CD28-ICOS costimulation enhances the therapeutic efficacy of L1CAM-targeted CAR-T cells against ovarian and gastric cancers.
BACKGROUND Ovarian and gastric cancers are associated with poor prognosis and derive limited benefit from current therapies, highlighting the need for novel immunotherapeutic approaches. The L1 cell adhesion molecule (L1CAM) is aberrantly expressed in several types of solid tumors and is associated with aggressive clinical behavior. This study aimed to develop and characterize optimized L1CAM-targeted chimeric antigen receptor-T (CAR-T) cells with improved efficacy and safety, using patient-derived preclinical models of ovarian and gastric cancer. METHODS L1CAM expression was assessed in tumor tissues from patients with ovarian cancer (OC;n=227) and gastric cancer (GC;n=132), followed by an analysis of its clinical impact. A panel of L1CAM-targeted CAR constructs incorporating distinct hinge and co-stimulatory domains was systematically evaluated. Functional activity was assessed by cytotoxicity and cytokine release assays using cancer cell lines and patient-derived tumor cells. Antitumor efficacy, persistence, and safety were evaluated in xenograft and patient-derived xenograft (PDX) models, including NanoLuc-based in vivo tracking of CAR-T cell distribution. RESULTS High L1CAM expression correlated with aggressive clinicopathological features and adverse prognosis in patients with OC. Among the constructs tested, CAR-T cells incorporating an IgD/IgG1 hybrid hinge and dual CD28-ICOS co-stimulatory domains demonstrated the most potent antigen-specific activity. These cells exhibited enhanced cytotoxicity, robust interferon-γ production, and sustained functional activity. In vivo, optimized L1CAM CAR-T cells achieved complete tumor regression in OC PDX models and significant tumor suppression in GC models, with durable tumor control and CAR-T persistence. Intraperitoneal delivery further improved therapeutic efficacy and CAR-T cell localization compared with intravenous administration. Importantly, no evidence of neurotoxicity or systemic toxicity was observed, supporting a favorable safety profile. CONCLUSIONS Optimized L1CAM-targeted CAR-T cells demonstrate potent, durable, and antigen-specific antitumor activity with a favorable safety profile in preclinical models of OC and GC. The combination of rational CAR design (CD28-ICOS signaling) and optimized delivery strategy provides a strong rationale for the clinical development of L1CAM-targeted CAR-T therapy, particularly in patients with L1CAM-positive solid tumors.