L1CAM-CAR T cells with enhanced potency overcome low-density antigen expression in rhabdomyosarcoma

Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, shows dismal survival in relapsed or metastatic alveolar disease. Chimeric antigen receptor (CAR) T cells are promising but limited by scarce tumor-selective antigens and suboptimal efficacy at low antigen density. We investigated L1 cell adhesion molecule (L1CAM) as a therapeutic target by profiling its expression by flow cytometry, immunoblotting, and immunohistochemistry in cell lines, patient-derived xenografts, and healthy tissues. Using the scFv derived from the CE7 antibody, we engineered L1CAM-CARs with distinct hinge and costimulatory domains and tested them in vitro and in orthotopic RMS mouse models against clinically tested CE7- and B7-H3-CARs. L1CAM was consistently expressed at moderate levels in RMS, especially alveolar subtypes, but very weakly expressed in healthy tissues. Flow cytometry revealed a moderate density typically limiting CAR activity. Among constructs, L1CAM.III (CE7-CAR with long hinge and CD28 domain) showed the strongest cytotoxicity and IFN-{gamma} release. In vivo, L1CAM.III-CAR T cells regressed tumors, prolonged survival, and persisted in orthotopic RMS models, showing greater efficacy in alveolar RMS and no off-tumor activity. These findings establish L1CAM as a rational RMS therapeutic target. Optimized L1CAM.III-CAR T cells overcome moderate antigen density, achieving potent and persistent antitumor activity comparable to B7-H3-CARs but with improved safety. This work supports CAR optimization for clinical translation to broaden pediatric sarcoma immunotherapy.