Abstract 3193: Overcoming glioblastoma heterogeneity and plasticity through the development of novel chimeric antigen receptor (CAR)-T cells targeting PTPRZ1

Abstract Glioblastoma (GBM) is an aggressive and heterogeneous brain tumor characterized by extensive plasticity, which contributes to resistance against conventional therapies. To address this challenge, we generated a unique single nuclei RNA sequencing atlas, integrating GBM, normal brain tissue, and developmental brain datasets. Utilizing single-cell RNA sequencing (scRNA-seq) analysis methods, we comprehensively characterized GBM heterogeneity and plasticity, focusing on identifying homogeneous cell surface antigens suitable for chimeric antigen receptor (CAR) T-cell targeting. scRNA-seq analysis revealed PTPRZ1 as a promising candidate antigen, uniformly expressed across tumor cells, offering a compelling therapeutic target for CAR-T cell therapy. Following antigen identification, we initiated mouse immunization campaigns to stimulate antibody production, coupled with phage display technology to generate a diverse library of single-chain variable fragments (scFvs). A rigorous cell-based positive and negative selection protocol was employed to isolate scFvs that specifically bind to PTPRZ1 while avoiding binding to closely related proteins from the same family, thereby minimizing off-tumor effects. Protein language models were used and experimental site-directed mutagenesis was performed to affinity mature and humanize selected scFv clones. Once PTPRZ1-specific scFvs were identified, they were incorporated into novel CAR constructs designed to overcome the low surface antigen density challenge commonly observed with GBM antigens. These CARs incorporated several signaling domains and structural modifications to enhance sensitivity and efficacy in low antigen-density settings. Our approach integrates cutting-edge single-cell sequencing, phage display technologies, and CAR-T cell design principles to address the heterogeneity of GBM and develop precision-engineered CAR-T cells targeting PTPRZ1. This study presents a step forward in overcoming GBM's plasticity, offering a potential therapeutic strategy with translational potential. Citation Format: Aditya A. Mohan, Barbara Lipes, Charla Gentry, Rushil Yerrabelli, Daniel Wilkinson, Kisha Patel, Emily Blandford, Andrew Hardigan, Peter Fecci, Michael Gunn, Anoop Patel. Overcoming glioblastoma heterogeneity and plasticity through the development of novel chimeric antigen receptor (CAR)-T cells targeting PTPRZ1 [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 3193.