Synthetic NKG2D receptor (SNR) armored CAR-T cells overcome antigen heterogeneity of solid tumor

Background: CAR-T cell therapy has demonstrated remarkable success in hematologic malignancies; however, its effectiveness against solid tumors remains limited due to tumor antigen heterogeneity. NKG2DLs, including MICA/B and the ULBP family, are stress-induced molecules frequently upregulated on the surface of tumor cells and components of the tumor microenvironment, providing attractive targets for immunotherapy. To broaden the targeting capability beyond conventional Claudin18.2-directed CAR-T cells, we engineered a Synthetic NKG2D Receptor (SNR). The SNR comprises the extracellular domain of NKG2D fused with the intracellular signaling domains of DAP10 and DAP12, enabling effective targeting of NKG2D ligands (NKG2DLs). Methods: Expression of NKG2DLs and CLDN18.2 were detected by immunohistochemistry on a gastric cancer tissue microarray. We designed SNR CAR-T cells by linking CLDN18.2 CAR with SNR by a 2A self-cleaving peptide. We assessed their cytotoxicity, tumor infiltration, persistence, and antitumor efficacy using in vitro assays, patient-derived xenograft (PDX) models, and murine syngeneic models. Additionally, transcriptomic analysis and flow cytometry were performed to evaluate exhaustion and memory markers. Results: SNR CAR-T cells demonstrated enhanced cytotoxicity against tumor cells with heterogeneous CLDN18.2 expression, effectively lysing both CLDN18.2-positive and NKG2DL-positive tumor cells in vitro. In PDX and murine models, SNR CAR-T cells exhibited superior antitumor efficacy, leading to significant tumor regression and CAR-T expansion compared to conventional CAR-T cells. Furthermore, SNR CAR-T cells displayed reduced expression of exhaustion markers and increased expression of memory-associated markers. Enhanced tumor infiltration, proliferation and cytotoxicity within the tumor microenvironment, and a reduced presence of myeloid-derived suppressor cells (MDSCs) and tumor neovasculature were observed. Importantly, SNR CAR-T cell therapy was well-tolerated, with no significant toxicity noted in all the treated animals. Conclusion: The SNR CAR-T cell approach addresses tumor antigen heterogeneity and suppressive tumor microenvironment, offering a promising therapeutic strategy for solid tumors and paving the way for its future clinical applications.