Background Malignant pleural mesothelioma (MPM) is an aggressive cancer with median survival of 18 months. Due to the lack of proper tumour targets, no cellular immunotherapy is currently available for MPM. New non-ablative radiation and surgery combination therapy programs for MPM are underway at the Princess Margaret Cancer Centre in Toronto. As part of a comprehensive multiomics study of tumor microenvironment, we implemented a computational method for discovery and prioritization of endogenous retroviral protein (ERV) particles as novel antigen-specific targets in MPM.
Methods We performed ERV gene-discovery based on identification of protein-coding open-reading frames with viral or bacterial origins.1 An extended human reference genome was constructed with novel open-reading frames and single-cell RNAseq sequencing (10Xgenomics Inc.) reads of tumor samples were aligned and gene-count tables were computed. ERVs with less than ten aligned reads or present in only one patient were removed from the analysis. ERVs with transcripts overlapping GTEx Consortium database (10K samples, 56 tissues) were removed to ensure no remaining ERV was constitutively expressed in normal tissues. The ERVs were further annotated with experimentally validated T and B cell epitopes in the Immune Epitope Database (IEDB).2 BLAST searches performed to identify humongous epitope of ERVs in human pathogen. A select set of identified transcripts were validated with PCR.
Results Single-cell RNAseq expression of tumor microenvironment of 19 MPM patients was analyzed. All patients expressed two ERVs annotated in the human reference genome (ERV3–1, ERVK3–1) with another four (ERVFRD-1, ERVH48–1, ERVMER34–1, ERVW-1) expressed in at least 5 patients. We identified 400 ERVs not curated in the human reference genome with lengths of 95–3935 amino acids (1520.9525 +- 1069). From this, 224 contained at least one T-cell epitope (with presentation at MHC class-I and class-II) or B epitope (antibody binding) record in IEDB. 135 ERVs had at least one epitope with 100% homology in human pathogens. We further measured the expression (in terms of TPM) of identified genes in 2708 publicly available cell lines of 14 cancers. All 400 ERVs were shared with at least one and some with all 14 other cancers.
Conclusions We identified several ERV proteins present in MPM tumors that are prominently encoded in intronic regions of but not curated in the human reference genome. Annotation of these proteins with homologues epitopes of human pathogen and IEDB epitopes provide opportunities for prioritization of these novel targets for T-cell therapies or antibody drugs.
Nakagawa S, Takahashi MU. gEVE: a genome-based endogenous viral element database provides comprehensive viral protein-coding sequences in mammalian genomes. Database, 2016; 2016 :1–8. https://doi.org/10.1093/database/baw087
Vita R, Mahajan S, Overton JA, Dhanda SK, Martini S, Cantrell JR, Wheeler DK, Sette A, Peters B. The Immune Epitope Database (IEDB): 2018 update. Nucleic Acids Research, 2019; 47 (D1):D339-D343. https://doi.org/10.1093/nar/gky1006
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