A Myeloid-Specific Endogenous TRP1-CAR Knock-in Mouse Model for Melanoma-Targeted Immunotherapy.

BACKGROUND: Malignant melanoma is highly aggressive and prone to therapeutic resistance. Chimeric antigen receptor macrophage (CAR-M) therapy is promising for solid tumors, but conventional ex vivo-engineered CAR-M has limited in vivo persistence, complicating longer-term safety evaluation. This study established a myeloid-specific, endogenously expressed tyrosinase-related protein 1 (TRP1)-CAR mouse model to assess antitumor activity and safety. METHODS: TRP1-CAR was conditionally knocked into the Rosa26 locus and crossed with Lysozyme 2 (Lyz2)-Cre mice to generate mice with myeloid-specific TRP1-CAR expression (TRP1-CARM). CAR signals and baseline observations were assessed by flow cytometry, tissue immunofluorescence, histopathology, and hematology. Phagocytosis, cytotoxicity, and inflammation-related gene expression were evaluated in vitro. A syngeneic B16-Fluc-GFP melanoma model was used in two independent cohorts to assess tumor growth, survival, intratumoral CD8+ T-cell infiltration, and histopathology. RESULTS: CAR signals were detected in peripheral blood and in F4/80-positive macrophage-containing areas of the lung, liver, and spleen. No overt histopathological abnormalities or hematological changes were observed in the tissues examined under baseline conditions. TRP1-CARM-BMDMs showed enhanced phagocytic and cytotoxic activity against B16-Fluc-GFP cells, accompanied by altered inflammation-related gene expression in the co-culture system. In vivo, TRP1-CARM mice showed delayed tumor growth (P < 0.0001) and prolonged survival (P = 0.0005), with increased intratumoral CD8+ T-cell infiltration. CONCLUSION: This myeloid-specific TRP1-CAR knock-in model showed in vitro antitumor activity and delayed melanoma progression in vivo. It provides a proof-of-concept framework for investigating antitumor activity and preliminary safety observations associated with sustained endogenous CAR expression.