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The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction., Targeting peptides from unmutated cancer drivers that are expressed in tumours but not in normal tissues using peptide-centric chimeric antigen receptors shows potential as treatment for cancer.

Background: The MHC presents a snapshot of the intracellular proteome for surveillance by T cells, including peptides from mutated proteins (neoantigens) and nonmutated but aberrantly expressed proteins. Though peptides derived from nonmutated oncoproteins may be presented on MHC, self-antigens are not normally immunogenic to native T cells. Neuroblastoma presents a unique combination of challenges in identifying and targeting tumor-specific antigens: low mutational burden and low MHC expression. Methods and Results: Using an immunogenomic and immunopeptidomics approach in 16 human neuroblastoma samples, we identified 265 novel antigens presented on MHC and prioritized 6 (including the PHOX2B master regulator) as lead preclinical candidates based on: 1) binding affinity to common HLA alleles, 2) extent of differential gene expression, 3) lack of MHC presentation in healthy tissue, 4) biological relevance to neuroblastoma, and 5) recurrence across multiple tumors. We validated PHOX2B binding to the predicted HLA allele A24 using crystallography of the refolded peptide-MHC (pMHC) complex, and confirmed the peptide sequence using LC/MS/MS of the synthetic peptide. Upon antigen validation, we engineered CAR receptors to induce immunogenicity to self-antigens. Phage display libraries were used to pan for tumor-specific scFv's, using predicted cross-reactive pMHCs as decoys, generating candidate scFv's that were cloned into CAR constructs. We developed an algorithm to predict cross-reactivity against normal tissue pMHCs and screened CARs for cross-reactivity, prioritizing constructs with high tumor antigen affinity and low cross-reactivity. Lead CARs demonstrate complete elimination of tumor cells in less than 24 hours using 1:1 E:T ratios in neuroblastoma cells, and not in other cancer lines expressing HLA-A24 but not PHOX2B, demonstrating highly specific and potent killing. Robust CAR killing was induced by pulsing HLA-A24+/PHOX2B- melanoma cells with PHOX2B peptide but not with potential cross-reactive peptides. Finally, two lead CAR constructs induced complete regression of established neuroblastoma HLA-A24+ SKNAS xenografts, with additional murine trials ongoing. Conclusion: Neuroblastomas present a unique ligandome, including a significant number of antigens derived from lineage-restricted oncoproteins. We demonstrate proof-of-concept using scFv-based CARs to target the previously undruggable PHOX2B transcription factor in in vitro and in vivo studies. These data provide a basis for targeting non-immunogenic lineage-restricted oncoproteins using CAR T cells in neuroblastoma and other human cancers. Citation Format: Mark Yarmarkovich, John M. Warrington, Quinlen F. Marshall, Helena Shen, Wei Li, Matt Beasley, Moreno Di Marco, Stefan Stevanovic, Nikolaos G. Sgourakis, Dimiter Dimitrov, Peter Smith, John M. Maris. Discovery and CAR T targeting of lineage-restricted neuroblastoma oncoproteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1493.