (GZMA, GZMB), perforin (PRF1), and natural killer receptor (NCR1). Intriguingly, the CD16 Hi group showed downregulation of the transcription factor-encoding gene RORC, which is related to the Th17-like phenotype. Th17-like Vδ2 T cells have been shown to promote cancer progression in several syngeneic cancer models, and there is evidence that they have a detrimental role in human malignancies 34-36 .

[0069] In addition, gene set enrichment analysis (GSEA) was performed 37To characterize the biological pathway features associated with CD16 expression ( Figure 2 d and Figure 9 b). High CD16 expression enriches features related to immune effector functions and activation, such as cytotoxicity, degranulation, Fcγ receptor signaling, and phagocytosis. Proliferation-related genes are also enriched in CD16 Hi samples ( Figure 2 d), although no differences in expansion were observed during in vitro generation of Vδ2T cells ( Figure 1 e). A gene ontology (GO) Cnet plot showing the connection between differentially expressed genes and biological processes 38 confirmed an increase in immune cell activation features in CD16 Hi Vδ2T cells ( Figure 2 e).

[0070] We also evaluated the relationship between CD16 expression in Vδ2T cell samples and the gene signatures of 24 immune cell types defined in ImmuCellAI 39 ( Figure 2 f). The enrichment score for each immune cell type in each sample was calculated using single-sample GSEA (ssGSEA) implemented in the gene set variation analysis (GSVA) software package 40 . The resulting correlation between the enrichment score and the CD16 expression level of the sample showed that CD16 expression enriches cytotoxicity, NK, and macrophage signatures and the absence of Tfh and Th17 signatures. CD16 expression also corresponds to the Th1 signature, which is supported by increased IFN-γ secretion in CD16 Hi Vδ2T cells. In summary, the bulk RNA-Seq results are consistent with the potent in vitro activity of CD16 Hi Vδ2T cells and provide future directions for cell product characterization, particularly regarding IL-17 production and expansion potential.

[0071] Development of CAR targeting mesothelin and IL-15-engineered CD16 Hi Vδ2T cells for the treatment of ovarian cancer. Mesothelin (MSLN) is a cell surface glycoprotein that is expressed at low levels in normal tissues but overexpressed in many solid cancers, including ovarian, lung, and pancreatic cancers, making MSLN a promising target for cancer therapies, including CAR-engineered cell therapies 41,42Due to the heterogeneous expression of MSLN in ovarian cancer, we hypothesized that MSLN-targeted CAR (MCAR)-engineered Vδ2 T cells might exhibit superior anti-tumor activity due to their potential multi-targeting ability. We also implemented cell engineering to produce IL-15 (termed MCAR15), as IL-15 signaling has been shown to enhance the persistence of innate / innate-like immune cells and, to our knowledge, has not been explored to regulate CAR Vδ2 T cells.

[0072] According to Figure 3 the schematic in Hi a, we produced MCAR- and MCAR15-engineered CD16 Hi Vδ2 T cells (MCAR-Vδ2 T and MCAR15-Vδ2 T), and included non-engineered CD16 Figure 3 Vδ2 T cells as a control (NT-Vδ2 T). The MCAR and MCAR15 constructs resulted in similar CAR expression, Vδ2 T cell expansion, and Vδ2 T cell purity, and routinely achieved a purity greater than 98% ( Figure 3 b-g). MCAR15-Vδ2 T cells expressed higher levels of the persistence-related proteins pSTAT5, BcL-xL, and BcL-2, as measured by intracellular staining and flow cytometry ( Figure 3 h). Significant IL-15 secretion was observed in the activated MCAR15 group after co-culture with OVCAR3-FG cells ( Figure 3 i), which is consistent with previously published data demonstrating increased IL-15 production by CAR / IL-15-engineered αβT 43 Vδ1 T 26 and NK cells 23 upon antigen stimulation. This may be due to increased metabolic activity and protein translation occurring during cell activation and the short half-life of IL-15. Although there are indeed various definitions of the memory state of Vδ2 T cells in the literature 44,45 , our analysis based on CD27 and CD45RA expression showed that the CAR15 + and CAR15 - populations from the MCAR15-Vδ2 T cell group were mostly of the central memory (~40%) and effector memory (~50%) phenotypes ( Figure 3 J-1).

[0073] MCAR15-Vδ2 T cells exhibited robust in vitro anti-tumor activity against multiple ovarian cancer cell lines. We performed in vitro cytotoxicity and cytokine production assays to evaluate the effector functions of CAR-engineered CD16 Hi Vδ2 T cells ( Figure 4)。A third ovarian cancer cell line, OVCAR8-FG, expressing the FG dual reporter gene was also included in several assays. MSLN expression on OVCAR3-FG, OVCAR8-FG, and SKOV3-FG was evaluated ( Figure 4 b), revealing variable MSLN expression in the three ovarian cancer models. Conventional αβCAR T cells (MCAR-T) targeting MSLN were included as controls, and Vδ2T cell groups were added in the presence and absence of ZOL, unless otherwise stated. After 24-hour co-culture, all effector cell groups exhibited efficient killing of OVCAR3-FG cancer cells ( Figure 4 c). However, for OVCAR8-FG cells, the Vδ2T cell group showed enhanced cytotoxicity compared to MCAR-T cells, and only in the presence of ZOL could Vδ2T cells kill CAR antigen-negative SKOV3-FG cells ( Figure 4 c). No differences were observed between the two engineered Vδ2T cell groups (with and without IL-15). In parallel, we evaluated the intracellular expression of granzyme B and perforin in MCAR15-Vδ2T cells co-cultured with OVCAR3-FG and SKOV3-FG cells in the presence or absence of ZOL ( Figure 4 d). We observed upregulation of effector molecule production in the presence of OVCAR3-FG cells with or without ZOL, while ZOL was required to increase effector molecule production in co-cultures with SKOV3-FG cells. We also compared the in vitro cytotoxicity of CAR- and IL-15-engineered CD16 Hi and CD16 Lo Vδ2T cells and witnessed a significant improvement in killing in the CD16 Hi group ( Figure 10 a, b).

[0074] The 24-hour co-cultures were also used to monitor IFN-γ production ( Figure 4 e). When co-cultured with OVCAR3-FG, CAR-engineered T and Vδ2T cells secreted sufficient IFN-γ, whereas unengineered control Vδ2T cells did not, highlighting the benefit of CAR engineering. To confirm the findings in terms of cytotoxicity, CAR-Vδ2T cells produced IFN-γ in response to SKOV3-FG only in the presence of ZOL. The cytotoxicity and IFN-γ results illustrate the tumor cell multi-targeting potential of CAR-Vδ2T cells through recognition by both CAR and TCR.

[0075] We performed in vitro repeated tumor attack assays to study the long-term function of CAR15- and CAR-Vδ2T cells ( Figure 4f, g). On days 0, 3, 6, 9, 12, and 15, effector cells were challenged with fresh tumor cells in 96-well plates with and without ZOL. Twenty-four hours after cancer cell addition, one of the 96-well plates was used for bioluminescence measurement to determine cancer killing. In repeated tumor challenges against OVCAR3-FG and OVCAR8-FG cells, IL-15 secretion significantly improved the cancer killing ability of the MCAR15 Vδ2T cell group.

[0076] To further illustrate CAR antigen-dependent and independent anti-tumor activities, we generated MSLN-negative OVCAR3-FG ( KO OVCAR3-FG) cells ( Figure 4 h) using CRISPR-Cas9 editing. As shown in the schematic diagram in Figure 4 f, in the repeated tumor challenge assay, KO OVCAR3-FG cells were co-cultured with CAR-engineered effector cells. After repeated tumor challenges, MSLN knockout led to reduced killing of CAR-Vδ2T cells in the absence of ZOL compared to using ZOL ( Figure 4 i), while no cytotoxicity difference was observed against the parental OVCAR3-FG cell line after 24-hour co-culture ( Figure 4 c). This indicates that in the absence of ZOL, killing of the parental OVCAR3-FG is driven by CAR-mediated killing. By day 7, after the 3rd tumor challenge, an KO OVCAR3-FG cell outgrowth occurred in all effector cell groups except for the MCAR- and MCAR15-Vδ2T cell cultures supplemented with ZOL. The MSLN knockout study demonstrated that CAR-antigen presentation can enhance the killing function of CAR-Vδ2T cells, and CAR-antigen escape may potentially be overcome by the Vδ2TCR-mediated killing mechanism.

[0077] Engineered CD16 Hi Vδ2T cells can target ovarian cancer cells via ADCC. For several anti-cancer monoclonal antibodies (mAbs) such as cetuximab and trastuzumab, Fc-mediated immune effector function ADCC is the main mode of action for eliminating tumor cells 46,47 . T- and NK cell-mediated ADCC is mainly attributed to the CD16a (FcγRIIIa) transmembrane receptor, which is expressed by many effector cells of the immune system, while CD16b (FcγRIIIb), a GPI-anchored protein, is only expressed by neutrophils 48Although CD32 (FcγRI) and CD64 (FcγRII) are expressed at low levels on Vδ2 T cells and may contribute to ADCC, they are mainly involved in myeloid-mediated ADCC. 49,50 Therefore, we focused our Vδ2 T cell studies on the canonical lymphocyte CD16a receptor, which we refer to as CD16.

[0078] To test the ADCC capabilities of unmodified and engineered CD16 Hi Vδ2 T cells, we performed in vitro tumor co-culture assays with a preclinical anti-HER2 mAb analogue of trastuzumab ( Figure 5 a). HER2 expression on OVCAR3-FG, OVCAR8-FG, and SKOV3-FG was evaluated by flow cytometry ( Figure 5 b). For unmodified CD16 Hi Vδ2 T cells, the isotype control had a minimal cytotoxic effect on Vδ2 T cells against OVCAR3-FG cells, while significant enhancement of tumor killing was observed at anti-HER2 mAb concentrations as low as 0.1 μg / mL ( Figure 5 c). The addition of anti-HER2 mAb also improved the killing efficacy of MCAR-Vδ2 T cells against OVCAR3-FG, OVCAR8-FG, and SKOV3-FG cells in the presence and absence of IL-15 secretion, while no benefit was observed for conventional MCAR-T cells ( Figure 5 d-f). We confirmed that CD16 Lo Vδ2 T cells lacked detectable ADCC function ( Figure 10 c, d). Importantly, ADCC enabled efficient killing of MSLN-negative SKOV3-FG cells by CD16 Hi MCAR-Vδ2 T cells ( Figure 5 f), and this was accompanied by significant IFN-γ secretion ( Figure 5 g). Finally, repeated tumor challenge assays were performed against KO OVCAR3-FG cells in the presence or absence of anti-HER2 mAb ( Figure 5 h). Antibody treatment greatly enhanced tumor killing by MCAR- and MCAR15-Vδ2 T cells, although only the combination of MCAR15-Vδ2 T cells and antibody established extended tumor control ( Figure 5 i).

[0079] MCAR15-Vδ2 T cells can kill tumor-associated macrophages in vitro. Macrophages Macrophages are large innate immune cells that phagocytose target cells in response to infection or injury, and recent work has highlighted the role of macrophages in both tumor elimination and progression. 51-54 Although existing on a continuum, the macrophage population that prevents cancer growth and activates anti-tumor immunity is commonly referred to as M1 type, while the macrophage population that promotes cancer growth and enhances immunosuppression is called M2 type. Most cancers are predominantly occupied by M2 macrophages, which represent a logical target for immunotherapy. We generated human monocyte-derived M2 macrophages ( by culturing PBMCs in the presence of macrophage colony-stimulating factor (M-CSF) to generate monocyte-derived macrophages (MDMs), and then culturing with IL-4 and IL-13 to generate M2 polarization Figure 11 a) 55 . FACS detection of CD11b and CD14 revealed successful generation of macrophages, and further characterization of the M2 markers CD163 and CD206 confirmed M2 polarization Figure 11 b, c). MCAR15-Vδ2T cells were cultured with M2 in vitro, and the resulting cytotoxicity was monitored by flow cytometry Figure 11 d). After 24-hour culture, 50% of the cells were killed at a ratio of 1:1 of MCAR15-γδT:M2 , and increased to 70% after addition of ZOL Figure 11 e). These results suggest that killing of tumor-associated macrophages (TAMs) may be another mechanism that MCAR15-Vδ2T cells can potentially utilize to mediate anti-tumor immunoreactivity.

[0080] MCAR15-Vδ2T cells are safe and effective in intraperitoneal and subcutaneous in vivo ovarian cancer models. The in vivo anti-tumor activity and safety of MCAR15-Vδ2T cells derived from CD16 Hi donors were evaluated in two xenograft tumor models. In the first model, NSG mice were inoculated intraperitoneally (i.p.) with 1×10 6 OVCAR3-FG cells and treated 14 days later with 4x10 6 MCAR-T, MCAR-Vδ2T or MCAR15-Vδ2T cells or vehicle (PBS) delivered i.p. Figure 6 a). Tumor growth was monitored by in vivo imaging of live animals using bioluminescence. Within one week of treatment, all effector cell groups showed significant tumor delay and effective tumor control compared to the control group Figure 6b - d). Due to the cancer burden, control mice reached the endpoint at approximately day 70 after tumor injection, and MCAR - T mice died shortly thereafter due to GvHD (as determined by weight loss, fur loss, and malaise). Figure 6 e). For mice treated with MCAR - Vδ2T cells, 2 / 5 of the mice survived until the end of the study, i.e., day 180, with one mouse having complete tumor elimination and one mouse having tumor recurrence, while the other three mice died of the recurrent tumor. MCAR15 - Vδ2T cells led to complete remission in all 5 / 5 mice until day 180 without any signs of GvHD, highlighting the benefit of IL - 15 engineering in long - term intraperitoneal tumor growth inhibition. Figure 6 e).

[0081] Then, we tested the therapeutic potential of MCAR15 - Vδ2T cells in a subcutaneous tumor model. OVCAR8 subcutaneous tumors were established in NSG mice, and when the tumors reached an average of 50 mm 3 in size, 10×10 6 effector cells were administered intravenously. Figure 7 a). All effector (cell) groups led to tumor growth inhibition, with MCAR15 - Vδ2T cells maintaining significantly enhanced tumor control compared to all other groups until day 57. Figure 7 b, c). On day 57, mice were sacrificed for terminal analysis. Tumors were resected and processed to evaluate human immune cell infiltration. Mouse tissues were also harvested to evaluate effector cell persistence and xenoreactivity (GvHD) throughout the pre - clinical model. MCAR15 - Vδ2T cells showed robust persistence, as demonstrated by their increased presence in tumors and all analyzed mouse organs. Figure 7 d). Excellent persistence was not associated with GvHD, as H&E - stained tissue sections collected from experimental mice on day 57 showed that MCAR15 - Vδ2T cells did not cause the accumulation of monocyte infiltration in the lung, liver, spleen, or kidney, while mice treated with MCAR - T cells showed a distinct manifestation of monocyte aggregates. Figure 7 e). Additional OVCAR8 subcutaneous studies showed that CD16 Lo MCAR15 - Vδ2T cells exhibited excellent in - vivo tumor control compared to CD16 Hi MCAR15 - Vδ2T cells. Figure 11 ). These in - vivo results highlight the potential of CD16 Hi MCAR15 - Vδ2T cells to treat intraperitoneal solid tumors without causing GvHD. The results also confirmed the benefit of engineering CAR - Vδ2T cells to secrete IL - 15.

[0082] Methods

[0083] Mouse. NOD.Cg-Prkdc SCID Il2rg tm1Wjl / SzJ (NOD / SCID / IL-2Rγ - / - , NSG) mice were maintained in the animal facilities at the University of California, Los Angeles (UCLA) under the following housing conditions: temperature range of 68°F to 79°F, humidity maintained at 30% to 70%, light cycle turned on at 6:00 am and turned off at 6:00 pm, and the room pressure set to negative pressure. Unless otherwise stated, all experiments were performed using 6- to 10-week-old female mice. Due to ethical reasons, we terminated the experiments before the mice developed severe ascites or the tumor volume exceeded 1000 mm 3 . All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at UCLA. All mice were housed and maintained under specific pathogen-free conditions, and all experiments were conducted in accordance with the animal care and use regulations of the Division of Laboratory Animal Medicine (DLAM) at UCLA.

[0084] Culture media, cytokines, and chemicals. Zoledronic acid monohydrate (ZOL) was purchased from Sigma (Catalog No. SML0223). Recombinant human IL-2, IL-4, IL-7, IL-15, IL-17, interferon γ (IFN-γ), and tumor necrosis factor α (TNF-α) were purchased from PeproTech. RPMI 1640 and DMEM cell culture media were purchased from Corning Cellgro. Fetal bovine serum (FBS) was purchased from Sigma. Culture medium supplements, including penicillin / streptomycin / glutamine (P / S / G), MEM non-essential amino acids (NEAA), HEPES buffer, and sodium pyruvate, were purchased from Gibco. β-mercaptoethanol (β-ME) was purchased from Sigma. Normocin was purchased from InvivoGen. CryoStor CS10 cell cryopreservation medium was purchased from Sigma (Catalog No. C2874). Complete lymphocyte medium (designated as C10 medium) was made from RPMI 1640 supplemented with FBS (10% vol / vol), P / S / G (1% vol / vol), MEM NEAA (1% vol / vol), HEPES (10 mM), sodium pyruvate (1 mM), β-ME (50 mM), and Normocin (100 mg / mL). The medium used to culture OVCAR3 and OVCAR8 tumor cell lines (designated as R10 medium) was made from RPMI 1640 supplemented with FBS (10% vol / vol) and P / S / G (1% vol / vol). The medium used to culture HEK-293T / 17 and SKOV3 tumor cell lines (designated as D10 medium) was made from DMEM supplemented with FBS (10% vol / vol) and P / S / G (1% vol / vol). The cryopreservation medium used for cryopreserving cell lines and PBMC-derived cells was made from CryoStor CS10 cell cryopreservation medium and complete basal medium at a ratio of 1:1.

[0085] Cell lines. Human embryonic kidney 293T / 17 (HEK-293T / 17, ATCC; Catalog No. CRL-11268) and human ovarian cancer cell lines OVCAR3 (ATCC; Catalog No. HTB-161), OVCAR8 (NIH; Catalog No. CVCL_1629), and SKOV3 (ATCC; Catalog No. HTB-77) were purchased from the American Type Culture Collection (ATCC) or obtained from the National Institutes of Health (NIH) via MTA. The HEK-293T / 17 and SKOV3 cell lines were maintained in D10 medium. The OVCAR3 and OVCAR8 cell lines were maintained in R10 medium.

[0086] To generate a stable tumor cell line overexpressing the dual reporter genes of firefly luciferase and enhanced green fluorescent protein (FG), the parental tumor cell line was transduced with the Lenti / FG vector encoding the FG dual reporter genes 92 . Seventy-two hours after lentiviral vector transduction, the cells were sorted by flow cytometry to isolate the genetically engineered cells for the preparation of stable cell lines. Three FG-labeled stable tumor cell lines were generated in this study, including OVCAR3-FG, OVCAR8-FG, and SKOV3-FG

[0087] This study generated an additional cell line overexpressing the FG dual reporter genes and knocking out the mesothelin (MSLN) tumor antigen KO OVCAR3-FG. Briefly, OVCAR3-FG cells were electroporated with the CRISPR-Cas9 / MSLN-sgRNA complex, which consisted of pre-mixed Cas9-NLS protein (6.5 μg / μL, 4 μL; UC Berkeley) and MSLN-sgRNA (100 μM, 1 μL). For electroporation, the tumor cells were pulsed twice at 1170 V for 30 ms in a Neon transfection system (Thermo Fisher Scientific; catalog number MPK5000) according to the manufacturer's protocol. Seventy-two hours after electroporation, the engineered OVCAR3-FG cells were sorted by flow cytometry to isolate the MSLN-KO OVCAR3-FG( KO OVCAR3-FG) cell line

[0088] Lentiviral vector construction. The lentiviral vectors used in this study were all constructed from the parental lentiviral vector pMNDW, which contained the MND retroviral LTR U2 region as an internal promoter and an additional truncated woodchuck response element (WPRE) to stabilize viral mRNA 92 . The 2A sequences derived from porcine teschovirus-1 (P2A) and Thosea asigna virus 2A (T2A) were used to link the inserted genes for co-expression

[0089] The Lenti / FG, Lenti / MCAR, Lenti / MCAR15, and Lenti / CD16 vectors were constructed by inserting the corresponding synthetic genes into the pMNDW parental vector: a bicistronic gene encoding the FG dual reporter genes, a gene encoding the MSLN-targeted CAR (MCAR), a bicistronic gene encoding the same MCAR and the secreted form of human IL-15 (MCAR15), and a gene encoding CD16a (CD16). MCAR consists of the SS1 scFv, CD8α hinge, CD28 transmembrane domain, CD28 signaling domain, and CD3ζ signaling domain. 93 The synthetic gene fragments were obtained from GenScript and IDT.

[0090] Lentiviruses were produced using HEK-293T / 17 cells according to a standard transfection protocol. Briefly, HEK-293T / 17 cells were co-transfected with three plasmids for 16 to 18 hours using the TransIT-Lenti transfection reagent (MirusBio; catalog number MIR6600): the lentiviral vector plasmid, the lentiviral glycoprotein plasmid (pCMV-VSVG), and the lentiviral packaging plasmid (pCMV-δR8.9). Then, the cells were treated with 10 mM sodium butyrate for 8 hours. Subsequently, virus-containing supernatant was produced in serum-free UltraCULTURE medium (Lonza Walkersville; catalog number BP12725F) for 48 hours. The supernatant was concentrated at 4000 rcf for 40 minutes at 4 °C using a 100 KDa Amicon Ultra-15 centrifugal filter unit (Millipore Sigma; catalog number UFC910024) and stored as aliquots at -80 °C. The lentiviral vector titer was measured by transducing HEK-293T / 17 cells with serial dilutions and performing flow cytometry according to an established protocol.

[0091] Antibodies and flow cytometry. Fluorochrome-conjugated antibodies specific for the following: human APC / Cy7-CD45 (catalog number 304014, clone H130, 1:100 dilution), PE / Cy7-TCRαβ (catalog number 306720, clone IP26, 1:25 dilution), FITC-CD3 (catalog number 317306, clone OKT3, 1:200 dilution), PB-CD3 (catalog number 317314, clone OKT3, 1:100 dilution), FITC-CD27 (catalog number 356403, clone M-T271, 1:100 dilution), APC / Cy7-CD45RA (clone HI100, 1:200 dilution), FITC-CD56 (catalog number 318304, clone HCD56, 1:10 dilution), APC-TCR Vγ9 (catalog number 331309, clone B3, 1:100 dilution), PerCP-TCR Vδ2 (catalog number 331410, clone B6, 1:100 dilution), APC-CD16 (clone 3G8, 1:250 dilution), PE-CCR2 (catalog number 357206, clone K036C2, 1:400 dilution), PE / Cy7-CCR4 (catalog number 359410, clone L291H4, 1:500 dilution), FITC-CCR5 (catalog number 359120, clone J418F1, 1:200 dilution), PE / Cy7-CXCR3 (catalog number 353720, clone G025H7, 1:100 dilution), APC / Cy7-IFN-γ (catalog number 502529, clone B27, 1:100 dilution), FITC-granzyme B (catalog number 372205, clone QA16A02, 1:1000 dilution), PE / Cy7-perforin (catalog number 308125, clone dG9, 1:50 dilution), PE-pSTAT5 (Tyr694, catalog number 936903, clone A17016B, 1:50 dilution), PE / Cy7-Bcl-2 (catalog number 633511, clone BCL / 10C4, 1:100 dilution), APC-HER2 (catalog number 324407, clone 24D2, 1:400 dilution), PB-CD14 (catalog number 301815, clone 63D3, 1:1000 dilution), FITC-CD11b (catalog number 301330, clone ICRF44, 1:10000 dilution), APC / Cy7-CD163 (catalog number 333622, clone GHI / 61, 1:500 dilution), APC-CD206 (catalog number 321110, clone 15-2, 1:500 dilution) and APC-streptavidin (catalog number 405207, 1:1000 dilution) for binding biotinylated human mesothelin for MCAR staining, all purchased from BioLegend.

[0092] Biotinylated human mesothelin (Catalog No. MSN-H82E9, diluted 1:400) was purchased from ACROBiosystems. Fluorescent dye-conjugated antibody specific for human APC-mesothelin (Catalog No. Fab32652A, clone 420411, diluted 1:100) was purchased from R&D Systems. Fluorescent dye-conjugated antibody specific for human FITC-Bcl-xL (Catalog No. MA5-28637, clone 7B2.5, diluted 1:100) was purchased from Thermo Fisher Scientific. Fluorescent dye-conjugated antibody specific for human FITC-TCRγ / δ (Catalog No. 347903, clone 11F2, diluted 1:15) was purchased from BD Biosciences. Fluorescent dye-conjugated antibody specific for human PE-TCRVδ1 (Catalog No. 130-120-580, clone REA173, diluted 1:500) was purchased from Miltenyi Biotec. Fixable viability dye eFluor506 (e506, Catalog No. 65-0866-18) was purchased from Affymetrix eBioscience. Mouse Fc blocker (anti-mouse CD16 / 32, Catalog No. 553142, clone 2.4G2, diluted 1:50) was purchased from BD Biosciences, and human Fc receptor blocker solution (TrueStain FcX, Catalog No. 422302, diluted 1:25) was purchased from BioLegend. In vivo SIM anti-human HER2 (trastuzumab biosimilar, Catalog No. SIM0005) was purchased from BioXCell.

[0093] Flow cytometry surface staining and intracellular staining were performed according to standard protocols and the specific instructions provided by the manufacturer for the specific antibodies. Intracellular staining of IL15-mediated prosurvival signaling pathway molecules (pSTAT5, Bcl-2, and Bcl-xL) was performed according to the Foxp3 / Transcription Factor Staining Protocol (Thermo Fisher Scientific; Catalog No. 50-112-8857). Stained cells were analyzed using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotech). FlowJo software version 10 (BD Biosciences) was used for data analysis.

[0094] Enzyme-linked immunosorbent assay for cytokines (ELISA). ELISA for detecting human IFN-γ was performed according to the standard protocol of BD Biosciences. The supernatant from the cell culture assay was collected and assayed to quantify IFN-γ. The capture (Catalog No. 551221, clone NIB42, 1:250 dilution) and biotinylation (Catalog No. 554550, clone 4S.B3, 1:500 dilution) for detecting IFN-γ were purchased from BD Biosciences. The HRP-avidin conjugate (Catalog No. 405103, 1:1000 dilution) and human IFN-γ ELISA standard (Catalog No. 570209) were purchased from BioLegend. One-step TM TMB ELISA substrate solution was purchased from Thermo Fisher Scientific (Catalog No. 34021). Human IL-15 was quantified using the Human IL-15 Quantikine ELISA kit (R&D Systems; Catalog No. D1500). The absorbance of the samples was analyzed at 450 nm using an Infinite M1000 microplate reader (Tecan).

[0095] Generation of PBMC-derived conventional αβ T cells and derivatives. Healthy donor human PBMCs were obtained from the UCLA / CFAR Virology Core Laboratory, and the identification information was removed according to federal and state regulations (USA). The protocol using these human cells was exempted by the UCLA Institutional Review Board (IRB), IRB#05-10-093, January 21, 2019. To generate PBMC-derived conventional αβ T (designated as PBMC-T) cells, 1×10 6 cells / mL of PBMCs were resuspended in C10 medium supplemented with 100 IU / mL of human IL-2 (T-medium) and stimulated with 50 ng / mL of anti-CD3 monoclonal antibody OKT3 (BioLegend; Catalog No. 317325). Two days after activation, the PBMCs were washed and passaged 3 times a week for 2 weeks to maintain the cell density at 0.5 - 1×10 6 cells / mL; fresh T-medium was added at each passage.

[0096] To generate MCAR-T cells, 1×10 6 cells / mL of PBMCs were stimulated with 50 ng / mL of anti-CD3 monoclonal antibody OKT3 in T-medium. Two days after activation of the PBMC culture, the cells were washed, resuspended in fresh T-medium, and then the concentrated MCAR lentiviral vector was added to the PBMC culture. The next day, the transduced cells were washed and passaged 3 times a week for 2 weeks to maintain the cell density at 0.5 - 1×10 6cells / mL; Fresh T-medium was added at each passage. The resulting MCAR-T cells were collected and cryopreserved for future use.

[0097] Generation of PBMC-derived Vδ2T cells and derivatives. Healthy donor human PBMCs were obtained from the UCLA / CFAR Virology Core Laboratory, and the identification information was removed in accordance with federal and state regulations (USA). The protocol using these human cells was exempted by the UCLA Institutional Review Board (IRB), IRB#05-10-093, January 21, 2019. To generate PBMC-derived Vδ2T (designated as Vδ2T) cells, 2.5×10 6 / mL of PBMCs were resuspended in C10 medium supplemented with 100 IU / mL of human IL-2 (T-medium) and stimulated with 5 μM ZOL. Three days after activation, Vδ2T cells were enriched by TCRγ / δ + T cell isolation kit (Miltenyi Biotech; catalog number 130-092-892) and then resuspended in fresh T medium. PBMCs were washed and passaged 3 times a week for 10 - 14 days to maintain the cell density at 1 - 1.5×10 6 cells / mL; Fresh T-medium was added at each passage.

[0098] To generate MCAR-Vδ2T and MCAR15-Vδ2T cells, 2.5×10 6 / mL of PBMCs were resuspended in T medium and stimulated with 5 μM ZOL. Three days after activation, PBMCs were washed, enriched by TCRγ / δ + T cell isolation kit, resuspended in fresh T medium, and then the concentrated MCAR or MCAR15 lentiviral vector was added to the PBMC culture. The next day, the transduced cells were washed and passaged 3 times a week for 2 weeks to maintain the cell density at 1 - 1.5×10 6 cells / mL; Fresh T-medium was added at each passage. The resulting MCAR-Vδ2T and MCAR15-Vδ2T cells were collected and cryopreserved for future use.

[0099] RNA-Seq analysis of Vδ2T cells. A total of 13 PBMC-derived Vδ2T cell samples were analyzed. Vδ2T cells were classified according to Figure 1a Amplification and purification. Cell samples were sorted using a FACSAria II flow cytometer (BD Biosciences). Total RNA was isolated from each cell sample using the miRNeasy Mini Kit (QIAGEN). cDNA was synthesized using the iScript cDNA Synthesis Kit (BioRad). Libraries were constructed using the Illumina TruSeq Stranded Total RNA Sample Prep Kit and sequenced on an Illumina HiSeq3000 with 50 bp single-end reads (targeting 20x10 6 reads per sample) according to the manufacturer's instructions and the TCGB Core standard protocol. The raw sequencing data was quality-checked using FastQC software (version 0.11.9), and sequencing adapters and low-quality bases were removed using fastp (version 0.23.2). The trimmed reads were mapped to the human reference genome (hg38) using STAR 2.7.9a, and gene count matrices were obtained using featureCounts from the Subread package (version 2.0.3). Batch effect removal was performed using Combat-Seq implemented in the sva package (version 3.44.0). Gene expression counts normalized by sequencing depth (cpm, counts per million) were obtained using edgeR (version 3.38.4). Log-transformations were performed after adding a pseudocount of 1 to the normalized counts for principal component analysis (PCA) and heatmap plotting. The PCA plot used the top 500 most variable genes. Differential expression analysis was performed using DESeq2 (version 1.36.0) based on the expression of CD16. For genes with an exact p-value of 0, the p-value was represented as the minimum p-value divided by 10. To control the false discovery rate (FDR), the Benjamini-Hochberg (BH) procedure was applied to correct the p-values, and genes with corrected p-values below the 0.05 threshold were identified as differentially expressed genes. Based on the differential analysis results of all genes, gene set enrichment analysis was performed using clusterProfiler (version 4.4.4). Gene ontology overrepresentation analysis was performed on the differentially expressed genes, and Cnet plots were used to show the connections between differentially expressed genes and enriched biological processes. Based on the gene signatures of 24 immune cell types from ImmuneCellI, the immune cell type scores for each Vδ2T cell sample were calculated using the ssGSEA method, and correlation plots were drawn using the R package corrplot (version 0.92) between the enrichment scores of the specified immune cell types and the CD16 expression levels of the samples.

[0100] In vitro phenotypic and functional analysis of Vδ2 T cells. The phenotypes of Vδ2 T cells and derivatives were studied using flow cytometry by analyzing the expression of cell surface markers (including MCAR and MCAR15), memory T cell markers (i.e., CD27 and CD45RA), chemokine receptors (i.e., CXCR3, CCR2, CCR4, and CCR5), and NK receptors (i.e., CD56). The ability of these cells to produce cytotoxic molecules (i.e., perforin and granzyme B) was studied using flow cytometry via intracellular staining. Intracellular staining of molecules in the IL15-mediated prosurvival signaling pathway (pSTAT5, Bcl-2, and Bcl-xL) was performed according to the Foxp3 / transcription factor staining protocol. The proliferation of Vδ2 T cells (identified as CD3 + TCR Vδ2 + ) over time was measured by cell counting and flow cytometry.

[0101] In vitro 24-hour tumor cell killing assay. In a Corning 96-well clear-bottom black plate, FG-labeled tumor cells (1×10 4 cells / well) were co-cultured with effector cells (at the specified ratios) in C10 medium with or without ZOL (5 μM) for 24 hours. At the end of the culture, viable tumor cells were quantified by adding D-luciferin (150 μg / mL; Caliper Life Science) to the cell culture and reading the luciferase activity using an Infinite M1000 microplate reader (Tecan).

[0102] In vitro repeated tumor attack assay. Panels Figure 4 f and 5h illustrate the experimental design employed in this study. Briefly, FG-labeled tumor cells (1×10 4 cells / well) were co-cultured with effector cells (at the specified ratios) in six Corning 96-well clear-bottom black plates containing C10 medium with or without ZOL (5 μM) or anti-HER2 Ab (0.1 μg / mL). After 24 hours, viable tumor cells from one plate (the 1st time point) were quantified by adding D-luciferin to the cell culture and reading the luciferase activity using an Infinite M1000 microplate reader. On day 3, the remaining 5 plates were centrifuged at 300 rcf for 5 minutes and the old medium was carefully replaced with fresh C10 medium. Subsequently, the cells from these 5 plates were resuspended and transferred to 5 newly seeded tumor cell plates. After 24 hours, viable tumor cells from one plate were quantified by measuring the luciferase activity (the 2nd time point). This process was repeated for a total of six time points over 16 days.

[0103] In vitro ADCC assay. FG-labeled tumor cells (1×104 Cells (at a density of [number of cells] cells / well) were seeded in a Corning 96-well clear-bottom black plate containing C10 medium and treated with anti-HER2 Ab (BioXCell; InVivoSIM anti-human HER2; catalog number SIM0005) at 37 °C for 30 minutes. Subsequently, effector cells were added to the antibody-treated tumor cells, and they were co-cultured for 24 hours. At the end of the culture, the luciferase activity was quantified by adding D-luciferin to the cell culture and reading the luciferase activity using an Infinite M1000 microplate reader (Tecan) to quantify viable tumor cells.

[0104] In vitro M2 polarized macrophage killing assay. Healthy donor human PBMCs were obtained from the UCLA / CFAR Virology Core Laboratory, and the identifying information was removed in accordance with federal and state regulations (USA). The protocol using these human cells was exempted by the UCLA Institutional Review Board (IRB), IRB#05-10-093, January 21, 2019. The PBMCs were cultured at a cell density of 1×10 7 cells / mL in serum-free RPMI 1640 medium (Corning cellgro, Manassas, VA, USA, #10-040-CV). Subsequently, 10 - 15 mL of the PBMC suspension was seeded into 10 cm culture dishes and incubated in a humidified incubator at 37 °C and 5% CO2 for 1 - 2 h. Then, the medium containing non-adherent cells was discarded, and the culture dishes were washed twice with PBS. The adherent monocytes were cultured in C10 medium and human M-CSF (10 ng / mL; PeproTech; catalog number 300-25) for 6 days to generate monocyte-derived macrophages (MDMs). On day 6, the generated MDMs were dissociated by 0.25% trypsin / EDTA (Gibco; catalog number 25200-056), collected, and re-seeded at a density of 0.5 - 1x10 6 cells / mL into 6-well plates with C10 medium in the presence of recombinant human IL-4 (10 ng / mL; PeproTech; catalog number 214-14) and human IL-13 (10 ng / mL; PeproTech; catalog number 214-13) for 48 hours to induce MDM polarization. Then, the polarized MDMs were collected and used for flow cytometry or for establishing in vitro co-culture experiments.

[0105] In vivo bioluminescence imaging of live animals (BLI). BLI was performed using a Spectrum Advanced Molecular Imaging (AMI) HTX imaging system (Spectrum Instrument Imaging). In vivo imaging of live animals was obtained 5 minutes after intraperitoneal (i.p.) injection of D-luciferin (1 mg / mouse) for whole-body bioluminescence. The imaging results were analyzed using AURA imaging software (Spectral Instrument Imaging).

[0106] In vivo anti-tumor efficacy study in an NSG mouse model of OVCAR3 human ovarian cancer xenografts (i.p. tumor inoculation simulating in situ growth of ovarian cancer). The experimental design is as shown in Main Figure 6 Figure a. Briefly, on day 0, NSG mice received intraperitoneal (i.p.) inoculation of OVCAR3-FG cells (1 × 10 6 cells / mouse). On day 14, the tumor burden of the experimental mice was determined using BLI, and then they were placed into 4 equivalent BLI expression groups. On the same day, the experimental mice either received an i.p. injection of vehicle (PBS) or an i.p. injection of effector cells (4 × 10 6 CAR + cells / mouse in PBS). The survival of all mice was monitored and their tumor burden was measured twice a week using BLI.

[0107] In vivo anti-tumor efficacy study in an NSG mouse model of OVCAR8 human ovarian cancer xenografts (s.c. tumor inoculation simulating solid tumor growth of ovarian cancer). The experimental design is as shown in Main Figure 7 Figure a. Briefly, on day 0, NSG mice received subcutaneous (s.c.) inoculation of OVCAR8 cells (1 × 10 6 cells / mouse). On day 7, the tumor burden of the experimental mice was determined, and then they were placed into 4 groups of equivalent tumor sizes. On the same day, the experimental mice either received an intravenous (i.v.) injection of vehicle (PBS) or an intravenous (i.v.) injection of effector cells (10 × 10 6 CAR + cells / mouse in PBS). The survival of all mice was monitored and their tumor burden was measured twice a week using a Fisherbrand TM Traceable TM Digital Caliper (Thermo Fisher Scientific). The tumor volume was calculated using the following formula: Volume (mm 3 ) = (Length x Width 2) / 2. At the end of the experiment, the mice were sacrificed. The solid tumors were retrieved, weighed using a PA84 precision balance (Ohaus), and then processed for flow cytometry analysis to detect tumor-infiltrating Vδ2 T cells (identified as hCD45 + Vδ2 + cells). Various mouse tissues (blood, heart, lung, liver, and kidney) were also harvested and processed according to established protocols for flow cytometry analysis to detect the tissue biodistribution of Vδ2 T cells 94 .

[0108] In vivo anti-tumor efficacy study comparison 16H MCAR15-Vδ2 T cells and 16L MCAR15-Vδ2 T cells. The experimental design was as Figure 12 shown in a. Briefly, on day 0, NSG mice received subcutaneous (s.c.) inoculation of OVCAR8 cells (1×10 6 cells per mouse). On day 14, the tumor burden of the experimental mice was measured and then they were placed into 4 groups of equivalent tumor sizes. On the same day, the experimental mice either received intravenous (i.v.) injection of vehicle (PBS) or intravenous (i.v.) injection of effector cells (5×10 6 CAR + cells / mouse) in PBS. The survival of all mice was monitored and their tumor burden was measured twice a week using calipers. The tumor volume was calculated using the following formula: Volume (mm 3 ) = (length x width 2 ) / 2.

[0109] Histopathological analysis

[0110] Tissues (i.e., spleen, lung, liver, heart, and kidney) were collected from the experimental mice and fixed in 10% neutral buffered formalin for up to 36 hours and embedded in paraffin for sectioning (5 μm thick). Tissue sections were prepared by the UCLA Translational Pathology Core Laboratory according to the core standard protocol and stained with hematoxylin and eosin (H&E). The stained sections were imaged using an Olympus BX51 upright microscope equipped with an Optronics Macrofire CCD camera (AU Optronics) at magnifications of 20x and 40x. The images were analyzed using Optronics PictureFrame software (AU Optronics).

[0111] Statistics

[0112] Statistical data analysis was performed using Rstudio and Graphpad Prism7 software (Graphpad). Paired comparisons were made using the Student's two-tailed t-test. Multiple comparisons were made using ordinary one-way ANOVA followed by Tukey or Dunnett multiple comparison tests. The Meier survival curve analysis was performed using the log-rank (Mantel-Cox) test corrected for multiple comparisons. Unless otherwise stated, data are presented as the mean ± standard error of the mean (SEM). In all figures and legends, "n" represents the number of samples or animals used in the indicated experiment. P values less than 0.05 were considered significant. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0113] Data availability

[0114] A large amount of RNAseq datasets generated in this study have been deposited in the GEO database under the accession code GSE235755 below.

[0115] As described above, we reported the generation and characterization of CAR- and IL-15-engineered CD16 Hi Vδ2 T cells for the development of allogeneic cellular immunotherapy. CD16 was identified as a biomarker for the selection of Vδ2 T cells with enhanced cytotoxicity. Engineered CD16 Hi Vδ2 T cells were generated with high yield and purity, targeted tumors through multiple mechanisms (such as CAR, TCR, and ADCC recognition), and exhibited durable preclinical in vivo tumor control and persistence without signs of GvHD.

[0116] To overcome the challenges in current conventional CAR-based αβ T cell therapies (including limited efficacy against solid tumors), the engineering of innate-like and innate immune cells (such as γδ T cells, iNKT cells, NK cells, and macrophages) is being actively investigated 56-59 . Similar to αβ T cells, innate / innate-like cell populations are heterogeneous mixtures of cells with distinct transcriptional programs, phenotypes, and functions, and certain subsets may be ideal for cellular therapies for cancer. Laskowski et al. recently highlighted the donor-to-donor variability in NK cell profiles and the need to thoroughly understand NK product characteristics to define biomarkers indicative of greater potency and persistence 60 . Our study suggests that CD16 may have the potential to serve as a biomarker for the selection of Vδ2 T cell donors.

[0117] Vδ2 T cells are known to have significant safety and intrinsic anti-tumor functions in an allogeneic setting, which is of great interest for the development of cancer therapies 22,61,62Although clinical studies of engineered Vδ2 T cells are still scarce 62 , preclinical studies have been conducted on CAR engineering and modifying culture conditions such as supplementing TGF-β to enhance the therapeutic potential of Vδ2 T cells 63-65 . Extensive studies on CD16 (FcγRIIIa) and its role in tumor control (through both therapeutic administration 47,66,67 and naturally occurring 68 antibody-mediated ADCC) have prompted us to characterize CD16 expression on Vδ2 T cells isolated from healthy human peripheral blood mononuclear cells and to evaluate the potential of CD16 as a biomarker for donor selection. Further support for focusing on CD16 to create cancer immunotherapies comes from several avenues: the development of high-affinity, non-cleavable CD16 incorporated into pluripotent stem cell-derived NK cells to enhance their anti-tumor capacity when combined with mAbs 69 , the use of CD56 dim CD16 + NK cells to improve dendritic cell vaccination responses 70 , and bispecific and trispecific killing conjugates (BiKE and TriKE) against tumor-specific antigens to enhance NK cell-mediated tumor rejection 74 . These data provide evidence that CD16 Hi Vδ2 T cells can be another strategy to realize the anti-tumor immune potential mediated by CD16

[0118] The expression and functionality of CD16 on Vδ2 T cells have been explored for over two decades 27-30 . One study hypothesized that the therapeutic potential of PBMC-derived CD16 + Vδ2 T cells might be limited by their poor expansion capacity 29 , and single-cell RNA-Seq (scRNA-Seq) studies have shown that PBMC-derived CD16 + Vδ2 T cells are fully differentiated 75 , but other researchers have demonstrated the high yield of CD16 + Vδ2 T cells 30 . In our study, using traditional ZOL and IL-2 expansion methods, the expanded CD16 Hi Vδ2 T cells had a proliferation and memory status similar to that of CD16 Lo Vδ2 T cells. From CD16 HiDonor-expanded Vδ2 T cells have enhanced cytotoxicity and cytokine production in response to ovarian cancer cell lines, and deep RNA-Seq revealed their activation, cytotoxicity, and phagocytic gene signatures. We further developed CD16 Hi Vδ2 T cells for cancer therapy by engineering with CAR and IL-15, and confirmed by in vitro and in vivo characterization that CAR15-Vδ2 T cells showed robust anti-tumor efficacy against ovarian cancer models. Although injection of IL-15 can increase NK and CD8 + T cells in the circulation, it is difficult to achieve sustained IL-15 signaling using soluble IL-15 due to its short serum half-life and limited bioavailability 76 . Autocrine IL-15 has the potential to provide sustained IL-15 to engineered immune cells and local delivery as well as simplify the treatment regimen and reduce systemic toxicity. In ovarian and other solid tumors, MSLN is a promising target, but antigen heterogeneity may reduce the effectiveness of single antigen-targeted forms. Using CD16 Hi MCAR-Vδ2 T cells, solid tumors can be killed by CAR- and TCR-mediated recognition and combination therapy with HER2 mAb.

[0119] The Vγ9Vδ2 TCR recognizes metabolic dysregulation caused by changes in the mevalonate pathway through conformational changes in BTN3A1 binding, which commonly occurs in solid tumors and may permit Vδ2 T cell killing 22,77 . Although addition of ZOL is required for Vδ2 TCR-mediated killing of ovarian cancer cell lines used in our study, cellular energy dysregulation is a budding hallmark of cancer 21 , and metabolic restrictions found in the tumor microenvironment may lead to Vδ2 TCR targeting of tumor cells in the absence of exogenous bisphosphonates 31,32 . Another γδ T cell subset being evaluated for cancer therapy carries Vδ1 TCR, and allogeneic CD20-targeted CAR-Vδ1 T cell therapy (ADI-100) had a promising 75% objective response rate (ORR) and 69% complete response rate (CR) in a small phase I study (n = 16) of adult relapsed / refractory advanced B cell lymphoma 78 . Importantly, ADI-001 showed a 100% CR rate (n = 5) in patients who relapsed after prior autologous anti-CD19 CAR T treatment. Vδ1 T cells mainly recognize glycolipids presented by class I MHC-like CD1 proteins, which are mainly expressed by antigen-presenting cells 79-81Thus, for harnessing the inherent TCR recognition of cancer cells, Vδ1 T cells are well-suited for hematological malignancies, while Vδ2 T cells can target both liquid and solid cancers. Other potential benefits of the Vδ2 subset include its stimulation by FDA-approved ZOL, higher starting cell numbers in peripheral blood (1 - 10% for Vδ2 vs 0.1 - 1% for Vδ1), and the fact that Vδ1 cells do not express CD16 82 While NK-mediated tumor-targeting antibody (Ab) enhancement remains a concern 83 our results suggest that CD16 Hi Vδ2 T cells can be used to achieve anti-tumor ADCC. Both Vδ1 and Vδ2 subsets exert NK-like activation and cytotoxicity through various natural killer receptors (NKRs) such as NKG2D and DNAM1, which can enhance the breadth and magnitude of their anti-tumor activity 82 .

[0120] Our exploration of CAR and IL-15 engineered CD16 Hi Vδ2 T cell therapy development has revealed several areas for further investigation to determine the potential of this population for cancer treatment, such as T cell memory state, in vivo polarization, immunogenicity, and CD16 Hi Vδ2 T cell pool formation. A great deal of effort has been focused on creating less differentiated memory-like cells for CAR-based therapies 84,85 . Activation and expansion of T cells may lead to terminally differentiated cell products, which may have reduced expansion potential. Intriguingly, although starting numbers are low, expanded Vδ1 T cells maintain an earlier memory state 26 , which may be a benefit of Vδ1 T cells compared to Vδ2, which predominantly present as T effector memory (T EM ) cells in our cultures. Methods to maintain the Vδ2 T cell memory state during expansion without compromising the expansion rate are actively being investigated. Other characterizations, including ex vivo analysis after murine tumor challenge, can be used to stress test the Th1 phenotype of CD16 Hi Vδ2 T cells and confirm the continued functionality of the CD16 receptor. Although γδ T cells have been identified as prognostic markers for better outcomes 16 and have recently been shown to be immunotherapy effectors for DNA mismatch repair-deficient cancers with class I HLA deficiency 86 , other studies have reported potential negative effects of IL-17-producing γδ T cells 34-36 . CD16 Hi Vδ2 T cells express more than their CD16 LoLow levels of ROR1 mRNA in counterparts, and single-sample GSEA indicated that CD16 was negatively correlated with Th17 polarization, but further experiments are needed to functionally examine CD16 Hi Th17 potential in Vδ2 T cells.

[0121] Addressing engineered CD16 Hi The immunogenicity of Vδ2 T cells is also important. Although lymphodepletion-based preconditioning is standard for adoptive cell therapy 87,88 , questions remain: whether host-mediated infused cell rejection affects its engraftment and precludes a therapeutic window for meaningful clinical benefit. If immunogenicity severely hinders engineered CD16 Hi Vδ2 T cells, then haploidentical or HLA-matched donors or masking strategies, such as HLA I / II knockout and / or HLA-E overexpression, may be necessary to escape immune rejection.

[0122] Studies on how the CD16 Hi Vδ2 T cell pool forms can deepen our understanding of CD16 Hi Vδ2 T cells as a distinct subset. We have shown that transgenic CD16 on CD16 Lo Vδ2 T cells does not affect cytotoxicity, supporting the view that CD16 is not just a functional enhancer of Vδ2 T cell tumor killing. Previous findings showed that FcRγ-deficient NK (g-NK) cells exhibit significantly more robust reactivity once stimulated with CD16, and subsequent studies revealed the formation and maintenance of the g-NK cell pool in individuals infected with human cytomegalovirus (CMV) through mechanisms involving both epigenetic modification and antibody-dependent expansion 89-91 . Additionally, the analysis of CD16 Hi Vδ2 T cells using scRNA-Seq and epigenomic sequencing can reveal its etiology and contribute to the creation of further enhanced CD16 Hi Vδ2 T cell products.

[0123] Successful CD16 Hi Vδ2 T cell-based therapies pose several potential challenges. In the development of CAR T cell therapy for solid tumors, tumor penetration is an important consideration. Although there is preliminary evidence that MCAR15-Vδ2 T cells infiltrate subcutaneous tumors in vivo, additional studies and ultimately clinical investigations are needed to show meaningful tumor penetration in patients. Once infiltrating the tumor, sufficient persistence and anti-tumor functionality are significant clinical challenges because immune rejection and immunosuppressive tumor microenvironments may impede CD16 HiTherapies for Vδ2 T cells. Additionally, although CAR-engineered γδ T cell clinical trials have been conducted and are ongoing, the use of CD16 Hi Large-scale production of MCAR15-Vδ2 T cells from donors will require significant manufacturing and process design optimization efforts. The findings of our study highlight the effectiveness of a combination of donor selection based on CD16 expression, CAR engineering, and IL-15 secretion in enhancing the cancer therapeutic potential of Vδ2 T cells. High CD16 expression on Vδ2 T cells allows these cells to be used in combination with therapeutic antibodies, while their cytotoxicity against M2-polarized macrophages provides additional anti-tumor properties. Given the heterogeneity of solid tumors and their complex immunosuppressive tumor microenvironment, a multi-faceted approach to tumor recognition and immune modulation may be required to achieve durable therapeutic outcomes in treatment-naive patients.

[0124] Example 2: Enhanced GAMMA DELTA T Cells for Immunotherapy

[0125] Cancer T cells are an innate-like subset of T cells that account for 1-5% of peripheral blood mononuclear cells, and Vδ2 T is the most common subset (80-90% of total γδ T cells). Vδ2 T cells do not cause graft-versus-host disease (GvHD) and have intrinsic cancer-killing capabilities, but their clinical application in cancer immunotherapy is limited by their scarcity, persistence, and tumor immunosuppressive mechanisms.

[0126] Chimeric antigen receptor-T (CAR-T) cells have revolutionized the treatment of hematological malignancies but have not had the same success against solid tumors. Additionally, all FDA-approved CAR-T cell therapies are autologous, which is a key bottleneck in their scalability, affordability, and accessibility and can lead to serious adverse events. To create an allogeneic "off-the-shelf" cell therapy that is safe and effective against both liquid and solid tumor types, we selected gamma delta (γδ) T cells as an alternative cell source for genetic engineering and cancer treatment. γδ functional readouts indicate that CD16-high Vδ2T (CD16 Hi Vδ2T) cells can exhibit more robust cytotoxic activity than CD16-low (CD16 Lo Vδ2T) Vδ2T cells and can perform antibody-dependent cell cytotoxicity (ADCC).

[0127] As described below, we have developed a CD16 Hi Vδ2 T cell expansion method that yields higher productivity than conventional expansion methods. Despite undergoing high levels of expansion, Vδ2 T cells maintain a less differentiated memory-like profile (e.g., memory markers: stem cell-like memory (TSCM): CD27 + CD45RA+ ; and / or central memory (TCM): CD27 + CD45RA - ; and / or effector memory (TEM): CD27 - CD45RA - ; and / or terminally differentiated effector memory (TEMRA): CD27 - CD45RA + ). Our progress in Vδ2 T cells may enable the large-scale production of potent, high-quality Vδ2 cells.

[0128] Exemplary inventive embodiments

[0129] Embodiments of the present invention include a donor screening method and a new amplification method (memory amplification) and related culture media, which can be used to generate Vγ9Vδ2 T (referred to as Vδ2 T) cells with enhanced anti-tumor properties.

[0130] The donor screening method includes selecting a donor based on CD16 expression on Vδ2 T cells.

[0131] The donor refers to a peripheral blood mononuclear cell (PBMC) donor. PBMCs can be isolated from leukapheresis (Leukopak) and used for the amplification of Vδ2 T cells.

[0132] Amplified CD16 Hi Vδ2 T cells can be conjugated with monoclonal antibodies to treat different disease targets for combination therapy.

[0133] Memory amplification includes 1) initially stimulating Vδ2 T cells from PBMCs; 2) in vitro amplifying Vδ2 T cells using the memory culture media disclosed herein. This amplification method can be used to amplify all types of Vδ2 T cells, including CD16 Hi Vδ2 T cell populations.

[0134] The resulting memory-like Vδ2 T ( Mem Vδ2 T) cells generated using memory amplification have central memory (T CM ) and stem cell-like memory (T SCM ) phenotypes and have better self-renewal potential and persistence than Vδ2 T cells amplified by conventional culture methods.

[0135] In addition to the screening and amplification methods, cultured Vδ2 T cells can be genetically engineered (e.g., gene overexpression, gene knockdown / knockout, gene disruption) to generate Vδ2 T cell derivatives with enhanced therapeutic potential. See the examples in the "Vδ2 T cell derivatives" section below.

[0136] Exemplary methods and media

[0137] Donor screening

[0138] Screen for CD16 expression on the PBMC Vδ2T cells of the donor. Select donors with high CD16 as PBMC donors.

[0139] Memory-like Vδ2T cell culture

[0140] Culture PBMCs to generate memory-like Vδ2T cells. PBMCs can be obtained from the selected donors with high CD16.

[0141] Stimulate and expand Vδ2T cells by supplementing the cell culture with Vδ2 stimulating reagents (including but not limited to TCR homologous antigens, phosphoantigens, small molecules, and / or antibodies). Examples of such reagents include isopentenyl pyrophosphate, zoledronate, pamidronate, risedronate, alendronate, ibandronate, tiludronate, etidronate, anti-γδTCR antibody, non-specific TCR stimulating reagents (anti-CD3 / anti-CD28 antibody or antibody-coated beads, concanavalin A, PMA / ionomycin, and artificial APC), anti-CD16 antibody, etc.

[0142] The memory-like characteristics of the cultured Vδ2T cells can be achieved by supplementing the cell culture with memory-promoting reagents, including but not limited to serum albumin, L-ascorbic acid, 2-mercaptoethanol, IL-2, IL-4, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, TNFα, SDF-1α, TGF-β, and Wnt activator or glycogen synthase kinase-3 (GSK-3) inhibitor such as Wnt3A, CHIR99021, AR-A014418, TWS119, LY2090314, 9-ING-41, lithium chloride (LiCl), BIO (6-bromoindirubin-3-oxime, 6-bromoindirubin-3'-oxime). The memory medium can also include tyrosine kinase inhibitors such as dasatinib, ibrutinib, acalabrutinib, zanubrutinib, etc. The memory medium can be used for culturing and expanding all types of Vδ2T cells, including CD16 Hi Vδ2T cells.

[0143] The cell culture basal medium can be, including but not limited to CTS OpTmizer, TexMACS, RPMI, DMEM, X-Vivo15, etc. The cell culture method can be serum-free and feeder-free.

[0144] Enrich CD16 by magnetic bead sorting or fluorescence-activated cell sorting (FACS) based on the CD16 expression of the culture at any stage (e.g., before, during, or after Vδ2T cell expansion)Hi Vδ2 T cells.

[0145] The resulting CD16 Hi The Vδ2 T cell product can be cryopreserved and stored for finished product distribution.

[0146] Vδ2 T cell derivatives

[0147] In some embodiments, Vδ2 T cells can be engineered to express transgenes. In one embodiment, such transgenes encode disease-targeting molecules, such as chimeric antigen receptors (CARs) and other natural or synthetic receptors / ligands. In another embodiment, such transgenes can encode T cell regulatory proteins, such as IL-2, IL-7, IL-15, IFN-γ, TNFα, CD28, 4-1BB, OX40, ICOS, FOXP3, etc. The transgenes can be introduced at different culture stages.

[0148] In some embodiments, gene editing tools (CRISPR, TALEN, zinc fingers, etc.) can be used to further engineer Vδ2 T cells to disrupt selected genes. In one embodiment, the disrupted genes encode T cell immune checkpoint inhibitors (PD-1, CTLA-4, TIM-3, LAG-3, etc.). The deletion of these negative regulatory genes may enhance the disease resistance of Vδ2 T cells, making them resistant to disease-induced ineffectiveness and tolerance.

[0149] In some embodiments, Vδ2 T cells can be further engineered to make them suitable for allogeneic adoptive transfer and thus suitable for use as a cellular product for finished products. In one embodiment, genes encode MHC molecules or MHC expression / display regulatory molecules [MHC molecules, B2M, CIITA (class II transactivator that induces the expression of MHC class II mRNA), etc.]. The lack of MHC molecule expression on Vδ2 T cells makes them resistant to allogeneic host T cell-mediated elimination. In another embodiment, MHC class I-deficient Vδ2 T cells will be further engineered to overexpress the HLA-E gene, which will confer resistance to host NK cell-mediated elimination on them.

[0150] Novel features and advantages of the embodiments of the present invention

[0151] When compared with conventional methods for generating Vδ2 T cell products for cancer immunotherapy, the present invention provides a method for screening PBMC donors and a method for cell culture that can generate Vδ2 T cells with enhanced anti-tumor properties.

[0152] The unique features of the present invention include:

[0153] 1) Select donors with high-performance Vδ2 T cells.

[0154] 2) The resulting Vδ2 T cells exhibit strong anti-tumor cytotoxicity and are less likely to polarize into pro-tumor or pathological (e.g., Th17-like) γδ T cells in patients.

[0155] 3) The resulting Vδ2 T cells have ADCC function and can be conjugated with monoclonal antibodies for combination therapy. This provides an opportunity to overcome tumor immune escape in cancer treatment.

[0156] 4) The resulting Vδ2 T cells have a higher yield! More than 10 11 high-performance memory-like Vδ2 T cells can be generated from a single apheresis of PBMC leukocytes, which can potentially be formulated into 100 - 1,000 doses (based on the estimated dose of CAR-T cell therapy of 10 8 - 10 9 cells / dose approved).

[0157] 5) The resulting Vδ2 T cells have a central memory (T CM ) and stem cell memory (T SCM ) phenotype and exhibit strong persistence and self-renewal potential.

[0158] 6) The resulting Vδ2 T cells can be effectively genetically engineered to produce immune-enhanced Vδ2 T cell products and derivatives!

[0159] Demonstrated high-yield donor screening and robust generation of CD16 Hi memory-like Vδ2 T cells. Such Vδ2 T cells can be effectively engineered to express tumor-targeting molecules (e.g., CAR) and immune-enhancing molecules (e.g., IL-15) without compromising the yield. In vitro and in vivo studies were performed, showing high safety, high anti-tumor efficacy, high persistence, and multiple tumor-targeting mechanisms to overcome tumor immune escape.

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[0255] Other publications

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[0261] All publications mentioned herein (e.g., U.S. Patent No. 10,316,289; U.S. Patent No. 11,111,478; WO2009083755; PCT Published International Application Nos. PCT / US21 / 65349; PCT / US19 / 36786 and PCT / US2020 / 037486; U.S. Patent Application Serial No. 15 / 320,037; and Zarin et al., Cell Immunol. 2015 Jul; 296(1):70-5. doi: 10.1016 / j.cellimm.2015.03.007. Epub 2015, those listed above, etc.) are incorporated by reference to disclose and describe aspects, methods, and / or materials related to the cited publications. Many of the techniques and procedures described or cited herein are well understood and commonly employed by those skilled in the art.

[0262] Unless otherwise defined, all professional terms, symbols, and other scientific terms or expressions used herein are intended to have the meanings commonly understood by those skilled in the art to which this invention pertains. In some instances, terms having commonly understood meanings are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference from what is commonly understood in the art.

Claims

1. A method for culturing mammalian cells, the method comprising: Obtaining a population of lymphocytes; Identifying, selecting, and / or purifying cells expressing the combination of CD16 and Vδ2 within the population of T lymphocytes; And Amplifying the T lymphocytes expressing the combination of CD16 and Vδ2.

2. The method according to claim 1, wherein the selected and / or purified T lymphocytes expressing the combination of CD16 and Vδ2 are amplified by placing the T lymphocytes in a cell culture medium comprising a combination of γδ T lymphocyte stimulants, a combination of cytokines, a combination of Wnt activators and GSK-3β inhibitors, and / or a combination of tyrosine kinase inhibitors.

3. The method according to claim 2, wherein: The T lymphocyte stimulant comprises a bisphosphonate; The cytokines comprise IL-2, IL-7, IL-15, and IL-21; The GSK-3β inhibitor comprises TWS119; and / or The tyrosine kinase inhibitor comprises dasatinib, ibrutinib, acalabrutinib, or zanubrutinib.

4. The method according to claim 1, wherein the population of lymphocytes is selected from one or more individuals identified as having at least 35% CD16-positive cells within the population of Vδ2 T lymphocytes obtained from the individual.

5. The method according to claim 1, wherein the population of lymphocytes is selected from one or more individuals identified as having less than 35% CD16-positive cells within the population of Vδ2 T lymphocytes obtained from the individual.

6. The method according to claim 1, wherein the method comprises examining the cells within the population of lymphocytes in a cellular cytotoxicity assay.

7. The method according to claim 1, wherein the population of lymphocytes is obtained from the peripheral blood or cord blood of one or more donors.

8. The method according to claim 1, wherein: The T lymphocytes are amplified to at least 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, and 10,000-fold; and / or The T lymphocytes are amplified in cell culture for more than one month and / or restimulated while maintaining a less differentiated memory state.

9. The method according to claim 8, which further comprises cryopreserving the amplified cells.

10. A method for preparing a cell culture medium for amplifying T lymphocytes expressing the combination of CD16 and Vδ2, the method comprising placing together: a combination of γδ T lymphocyte stimulants, a combination of cytokines, a combination of Wnt activators; a combination of GSK-3β inhibitors, and / or a combination of tyrosine kinase inhibitors.

11. The method according to claim 10, wherein: The T lymphocyte stimulant comprises a bisphosphonate; The cytokines comprise IL-2, IL-7, IL-15, and IL-21; The GSK-3β inhibitor comprises TWS119; and / or The tyrosine kinase inhibitor comprises dasatinib, ibrutinib, acalabrutinib, or zanubrutinib.

12. The method according to claim 10, further comprising placing T lymphocytes selected to express the combination of CD16 and Vδ2 in the cell culture medium.

13. The method according to claim 13, wherein the lymphocyte population is selected from one or more individuals identified as having at least 35% CD16-positive cells within the Vδ2 T lymphocyte population obtained from the individual.

14. A cell culture medium for amplifying T lymphocytes expressing the combination of CD16 and Vδ2, the medium comprising: a bisphosphonate, a cytokine, and optionally a GSK-3β inhibitor.

15. The cell culture medium according to claim 15, wherein the cell culture medium comprises zoledronic acid, IL-2, IL-7, IL-15, and / or IL-21.

16. The cell culture medium according to claim 15, wherein the cell culture medium is a serum-free cell culture medium.

17. The cell culture medium according to claim 15, wherein the cell culture medium comprises a tyrosine kinase inhibitor.

18. The cell culture medium according to claim 15, further comprising a lymphocyte population selected from one or more individuals identified as having at least 35% CD16-positive cells within the Vδ2 T lymphocyte population obtained from the individual.

19. The cell culture medium according to claim 19, wherein the lymphocyte population has been enriched using an antibody that specifically binds CD16, an antibody that specifically binds Vδ2 TCR; magnetic bead sorting; and / or fluorescence-activated cell sorting (FACS).

20. The cell culture medium according to claim 19, wherein the lymphocyte population has been modified to regulate the expression of one or more endogenous genes and / or the cell population expresses one or more exogenous transgenes.

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