Abstract B008: Development of intravesical adoptive NECTIN4 chimeric antigen receptor (CAR) T cell therapy for non-muscle invasive bladder cancer

Abstract Bladder cancer is one of the most common genitourinary tract cancers. Non-muscle-invasive bladder cancer (NMIBC) represents the most common type. Intravesical immunotherapy with Bacillus Calmette-Guerin has been used to treat patients with high-risk NMIBC after surgery. However, 50-70% of patients with NMIBC experience disease recurrence and progression. Therefore, identifying novel therapeutic strategies for bladder cancer patients is needed. Nectin cell adhesion molecule 4 (NECTIN4) is an immunoglobulin-like transmembrane protein which is involved in the formation and maintenance of adhesion junctions. High expression of NECTIN4 in bladder cancer makes it a potential drug target for cancer therapy. While Enfortumab vedotin, an antibody-drug conjugate targeting NECTIN4, has been approved for patients with locally advanced or metastatic urothelial carcinoma, alternative therapeutic strategies to target NECTIN4 in bladder cancer are still urgently needed. Here we develop intravesical NECTIN4 chimeric antigen receptor (CAR) T cell therapy for NMIBC, which may overcome the barriers related to T cell infiltration and activation in the tumor as well as the on-target off-tumor effects associated with systemic CAR T cell therapy. We generate a second-generation CD28-based NECTIN4 CAR and demonstrate that our NECTIN4 CAR T cells possess potent and antigen-specific anti-tumor activity against NECTIN4-expressing bladder cancer cells in the co-culture assay with the release of interferon-gamma (IFN-γ), an indicator of T cell activation. To confirm the anti-tumor activity of NECTIN4 CAR T cells in vivo, we establish a subcutaneous xenograft bladder tumor model with the systemic administration of T cells by intravenous injection. We demonstrate that NECTIN4 CAR T cells suppress tumor growth in mice. Next, we develop an orthotopic bladder tumor model to determine if intravesical delivery of NECTIN4 CAR T cells in mouse bladder exhibits an efficient anti-tumor effect. Our results demonstrate that local delivery of NECTIN4 CAR T cells into the bladder possesses strong anti-tumor activity, and tumor growth inhibition can be observed three days post-T cell injection. Of note, intravesical NECTIN4 CAR T cells remain in the bladder and do not enter the bloodstream, ensuring their safety and minimizing off-target effects. Taken together, our study provides novel CAR T cell therapeutic approaches for NMIBC patients. Future studies using human NECTIN4 Knock-in mice models will be conducted to investigate the preclinical safety and efficacy of NECTIN4 CAR T cell therapy. Citation Format: Sheng-You Liao, Li-Ting Wu, Jonathan Chou, John K Lee. Development of intravesical adoptive NECTIN4 chimeric antigen receptor (CAR) T cell therapy for non-muscle invasive bladder cancer [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B008.