Abstract 4814: Development of a novel claudin-6-targeting CAR-T cell

Abstract Chimeric antigen receptor (CAR) T cell therapies have achieved remarkable efficacy in treating B cell malignancies but have shown limited clinical success in patients with solid tumors. A key challenge in advancing these therapies lies in the identification and targeting of highly tumor-specific antigens to ensure both safety and efficacy. In this study, we employed HybriFree B-cell cloning technology and phage display to discover antibodies targeting Claudin-6 (CLDN6), an oncofetal protein selectively expressed in subsets of ovarian, lung, endometrial, and testicular cancers, with minimal or no expression in healthy adult tissues. To generate a robust immune response, chickens were immunized with virus-like particles (VLPs) displaying a high-density presentation of transmembrane CLDN6. Monoclonal antibodies specific to CLDN6 were subsequently screened for their specificity towards CLDN6 using VLPs bearing other Claudin family members. The most promising specific binders that were able to discriminate between binding of CLDN6 and CLDN9 were humanized via the Qumanize platform and incorporated into a second-generation CAR construct with a 4-1BB costimulatory domain and CD3ζ signaling domain. CLDN6-specific CAR T cells were then generated and evaluated for their cytotoxicity in tumor models of ovarian, lung, and gastric cancers. Two CLDN6-specific CARs exhibited highly specific recognition and efficient lysis of cancer cells endogenously expressing CLDN6. Within 24 hours of co-culture, CLDN6 CAR T cells eliminated 80% of PA-1 cells and 60% of OV-90 cells. To assess T cell immune activation, cytokine secretion was measured upon co-culture with various target cells. Notably, IFN-γ secretion was observed exclusively when CLDN6 CAR T cells were co-cultured with CLDN6-positive targets, confirming their antigen specificity. This study underscores the utility of HybriFree technology and the Qumanize platform in generating highly specific and high-affinity CARs for tumor targeting. These findings present a promising avenue for developing next-generation immunotherapies for CLDN6-expressing solid tumors. Citation Format: Ana Shahpazir, Siret Tahk, Paule Hermet, Anu Ustav, Robin Pau, Gaily Kivi, Mario Plaas, Andres Männik, Joan Teyra, Mart Ustav, Tanel Mahlakōiv. Development of a novel claudin-6-targeting CAR-T cell [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 4814.