Multiscale engineering of PTPRZ1 CAR T cells through affinity-tuned binders and modular architecture optimization

Glioblastoma remains universally lethal, with rapid recurrence and a median survival of less than two years. Current CAR T approaches in glioblastoma have been limited by a lack of uniformly expressed and therapeutically actionable antigens. Using integrated single-cell and single-nucleus atlases, we identify PTPRZ1 as a consistently and highly expressed surface antigen across malignant glioblastoma states with minimal expression in non-neoplastic neural lineages. To translate this target, we developed a binder engineering pipeline combining mouse immunization, phage display with counter-selection against PTPRG, and yeast based directed evolution to generate a panel of scFv and nanobody binders with a spectrum of affinities that recognize the same PTPRZ1 epitope. This panel enables systematic tuning of CAR antigen sensitivity and functional thresholds and allows direct comparison of how binder format and affinity shape CAR activity against endogenous antigen. We incorporated these affinity-graded binders into a modular CAR backbone and constructed a combinatorial CAR library that varies hinge domains, transmembrane regions, and costimulatory modules. Primary human T cells transduced with pooled CAR libraries were screened against patient-derived glioma stem cells to quantify how each architectural module influences CAR function as an isolated component and how combinations of modules synergize to enhance activation, cytotoxicity, proliferation, resistance to exhaustion, and durability under chronic stimulation. This screening strategy also enabled the identification of recurrent architectural features that support persistence and metabolic fitness in solid tumor settings. Individual validation studies confirmed that both scFv and nanobody CARs targeting the identical epitope exhibit distinct activation and persistence profiles, and that optimal performance arises from specific pairings of hinge, transmembrane, and costimulatory domains. Safety studies using human epilepsy-derived cortical slice cultures reveal minimal off-tumor cytotoxicity and preserved neural architecture following exposure to PTPRZ1-targeting CAR T cells. Collectively, these data nominate affinity-tuned PTPRZ1 binders and optimized CAR architectures as strong candidates for translation and provide generalizable design principles for improving CAR T therapy in solid tumors.