Efficient multiplex non-viral engineering and expansion of polyclonal γδ CAR-T cells for immunotherapy

Abstract

Gamma delta (γδ) T cells are defined by their unique ability to recognize a limited repertoire of non-peptide, non-MHC-associated antigens on transformed and pathogen-infected cells. In addition to their lack of alloreactivity, γδ T cells exhibit properties distinct from other lymphocyte subsets, prompting significant interest in their development as an off-the-shelf cellular immunotherapeutic. However, their low abundance in circulation, heterogeneity, limited methods for ex vivo expansion, and under-developed methodologies for genetic modification have hindered basic study and clinical application of γδ T cells. Here, we implement a feeder-free, scalable approach for ex vivo manufacture of polyclonal, non-virally modified, gene edited chimeric antigen receptor (CAR)-γδ T cells in support of therapeutic application. Engineered CAR-γδ T cells demonstrate high function in vitro and and in vivo. Longitudinal in vivo pharmacokinetic profiling of adoptively transferred polyclonal CAR-γδ T cells uncover subset-specific responses to IL-15 cytokine armoring and multiplex base editing. Our results present a robust platform for genetic modification of polyclonal CAR-γδ T cells and present unique opportunities to further define synergy and the contribution of discrete, engineered CAR-γδ T cell subsets to therapeutic efficacy in vivo.

Figure 1.. Antibody-based stimulation yields robust outgrowth of polyclonal γδ T cell populations.

(A) Fold expansion after 11 days. Human peripheral blood γδ T cells were stimulated with either zoledronate or plate-bound αPan-γδ TCR and soluble αCD28 (n=11, paired T test). (B) Fold expansion at day 22 after two rounds of stimulation with αPan-γδ TCR and soluble αCD28 antibodies, occurring on day 0 and day 11 (n=11). (C) γδT cell subset frequency, as measured by flow cytometry, in zoledronate or αPan-γδ TCR plus soluble αCD28 stimulated γδ T cells and days 0, 11, and 22 (n=2 technical replicates of 2 independent biological donors). (D) Bar graphs showing Shannon entropy, a measure of TCR clonal diversity, of γδ T cell TCR diversity for TRD chains (red bars) and TRG chains (blue bars) at day 0, 11, and 22 (n=3). (E) Naive, T_CM, T_EM, and T_EMRA frequencies in Vδ1+ (top left panel), Vδ2+ (top right panel), and Vδ1-Vδ2-αβ- (bottom panel) γδ T cells subsets as determined by CD27 and CD45ra expression (n=4). (****p<0.0001, **p<0.01, and *p<0.05)

Figure 2.. Non-virally engineered CAR-γδ T cells display potent in vitro cytotoxicity.

(A) Diagram of construct containing CD19-CAR, mutant DHFR, conferring resistance to MTX in engineered cells, and GFP, used to manufacture CD19-CAR γδ T cells. (B) Diagram of our clinically scalable platform for γδ T cell engineering and expansion. Human γδ T cells are isolated by immunomagnetic separation from healthy donor PBMCs, then stimulated with plate-bound αPan-γδ TCR and soluble αCD28. γδ T cells are then electroporated with genome engineering reagents at day 2, re-stimulated on day 11, and collected at day 22. (C) Efficiency of non-viral construct delivery, as measured by GFP expression after stimulation and transfection of DNA-based CAR constructs alongside hyperactive Tc Buster transposase mRNA (n=8 for 48-hour time point, n=4 for 72- and 96-hour time points). (D) Frequency of CAR-γδ T cells, as measured by GFP expression, and (E) fold expansion on day 22 following MTX selection (n = 4). (F) CAR copy number per γδ T cell genome following non-viral integration as measured by ddPCR. Each point represents the average of three technical replicates (n=3). (G) In vitro cytolysis of Raji-luc cells after coculture with CD19-CAR γδ T cells. Assays were performed at 3:1, 1:1, and 1:3 E:T ratios in technical triplicate for two human donors. Luminescence intensity was used to quantify cytolysis of Raji-Luc target cells, with Triton X-100 serving as a positive control for max killing (n=3). (H) Cytotoxicity of CD19-CAR γδ T cells after repeated rounds of exposure to Raji-Luc target cells (n=6) (****p<0.0001, ***p<0.001 **p<0.01, and *p<0.05)

Figure 3.. Multiplex gene knockout enhances γδ T cell function in immunosuppressive environments.

(A) Diagram highlighting the roles of CISH, PDCD1, and FAS in suppressing γδ T cell activity in vivo. (B) Editing efficiency of CISH KO gRNA, PDCD1 KO gRNA, dnFAS gRNA, a combination of CISH and PDCD1 gRNA, and all three gRNAs, as well as a no-gRNA control in γδ T cells when cotransfected with CBE (n=4). Normalized survival of (C) unstimulated and (D) αPan-γδ TCR antibody-stimulated control, dnFAS, and triple edit CAR-γδ T cells either untreated or treated with soluble Fas ligand. Live cell counts were quantified after 24 hours and normalized to untreated control CAR γδ T cells (n=2). (E) Cytolysis of PDL1^high Raji-Luc target cells following coculture with MTX-selected CAR γδ T cells, MTX-selected CAR γδ T cells with a PD1 KO, and MTX-selected CAR γδ T cells with PD1 KO, CISH KO, and Fas dn (n=6). (F) Fold expansion of engineered γδ T cells cultured in in decreasing amount of IL2 (left panel) or IL15 (right panel) (n=2). (G) Expression of 4–1BB, as measured by flow cytometry, in gene edited γδ T cells following stimulation with αPan-γδ TCR and soluble αCD28 for 24 hours (n=3).

Figure 4.. Engineered polyclonal γδ T cells exhibit potent anti-tumor activity in vivo.

(A) Diagram of in vivo tumor challenge model. Mice were injected intravenously with 5×10⁶ Raji-luc cells on day −4 and treated with 10×10⁶ γδ T cells on day 0. Mice that received two doses were injected again with 10×10⁶ γδ T cells on day seven. Tumor growth and overall survival was measured and flow cytometry on blood was done weekly until they reached endpoint. (B) Tumor growth and (C) Kaplan-Meier survival curve for NSG mice injected with Raji-Luc cells and treated with the indicated γδ T cells (n=5). (D) Immunohistochemistry staining of CD3 on paraffin embedded skin, intestines, stomach, brain, and ovary in two animals treated with γδ T cells engineered with CD19-CAR and IL15.

Figure 5.. Effect of gene engineering on γδ T cell subset frequency and persistence in vivo.

Weekly frequency of Vδ1+, Vδ2+, and Vδ1-Vδ2-αβ- subsets (A) as a percent of total CD3+ cells and (B) as a percent of total PBMC as measured by flow cytometry. “Infusion product” indicates the frequency before injection while “Endpoint” indicates the frequency at necropsy (n=10). (C) Frequency of γδ T cell subsets as a percent of total CD3+ cells and (D) as a percent of total PBMCs at necropsy as measured by flow cytometry (n=5). (E) Frequency of γδ T cell subsets as a percent of total CD3+ cells and (F) as a percent of total PBMCs at endpoint in groups that received a single dose of cells engineered with a constructs that did not contain IL15 compared with groups that received a single dose of cells engineered with constructs that contained IL15 (n=10).