Optimized L1CAM-CAR T cells enhance activity against moderate-antigen-density rhabdomyosarcoma models

Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, remains difficult to treat in relapsed, metastatic, or refractory disease. Chimeric antigen receptor (CAR) T cell therapy has demonstrated promising results in hematological diseases, but its application to solid tumors including RMS is limited by antigen heterogeneity, on-target/off-tumor toxicity, and insufficient activity against moderate antigen density. We investigated L1 cell adhesion molecule (L1CAM) as a candidate CAR T cell target in RMS by profiling expression in cell lines, patient-derived xenografts, and healthy tissues. Using the CE7-derived single-chain variable fragment, we engineered and compared L1CAM-CAR constructs differing in hinge and costimulatory domains, including the clinically tested 4-1BB-based CE7-CAR configuration. Functional activity was assessed across fusion-positive and fusion-negative RMS models in vitro and in orthotopic mouse models, with B7-H3-CAR T cells included as a benchmark. L1CAM was expressed at variable but specific levels across RMS models, with more prominent expression in fusion-positive RMS and limited expression in healthy tissues. Among constructs, the CD28-based L1CAM.III-CAR showed the strongest cytotoxicity and IFN-γ release, including partial activity in a low-L1CAM model. In vivo, L1CAM.III-CAR T cells improved expansion, delayed tumor progression, and prolonged survival compared with the clinical-reference L1CAM.CT construct, although responses were incomplete and less pronounced than those achieved with B7-H3-CAR T cells. These findings support L1CAM as a rational target for L1CAM-positive RMS cases and demonstrate that CAR optimization can enhance activity against moderate-density antigens. The potent antitumor activity and favorable selectivity profile of L1CAM.III-CAR T cells support their development for pediatric sarcoma immunotherapy.