Chimeric antigen receptor (CAR)-T cell therapy is highly effective in hematologic malignancies but remains limited in solid tumors due to poor tumor infiltration, immunosuppressive tumor microenvironments, and off-tumor toxicity. To address these challenges, we developed a dual immune-cell engineering platform combining tumor-tropic macrophages with AND-gate synthetic Notch (SNIPR) CAR-T cells.
RAW264.7 macrophages were engineered with a tumor-conditional Arg1 promoter redesigned to reduce inflammation-induced background activation, resulting in enhanced tumor selectivity under tumor-conditioned media. The promoter drives expression of a CD19-decorin fusion payload, in which decorin functions as a broad RTK inhibitor and collagen-binding molecule, while CD19 serves as a synthetic neoantigen for SNIPR CAR-T activation.
Conditioned media from engineered macrophages significantly reduced CT-26 viability, and collagen-binding assays demonstrated prolonged retention of CD19-decorin within the tumor microenvironment. SNIPR CAR-T cells specifically recognized collagen-bound CD19-decorin, while Transwell assays confirmed selective macrophage migration toward tumor-derived signals and activation of the conditional promoter.
Together, these findings demonstrate a modular platform that integrates macrophage tumor tropism, tumor-selective transcriptional control, and neoantigen-gated CAR-T activation to overcome key barriers in solid tumor immunotherapy.