Durable remission of high-risk and refractory neuroblastoma by PHOX2B derived peptide-HLA directed CAR T cells: An IND enabling study

Effective cellular therapies for solid tumors are limited by the lack of tumor-specific antigens. We previously showed that non-mutated self-peptides from essential intracellular neuroblastoma (NB) oncoproteins are presented by common HLA allotypes, enabling selective targeting (Nature 2023). Here, we report IND-enabling studies of second-generation PHOX2B PC-CAR T cells incorporating a 4-1BB costimulatory domain. A GMP-grade lentiviral vector encoding the PHOX2B peptide-HLA-specific CAR was produced at CHOP and used to transduce healthy donor and patient T cells. PHOX2B expression and epitope heterogeneity were analyzed across solid tumors, NB xenografts, and cell lines by RNA-seq, IHC, and a PHOX2B scFv assembled with klickmer detecting the QYNPIRTTF/HLA complex. Safety was evaluated using the X-scan and sCRAP cross-reactivity algorithms integrated with experimental testing across 25 normal HLA-A24/23 primary cell lines and HLA-matched PHOX2B negative cancers. Functional activity was assessed via IncuCyte based cytotoxicity, multiplex cytokine profiling, and T-cell activation/proliferation by flow cytometry. Efficacy was tested in four HLA-A24/23 NB xenografts (including chemotherapy-resistant models) and one non HLA-A*24:02/23:01 NB control in NSG MHC-I/II deficient mice receiving a single ~7×10⁶ PC-CAR T-cell infusion. Longitudinal expansion, immunophenotype, and transcriptional states were characterized by flow cytometry and single-cell RNA-seq. PHOX2B was highly expressed in all neuroblastomas and pheochromocytomas and not expressed in other cancers. Klickmer staining revealed variable epitope density correlating with PC-CAR T cell activation. PHOX2B PC-CAR T cells mediated potent, dose-dependent killing of HLA-A 2 4/23 neuroblastomas, sparing antigen-negative non-HLA-A24/23 cancer cells unless peptide-pulsed. Antigen engagement triggered proliferation, effector cytokine release, and IFN-gamma dependent upregulation of HLA-I, significantly increasing antigen density. No cross-reactivity was detected in co-culture assays. A single dose of PC-CAR T cells induced complete regression (>50 days) across all xenografts. One relapse in each of two models either regressed spontaneously with re-emergence of PC-CAR T cells or after re-treatment. Responses were durable >120 days, whereas PHOX2B+ non-HLA-A24:02/23:01 controls showed progressive disease. Expansion peaked at day 14 with CD8⁺ effector-memory predominance transitioning to central-memory persistence. PHOX2B-directed PC-CAR T cells show stringent specificity, potent cytotoxicity, and curative efficacy in preclinical patient-derived NB models. These IND-enabling data supported the ongoing first-in-human/child Phase 1 trial (NCT07007117), validating PHOX2B as a lineage-restricted immunotherapeutic target in high-risk neuroblastoma.