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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.

T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have demonstrated impressive activity against relapsed or refractory B cell cancers yet fail to induce durable remissions for nearly half of patients treated. Enhancing the efficacy of this therapy requires detailed understanding of the molecular circuitry that restrains CAR-driven anti-tumor T cell function. We developed and validated an in vitro model that drives T cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains, central components of CAR structure and function, contribute to T cell failure. We found that chronic activation of CD28-based CARs results in activation of classical T cell exhaustion programs and development of dysfunctional cells that bear the hallmarks of exhaustion. In contrast, 41BB-based CARs activate a divergent molecular program and direct differentiation of T cells into a novel cell state. Interrogation of CAR T cells from a patient with progressive lymphoma confirmed activation of this novel program in a failing clinical product. Further, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is directly responsible for impairing CAR T cell function. These findings identify that costimulatory domains are critical regulators of CAR-driven T cell failure and that targeted interventions are required to overcome costimulation-dependent dysfunctional programs.

Summary Here we propose a SARS-CoV-2 vaccine design concept based on identification of highly conserved regions of the viral genome and newly acquired adaptations, both predicted to generate epitopes presented on MHC class I and II across the vast majority of the population. We further prioritize genomic regions that generate highly dissimilar peptides from the human proteome, and are also predicted to produce B cell epitopes. We propose sixty-five 33mer peptide sequences, a subset of which can be tested using DNA or mRNA delivery strategies. These include peptides that are contained within evolutionarily divergent regions of the spike protein reported to increase infectivity through increased binding to the ACE2 receptor and within a newly evolved furin cleavage site thought to increase membrane fusion. Validation and implementation of this vaccine concept could specifically target specific vulnerabilities of SARS-CoV-2 and should engage a robust adaptive immune response in the vast majority of the population., Graphical Abstract, Highlights • Selecting optimal epitopes is essential for vaccine safety and efficacy • We report 65 vaccine peptides predicted to drive long-term immunity in most people • Epitopes contain domains conserved in 15 coronaviruses and newly evolved SARS2 regions • Epitopes can be used to generate B and/or T cell vaccines (RNA and DNA), Yarmarkovich et al. report SARS-CoV-2 peptides for use in multi-epitope vaccines. These peptides are predicted to activate CD4 and CD8 T cells, are highly dissimilar from the self-proteome, and are conserved across 15 related coronaviruses. Presented epitopes are expected to drive long-term immunity in the majority of the population.

Here we propose a vaccination strategy for SARS-CoV-2 based on identification of both highly conserved regions of the virus and newly acquired adaptations that are presented by MHC class I and II across the vast majority of the population, are highly dissimilar from the human proteome, and are predicted B cell epitopes. We present 65 peptide sequences that we expect to result in a safe and effective vaccine which can be rapidly tested in DNA, mRNA, or synthetic peptide constructs. These include epitopes that are contained within evolutionarily divergent regions of the spike protein reported to increase infectivity through increased binding to the ACE2 receptor, and within a novel furin cleavage site thought to increase membrane fusion. This vaccination strategy specifically targets unique vulnerabilities of SARS-CoV-2 and should engage a robust adaptive immune response in the vast majority of the human population. Funding: This work was supported by a St. Baldrick's-Stand Up To Cancer Pediatric Dream Team Translational Research Grant (SU2C-AACR-DT2727) and the Beau Biden Cancer Moonshot Pediatric Immunotherapy Discovery and Development Networ (NCI Grant U54 CA232568). Stand Up To Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research. This work was also supported by NIH R35 CA220500 and the Giulio D'Angio Endowed Chair and the Quod Erat Demonstrandum (QED) program at the Science Center in Philadelphia.

Chimeric antigen receptor (CAR) T cells have revolutionized the management of B cell malignancies. These synthetic molecules are composed of peptide fragments from several distinct immune cell proteins and link highly-specific antigen recognition with potent T cell activation. Despite impressive results in many, less than half of patients treated will achieve durable remission after CAR therapy. Recent studies have identified the central role that each structural component of the CAR molecule plays in regulating T cell function. Significant effort has been dedicated to exploring strategies to improve the design of CARs themselves or integrate their activity with other regulatory circuits to enable more precise function. In this review, we will summarize recent pre-clinical and clinical studies that have evaluated novel CAR design formats.

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.

The current SARS-CoV-2 pandemic has precipitated an urgent need for a safe and effective vaccine to be developed and deployed in a highly accelerated timeframe as compared to standard vaccine development processes. Upfront selection of epitopes most likely to induce a safe and effective immune response can accelerate these efforts. Optimally designed vaccines maximize immunogenicity towards regions of proteins that contribute most to protective immunity, while minimizing the antigenic load contributed by unnecessary protein domains that may result in autoimmunity, reactogenicity, or enhanced infectivity. Adopting tools developed for population-scale characterization of HLA presentation of tumor antigens and cross-reactivity of TCRs with tumor self-antigens, we have generated an immunogenicity map of SARS-CoV-2 to inform vaccine design based on analyses across five parameters: 1) stimulation of CD4 and CD8 T cells; 2) immunogenicity across the majority of human HLA alleles; 3) targeting both evolutionarily conserved regions, as well as newly divergent regions of the virus that increase infectivity; 4) targeting linear and conformational B-cell epitopes; and 5) targeting viral regions with the highest degree of dissimilarity to the self-immunopeptidome such as to maximize safety and immunogenicity. Using these analyses, we have generated 11 SARS-CoV-2 vaccine constructs optimized for long-term immunity across the majority of the population. These constructs contain combinations of epitopes selected from our analysis such as to drive affinity-enhanced memory response in combination with current spike protein vaccine strategies, for use as T-cell vaccines, and a stand-alone vaccine designed to drive memory B- and T-cell responses in the majority of the population. Epitopes were optimized using our immunogenicity algorithm to minimize immunogenicity across the junctions between epitopes and cloned into pVAX vectors, using signal peptides targeting the lysosome, ER, and cytoplasmic secretion, such as to promote presentation to CD4, CD8, and B cells, respectively. Finally, we describe methods for identifying immunodominant epitopes arising from vaccination using barcoded, multiplexed tetramers. Vaccine constructs are currently undergoing testing in transgenic mice expressing human HLA-A2. We expect that these constructs will help drive long-term immunity across the population, targeting conserved regions across multiple coronaviruses. Citation Format: Mark Yarmarkovich, John M. Warrington, Alvin Farrel, John M. Maris. SARS-CoV-2 multiepitope vaccine constructs designed to drive long-term immunity in the majority of the population [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr PO-049.