Synergistic enhancement of γδ T cell cytotoxicity against tumor spheroids through B7H3 CAR and zoledronate sensitization

Abstract Despite significant progress in treating hematological malignancies with chimeric antigen receptor (CAR) T cell therapies, their application in solid tumors remains challenging. The use of autologous T cells in solid tumor patients is often limited by prior chemotherapy and advanced disease stage at treatment initiation. Unlike hematologic cancers targeted by CARs against well-defined antigens, solid tumors present limited suitable targets, underscoring the need for combination approaches that engage tumor cells through multiple mechanisms. Gamma delta (γδ) T cells have emerged as a promising platform for allogeneic CAR therapies due to their innate antitumor activity, MHC-independent tumor antigen recognition, and multiple tumor-sensing mechanisms, all combined with a low risk of graft-versus-host disease (GvHD). Vγ9Vδ2 T cells, the main γδT cell subset in peripheral blood has been preferred for initial engineering given their robust expansion via bisphosphonates and cytokines. Zoledronate, a third-generation amino-bisphosphonate inhibits the farnesyl pyrophosphate synthase enzyme which metabolizes isopentenyl pyrophosphate (IPP), leading to increased intracellular IPP levels in monocytes that activates Vγ9Vδ2 T cells through butyrophilin family molecules. This activation mechanism resembles the γδ T cell response to intracellular bacterial infections, where microbial phosphoantigens like (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) drive γδ T cell expansion. However, IPP in human cells normally does not activate this activation mechanism except under malignant transformation where phosphoantigens accumulate. We expanded Vγ9Vδ2 T cells with zoledronate, IL-2, and IL-15, followed by transduction with a lentiviral vector (LV) manufactured with a baboon endogenous virus envelope protein (BaEV) pseudotyped LV encoding a B7H3 (CD276)-specific third generation CAR. This approach achieved consistent CAR expression at extremely low multiplicity of infection (MOI), while preserving an appropriate γδ T cell phenotype. We evaluated the activity of gamma-delta (γδ) CAR-T cells expanded using this method against tumor spheroids derived from various CD276+ cancer cell lines: MDA-MB-468 (triple-negative breast cancer), U-87MG (glioblastoma), SK-OV-3 (ovarian carcinoma), and RD (rhabdomyosarcoma) at multiple effector-to-target (E:T) ratios. Motivated by prior evidence that bisphosphonates activate γδ T cells in cancer patients, we pre-sensitized target cells for 16 hours with 500 ng/mL zoledronate prior to co-culture with effector cells. Untransduced γδ T cells displayed low baseline cytotoxicity, which significantly increased following zoledronate treatment of target cells. CAR-modified γδ T cells demonstrated potent cytotoxicity early in culture, with a modest but sustained enhancement observed upon zoledronate sensitization. Furthermore, comparison of wild-type U87MG cells with a B7H3 knockout variant revealed comparable cytotoxic effects, indicating that target antigen loss did not significantly diminish antitumor activity. From this we can conclude that armoring γδ T cells imparts antigen specificity, but this must be analyzed in the context of the strong cytotoxic activity mediated by the innate immune receptors present on activated γδ T cells. These findings support further investigation into the combined use of pre infusion of zoledronate and CAR γδ T cells as a strategy to potentiate cytotoxic responses against human solid tumors.