56 Identification and Prioritization of Human Endogenous Retrovirus–Derived Antigens for mRNA Vaccine Development in Renal Cell Carcinoma

Abstract Background Immune checkpoint inhibitors improve outcomes in clear cell renal cell carcinoma (ccRCC), yet most patients do not achieve durable responses due in part to insufficient tumor antigenicity. While neoantigen vaccines have shown promising early-phase results in ccRCC, their patient-specific design limits broad applicability and poses additional challenges in low mutational burden tumors, where targetable epitopes are scarce. Human endogenous retroviruses (hERVs), which comprise ∼8% of the human genome, can become aberrantly expressed in cancer and represent a source of shared, tumor-specific antigens; however, the extent to which they expand the limited antigenic landscape of not only ccRCC, but non-ccRCC subtypes, remains largely unexplored. We hypothesized that a subset of these aberrantly expressed hERVs provide a new public source of immunogenic peptides that can be prioritized as candidates for mRNA vaccine development. Methods We performed a pan-cancer (TCGA) analysis of 3,173 putative hERV loci from the Vargiu et al (Retrovirology, 2016) reference to identify hERVs upregulated across all cancer types, with a focus on kidney cancer subtypes. Candidates were prioritized using a scoring framework that enriched for hERVs with high tumor expression and minimal expression in normal tissues. From prioritized loci, six-frame translation was used to generate candidate open reading frames (ORFs), which were filtered and integrated with polysome sequencing data from RCC cell lines to identify translation-supported regions. Candidate peptides were evaluated for HLA binding using complimentary antigen prediction pipelines (e.g., HLAthena, netMHCpan) for various HLA alleles. To assess immunogenicity, we developed a multi-epitope hERV-targeting mRNA vaccine and evaluated antigen-specific immune responses in HLA-A11 transgenic mouse models. Results TCGA analysis combined with our scoring framework identified ≥25 unique hERV vaccine candidates per RCC subtype, with substantial overlap between ccRCC and papillary RCC (pRCC) and a distinct profile in chromophobe RCC (chRCC). Within ccRCC-prioritized hERV candidates, ORFs were generated via six-frame translation, filtered for canonical structure, and integrated with polysome sequencing data from three ccRCC cell lines to identify regions with strong translational support. This reduced 294,927 candidate ORFs to 805 high-confidence ORFs from 18 unique hERV loci, including recently described HIF-2α–regulated hERVs 4818 and 5875. HLA binding prediction identified numerous high-affinity candidate peptides across all HLA alleles (e.g., HLA-A*02, HLA-A*11), with some loci yielding >80 predicted binders for HLA-A*11 alone. Reanalysis of published immunopeptidomics datasets identified multiple peptides mapping to prioritized hERV loci, supporting endogenous processing and presentation. A pilot study using a multi-epitope mRNA vaccine encoding previously described hERV-derived peptides in a prophylactic setting elicited robust tetramer-positive, antigen-specific T cell responses in HLA-A11 transgenic mice. Compared with a peptide-based counterpart, mRNA vaccination generated superior responses, with approximately threefold and twofold higher frequencies of tetramer-positive CD8+ T cells in the spleen and vaccine-draining lymph nodes, respectively. Conclusions We present an integrative framework to identify translated hERV-derived antigens across RCC, expanding on prior work in ccRCC by identifying both known and novel immunogenic candidate peptides with evidence of translation and strong predicted HLA binding. mRNA vaccination using previously described hERV-derived peptides elicited robust antigen-specific CD8+ T cell responses, outperforming peptide vaccination. Our findings also provide initial insight into the hERV landscape in pRCC and chRCC, supporting further investigation. Future work will evaluate newly predicted peptides in multi-epitope mRNA vaccines and test their anti-tumor efficacy in vivo. DOD CDMRP Funding yes