Development of CD70 CAR-NK Cells with TGF-β Sensor-Controlled and Tumor-Directed IL-12 Expression against Clear Cell Renal Cell Carcinoma

Abstract

Chimeric antigen receptor (CAR)-engineered T and NK cell therapies have shown promising success in treating hematological malignancies, yet their efficacy in solid tumor remains limited due to poor infiltration and reduced expansion and persistence in the tumor microenvironment (TME). To address these major challenges, we engineered a novel TGF-β sensor-controlled and tumor matrix-directed IL-12 secreting CD70 CAR-NK cell and evaluated it against clear cell renal cell carcinoma (ccRCC). In vitro studies confirmed that CD70 CAR-NK cells exhibited more potent tumor antigen-specific cytotoxicity, and both secretory and collagen-binding IL-12 enhanced killing efficiency by two folds against ccRCC cell lines. IL-12 secreting CD70 CAR-NK cells also demonstrated increased IFN-γ production and degranulation. Mice study indicated that these engineered NK cells effectively suppressed tumor growth in vivo. To further counteract TGF-β-induced immunosuppression within the TME and ensure controlled IL-12 release for improved clinical safety, we designed a novel TGF-β sensor based on SMAD-binding motifs. We showed that the TGF-β sensor successfully upregulated EGFP reporter protein expression in a TGF-β dose-dependent manner. Positioning the sensor on the reverse strand with IL-12 secretion domain precisely regulated IL-12 release upon TGF-β1 stimulation with minimal leakage. In vitro studies revealed that TGF-β sensor-controlled IL-12 secreting CD70 CAR-NK cells displayed superior tumor-killing capacity comparable to IL-12 secreting CD70 CAR-NK cells even at a low transduction rate. Overall, this study described a novel strategy to overcome the immunosuppressive TME and enhance tumor-specific killing in CAR-NK cell therapy while ensuring safety, supporting it as an effective therapeutic approach against ccRCC.