EXTH-68. Targeting Tumor Heterogeneity And Plasticity in Glioblastoma With Novel CAR T Cells Targeting PTPRZ1

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Abstract Glioblastoma (GBM) remains among the most aggressive and intractable human cancers, driven by profound intratumoral heterogeneity, dynamic cellular plasticity, and robust mechanisms of immune escape. To overcome these barriers, we developed an integrated discovery and engineering pipeline that combines single-nucleus RNA sequencing (snRNA-seq), antibody generation through both immunization and generative AI design, yeast-based directed evolution, and rationally engineered CAR-T cell architectures. We first assembled a high-resolution snRNA-seq atlas spanning malignant GBM, adult brain, and developmental brain tissues. Cross-comparative analysis revealed PTPRZ1 as a consistently expressed, tumor-enriched surface antigen with minimal expression in normal adult tissues. Further analysis across developmental datasets confirmed PTPRZ1 expression during neurodevelopment, particularly in glioma stem cells and oligodendrocyte progenitor-like states, and showed retention of expression across all major GBM differentiation trajectories. These findings suggest that PTPRZ1 marks a persistent cellular state within the glioma hierarchy, making it a stable and tractable immunotherapeutic target despite GBM’s plasticity. To generate high-affinity binders against PTPRZ1, we pursued two parallel strategies: (1) phage display from immunized hosts followed by affinity maturation via yeast-based directed evolution, and (2) de novo nanobody design using structure-aware generative models trained on antibody sequence and structural datasets. Candidate binders were refined through site-directed mutagenesis to enhance specificity and binding kinetics. Given the known challenges of targeting PTPRZ1, including extracellular domain shedding and glycosylation-dependent surface stability, we engineered a suite of TCR-mimetic CAR-T cells incorporating modular costimulatory domains to enable tunable activation thresholds and improved resistance to exhaustion. These constructs emulate core features of physiological TCR signaling, offering increased antigen sensitivity, functional persistence, and reduced tonic signaling in hostile tumor contexts. This work establishes a modular platform for precision antigen discovery and CAR-T cell engineering in GBM and validates PTPRZ1 as a compelling, developmentally anchored immunotherapeutic target. More broadly, it offers a scalable roadmap for rational design of next-generation cellular therapies against solid tumors that evade conventional targeting strategies.

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