Abstract 6110: Improving CAR T cell potency against low-density targets in rhabdomyosarcoma

Abstract Engineering of L1CAM-directed CAR T cells aims to demonstrate high efficacy and better persistence in rhabdomyosarcoma (RMS) models. RMS is the most common pediatric soft tissue sarcoma, and the low overall survival rates for metastatic RMS emphasize the urgent need for improved therapies. Chimeric Antigen Receptor (CAR) Ts have shown remarkable efficacy against hematological malignancies; however, their application in solid tumors remains challenging, mainly due to the absence of ideal targets and to the immunosuppressive nature of the tumor microenvironment (TME). To optimize antitumor activity against RMS and increase CAR T cell persistence in vivo, we investigated novel CAR constructs targeting newly identified RMS surface proteins. Through comprehensive RMS surfaceome profiling and validation in patient-derived tumor microarrays (TMAs), we identified L1CAM as attractive target. To evaluate the efficacy of L1CAM-directed CAR T cells against RMS, we generated a panel of anti-L1CAM.CE7 CARs expressing either a long (IgG4CH2CH3, 229aa) or a short (CD28, 35aa) hinge moieties, each paired with either a CD28- or 4-1BB-derived costimulatory domain (CSD).48h co-incubation experiments revealed that anti-L1CAM CAR T cells with long hinge and CD28 CSD showed superior efficacy, killing up to 60% of RMS cells at an Effector-to-Target (E:T) ratio of 1:1 and releasing double amount of IFN-γ compared to other constructs. To corroborate these findings in vivo, the selected anti-L1CAM.IgG4CH2CH3.28.3ζ CAR Ts were tested against the orthotopic RMS model Rh30, expressing low copy numbers of L1CAM (∼10’000). Despite limited efficacy following the initial CAR T dose, we observed tumor eradication in 2/5 mice and survival benefit in the remaining cohort after administration of a second dose on day 12. Furthermore, by bioluminescence of CAR T cells, we observed long-term persistence of CAR T cells in murine lymph nodes and the abdominal region for up to 50 days post-injection. To enhance therapeutic outcomes and achieve complete remission in 5/5 mice, we modified the anti-L1CAM.IgG4CH2CH3.28.3ζ construct to co-express an additional CD3ζ-mock CAR targeting CD276 - highly expressed on RMS - designed to boost the activation of anti-L1CAM CAR Ts (termed “IF-BETTER” CAR). In vitro screening identified 4-1BB as the optimal CSD for the CD276-associated CAR, which conferred enhanced activity to the L1CAM.IgG4CH2CH3.28.3ζ construct against RD and Rh30 cells, reaching up to ∼80% at the E:T ratio of 1:2. Furthermore, the selected IF-BETTER anti-L1CAM CAR Ts improved potency against the challenging Rh4 cells, which express very low copies of L1CAM (∼2’000). The selected IF-BETTER CAR Ts are currently tested in preclinical studies. These findings highlight the potential of the IF-BETTER anti-L1CAM CAR T cells as a promising therapeutic strategy for L1CAMdim tumors like RMS. Citation Format: Caroline Piccand, Andrea Timpanaro, Dila Handan Kaymaz, Jochen Karl Rössler, Michele Bernasconi. Improving CAR T cell potency against low-density targets in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6110.