Endoglin-directed CAR-T cells Comprehensively Target Tumors in Advanced Sarcomas

Abstract Advanced sarcomas have dismal outcomes in children and adults, with limited therapeutic options. While chimeric antigen receptor T cells (CAR-T) hold promise for treating advanced sarcomas, it is constrained by a paucity of effectual targets. Our previous clinical study identified endoglin (ENG/CD105), a TGF-β co-receptor, as a target of the endogenous immune response in a sarcoma patient who exhibited an exceptional response to HER2 CAR-T therapy. ENG is expressed on various sarcomas, cancer-associated fibroblasts, and neoangiogenic vessels – offering comprehensive tumor targeting. Further, ENG knockout in sarcoma cells reduced their invasiveness, highlighting its potential as a therapeutic target. Accordingly, we designed a second-generation human ENG targeting CAR molecule signaling through the CD28 endodomain and retrovirally transduced primary human T cells. The ENG CAR-T exhibited strong antigen-specific cytokine release, robust proliferation, memory formation, and cytotoxic function against various sarcoma cell lines. Their cytotoxicity remained unaffected by the presence of soluble ENG or its natural ligand bone morphogenetic protein-9. Further, ENG CAR-T disrupted multicellular tumor spheroids in vitro, overcoming tumor compactness and the stromal barrier created by cancer-associated fibroblasts – critical challenges in sarcoma CAR-T therapy. In orthotopic murine models of sarcomas, ENG CAR-T treatment resulted in control of tumor growth and metastasis leading to survival extension. In summary, our study describes the involvement of ENG in sarcoma metastasis and validates our human ENG CAR-T as a potential therapeutic for advanced sarcomas.