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.
Peptide-Centric Chimeric Antigen Receptors (PC-CARs), which recognize oncoprotein epitopes displayed by human leukocyte antigens (HLAs) on the cell surface, offer a promising strategy for targeted cancer therapy. We have previously developed a PC-CAR targeting a neuroblastoma-associated PHOX2B peptide, leading to robust tumor cell lysis restricted by two common HLA allotypes. Here, we determine the 2.1 Angstrom structure of the PC-CAR:PHOX2B/HLA-A*24:02/beta2m complex, which reveals the basis for antigen-specific recognition through interactions with CAR complementarity-determining regions (CDRs). The PC-CAR adopts a diagonal docking mode, where interactions with both conserved and polymorphic HLA framework residues permit recognition of multiple HLA allotypes from the A9 serological cross-reactivity group, covering a combined American population frequency of up to 25.2%. Comprehensive characterization using biochemical binding assays, molecular dynamics simulations, and structural and functional analyses demonstrate that high-affinity PC-CAR recognition of cross-reactive pHLAs necessitates the presentation of a specific peptide backbone, where subtle structural adaptations of the peptide are critical for high-affinity complex formation and CAR-T cell killing. Our results provide a molecular blueprint for engineering CARs with optimal recognition of tumor-associated antigens in the context of different HLAs, while minimizing cross-reactivity with self-epitopes.
BACKGROUND High-risk neuroblastoma is a pediatric cancer arising from the developing sympathetic nervous system with a 50% relapse rate that is typically fatal. At least two subpopulations of neuroblastoma cells exist that can transdifferentiate, adrenergic and mesenchymal, the latter being more resistant to chemotherapy. Mechanisms of therapy resistance are largely unknown and the cells responsible for relapse have not been identified. METHODS We used single nucleus RNA and ATAC sequencing to identify and characterize the cells that survive chemotherapy, termed here “persister cells”, from a cohort of 20 matched diagnostic and post induction chemotherapyhigh-risk neuroblastoma patients and two patient derived xenograft (PDX) models from diagnostic tumors. Eight representative cell lines derived from neuroblastomas at diagnosis were treated with standard-of-care chemotherapy, and flow cytometry was used to sort for live cells. ML120B and CRISPR-CAS9 were used to modulate NF-kB signaling. An RNA-seq dataset of 153 high-risk neuroblastoma patients was used to determine differentially activated pathways between adrenergic and mesenchymal tumors. RESULTS Residual malignant cells in the post-chemotherapy tumor samples clustered into three main groups separated by the response to therapy. The most prevalent group of persister cells in responders (N=16/20) displayed low MYC(N) activity even in the presence of MYCN amplification. This group also demonstrated decreased expression of the adrenergic core regulatory circuit genes including PHOX2B, ISL1, HAND2, along with marked activation of TNF-alpha via NF-kB signaling. High NF-kB activity was found in a subpopulation of diagnostic cells in two chemo-refractory patients. We validated decreased expression of MYCN (2-fold decrease, p<0.0001) and PHOX2B (3.13-fold decrease, p<0.0001) in PDXs following chemotherapy. MYCN protein levels were decreased and nuclear p65 levels were increased in cell lines treated with chemotherapy. Pharmacologic inhibition of NF-kB signaling and genetic depletion of p65 resulted in increased killing (3.58-fold increase, p=0.0012) of neuroblastoma cell lines in response to chemotherapy. Finally, we classified 153 diagnostic high-risk neuroblastomas as predominantly adrenergic or mesenchymal using RNA-seq, showing that mesenchymal tumors were enriched with NF-kB pathway activation signatures. We then validated high nuclear p65 levels in 3 mesenchymal cell lines. We tested 6 adrenergic lines, 4 of which had no detectable nuclear p65. Notably, the 2 cell lines with detectable nuclear p65 were derived from diagnostic specimens that showed de novo chemotherapy resistance. CONCLUSIONS NF-kB activation is a major mediator of de novo and acquired chemotherapy resistance in high-risk neuroblastoma. We postulate that concomitant silencing of this pathway could eliminate persister cells and prevent disease relapse. Citation Format: Liron D. Grossmann, Yasin Uzun, Jarrett Lindsay, Chia-Hui Chen, Catherine Wingrove, Peng Gao, Anusha Thadi, Quinlen Marshall, Nathan M. Kendsersky, Lea Surrey, Daniel Martinez, Emily Mycek, Colleen Casey, Kateryna Krytska, Matthew Tsang, Adam Wolpaw, David N. Groff, Erin Runbeck, Jayne McDevitt, Dinh Diep, Tasleema Patel, Kathrin M. Bernt, Chi Dang, Kun Zhang, Yael P. Mosse, Kai Tan, John M. Maris. NF-kB is a master regulator of resistance to therapy in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 699.
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.
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