Transduction of γδ T cells with Baboon envelope pseudotyped lentiviral vector encoding chimeric antigen receptors for translational and clinical applications

Abstract

γδ T cells represent a promising cell platform for adoptive cell therapy. Their natural anti-tumor reactivity and HLA-independent target cell recognition make them an attractive platform for allogeneic adoptive immunotherapy clinical interventions. Initial clinical trials exploring allogeneic γδ T-cell therapies have demonstrated encouraging safety profiles. However, their therapeutic efficacy, especially against solid tumors, remains limited. This highlights the need for further optimization of γδ T cell products to improve anti-tumor potency, such as the increased targeting induced by the expression of a chimeric antigen receptors (CAR). However, a critical challenge in the development of CAR-γδ T cell therapies has been optimizing transduction efficiency with standard vector formats allowing for optimal CAR transgene expression that then produces an optimal therapeutic product. Here we present an effective method for enhancing CAR transgene expression in γδ T cells using a Baboon-pseudotyped lentiviral vector (BaEV-LV), comparing it to the conventional vesicular-stomatitis-virus-G protein (VSV-G) LVs. BaEV-LV significantly enhanced the transduction efficiency of γδ T cells with CARs, while conserving the beneficial cell product composition and phenotype of untransduced γδ T cells. The γδ T cells transduced with BaEV-LV CARs demonstrated significantly enhanced cytotoxicity against B7H3-expressing tumor cells in both 2D and 3D in vitro models. Our findings represent a significant advancement in CAR-γδ T cell engineering, offering a promising new avenue for cancer immunotherapy that combines the unique properties of Vγ9Vδ2 T cells with the targeted specificity of CAR technology. This method is compatible with automated closed-system platforms such as the CliniMACS Prodigy^®, facilitating Good Manufacturing Practice (GMP)-compliant production for clinical trials. This feature significantly enhances the translational potential of engineered γδ T cells, paving the way for the development of next-generation γδ T cell-based immunotherapies.

Keywords: CAR gd T cells; allogeneic; chimeric antigen receptor; immunotherapy; lentiviral transduction; γδ T cells.

Figure 1

γδ T-cell CAR transduction. (A) After three days of expansion, cells were transduced with BaEV or VSV-G pseudotyped LV encoding a B7H3 CAR. VF-1 was used in UTD and BaEV samples. Transduction efficiency (B) and the CAR MFI (C, D) were measured by flow cytometry at the end of culture. (E) After 3 days of expansion, cells were transduced with BaEV-pseudotyped LV encoding either a CD19 or a CD33 CAR and VF-1. Transduction efficiency was measured by flow cytometry at the end of the culture. (F) The final cellular product was also analyzed with flow cytometry to determine its cell composition. ns = non-significant, * = p<0.05, ** = p<=0.01, *** = p<=.001 and **** = p<0.0001. Each data point is an individual donor.

Figure 2

γδ CAR- T cell phenotype and activation profile. (A) γδ T cells were analyzed by flow cytometry at the end of culture to determine phenotype based on expression of CD45RA and CD27. (B, C) The expression of activation (B) and inhibition (C) markers by γδ T cells was analyzed by flow cytometry after expansion. Populations analyzed: starting population (Day 0), untransduced γδ T cells (UTD), untransduced γδ T cells supplemented with VF-1 (+VF-1), CAR+ γδ T cells transduced by either BaEV-LV (BaEV CAR+) or VSV-G (VSV-G CAR+) and CAR- γδ T cells in samples transduced by either BaEV-LV (BaEV CAR-) or VSV-G (VSV-G CAR-). * = p<0.05.

Figure 3

γδ CAR T cell cytotoxicity. B7-H3 CAR γδ T cells were co-cultured with indicated tumor cell lines (ea. row). (A-D) Overnight viability of luciferase expressing lines MCF-7 (A, n=6), MDA-MB-468 (B, n≥6), U87-MG (C, n=6) and U87-MG B7-H3 KO (D, n≥3) was measured after 24h of coculture with UTD and B7-H3 CAR γδ T cells. (E-H) Incucyte analysis with and without γδ T cells at a 1:1 E:T ratio, error bars = SEM. (I-L) The expression of CD107as measured by flow cytometry after 2h of co-culture. * = p<0.05 and ** = p<=0.01. Each data point is an individual donor.

Figure 4

γδ CAR T cell cytokine production. B7-H3 CAR γδ T cells were co-cultured with tumor cell lines (x-axis) for 24h at an E:T ratio of 4:1. Supernatant was collected and analyzed with a T/NK MACSplex kit. Granzyme B (A), perforin (B), IFN-γ (C) and TNF-α (D) concentrations were detected in samples containing γδ CAR T cells and not in samples with targets alone. n=5. * = p<0.05, ** = p<=0.01. Each data point is an individual donor.

Figure 5

B7-H3 CAR γδ T cells clear U87-MG tumor spheroids. B7-H3 CAR γδ T cells were co-cultured with U87-MG WT or B7H3 KO in an Incucyte device. (A, B) GFP integrated total intensity was measured every two hours for both U87-MG (A) and U87-MG B7-H3 KO (B) with and without γδ T cells at a 1:1 E:T ratio. (C) Images for U87-MG GFP expression are shown every 24h for both UTD and B7-H3 CAR γδ T cells co-cultures.