Clathrin mediated endocytosis targeting chimera for targeted membrane proteins degradation and enhance CAR-T cell anti-tumor therapy.

Rationale: T cell exhaustion, mediated by the expression of inhibitory receptor proteins, significantly reduces their anti-tumor efficacy. Therefore, strategies aimed at degrading membrane proteins have emerged as promising approaches for enhancing the therapeutic effectiveness of Chimeric Antigen Receptor (CAR) T cells and improving cancer treatment outcomes. Methods: In this study, we developed a Clathrin-Mediated Endocytosis Targeting Chimera (CleTAC), an innovative platform designed to facilitate membrane protein degradation via the clathrin-mediated endocytosis pathway. CleTAC employs the YVKM motif to interact with the AP2 complex, driving the internalization of targeted membrane proteins (proteins of interest, POI) along with associated cell membrane components. These internalized complexes are subsequently trafficked to lysosomes for degradation. We conducted multiple validations using flow cytometry, Western blotting, and in vitro and in vivo experiments to verify its degradation efficiency. Additionally, we integrated CleTAC targeting CTLA4 membrane proteins into a CAR construct and evaluated its impact on CAR-T cell functionality and tumor suppressive efficacy using both cellular assays and animal tumor models. Results: We demonstrated that CleTAC effectively and specifically mediates the degradation of EGFP and CTLA4 proteins on the cell surface. When incorporated into a CAR construct, CleTAC targeting CTLA4 enhanced CAR-T cell anti-tumor activity, as evidenced by improved functional assays and tumor suppression in animal models. These findings establish CleTAC as a versatile and effective tool for modulating membrane protein levels and enhance anti-tumor efficacy in CAR-T cells. Conclusions: Our results highlight CleTAC as a promising therapeutic tool with substantial clinical potential to significantly enhance CAR-T cell therapy and advance current oncology treatment strategies.