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Screening for Cross-Reactivity and Validation of Peptide-Centric CAR-T Cells for Neuroblastoma using MHC Dextramer®
Yarmarkovich M, et al. Cross-HLA targeting of intracellular oncoproteins with peptide-centric CARs. Nature 2021. doi.org/10.1038/s41586-021-04061-6
Neuroblastoma (NB) is an aggressive paediatric cancer. Yamarkovich et al. discovered that the NB immunopeptidome is enriched with intracellular peptides derived from essential oncoproteins. These antigens cannot be targeted using traditional Chimeric Antigen Receptor (CAR)-T cells limited to surface antigens. Since NB is driven by epigenetically deregulated transcriptional networks rather than mutations, unmutated self-peptides presented on tumor cells were also included in the antigen-discovery process.
The authors engineered peptide-centric (PC)-CARs with a single-chain antibody variable fragment (scFv) region, capable of binding MHCs presenting intracellular peptides. The resulting PC-CARs induced potent tumor killing across multiple HLA alleles in NB cells in vitro, and complete tumor regression in mice (Fig. 1).
The unmutated peptide QYNPIRTTF on HLA-A*2402 encoded by PHOX2B was identified by immunopeptidomics and selected for further studies based on pMHC binding affinity expression data. Normal donor-matched CD8+ T cells were pulsed with peptide, enriched using MHC Dextramer® reagents, and incubated with pulsed dendritic cells. Expanded T cells were validated for antigen-specificity by staining with NB-specific MHC Dextramer® reagents and analyzed by flow cytometry. Candidate PC-CARs were screened with predicted cross-reactive peptides to minimize off-target effects against MHC or the normal immunopeptidome.
Staining with MHC Dextramer® showed significant cross-reactivity to the MHC in first generation PC-CARs (Fig. 2). Cross-reactive binding was later abrogated using saturation mutagenesis. Following selection via protein display, and further cross-reactivity screening, PC-CARs capable of selective binding were successfully engineered.
Fig.1: Cross-reactive binding of first-generation PC-CARs identified using MHC Dextramer®. Flow cytometry of Jurkat cells transduced with A7 CAR stained with PHOX2B MHC Dextramer® on x-axis and mismatched PBK peptide on matched HLA-A*2402 on y-axis.
- MHC Dextramer® reagents were successfully used to characterize target interaction, peptide cross-reactivity, and allele specificity of PC-CAR T cells, leading to complete tumor regression in mice.
- PC-CARs provide a roadmap to target non-immunogenic intracellular oncoproteins in cancer.
- PC-CARs recognized identical peptides on multiple HLA allotypes, expanding the patient population that could potentially benefit from PC-CAR T-cell therapy.
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