35 Targeting HIF2-Driven Endogenous Retroviral Antigens for Immunotherapy in Kidney Cancer

Abstract Background Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer and remains a major clinical challenge. Although treatment options have expanded to include VEGF pathway inhibitors, immune checkpoint inhibitors (ICIs), and more recently a HIF2 inhibitor, many patients with metastatic disease eventually relapse, and overall survival rates remain below 50%. While ccRCC is considered an immunogenic tumor, the antigenic targets driving effective immune responses remain poorly understood. A hallmark of ccRCC is loss of the VHL gene, which leads to stabilization of hypoxia-inducible factor 2 (HIF2), a transcription factor that promotes tumor growth and survival. A key insight came from a patient who achieved complete remission following allogeneic stem cell transplantation (allo-SCT). Analysis revealed donor-derived T cells that recognized a peptide derived from a HIF2-induced endogenous retrovirus (ERV), specifically ERVE-4. This finding provided the first evidence that HIF2-driven ERV expression in ccRCC can generate tumor-specific epitopes capable of eliciting strong antitumor immune responses, suggesting a previously underappreciated source of immunogenic targets. Supporting this concept, clinical studies have shown that ERV expression correlates positively with responses to immunotherapy, further indicating that ERVs may serve as tumor-specific antigens in ccRCC. Building on this rationale, we used a multi-omics approach to systematically identify HIF-regulated ERVs. Methods To improve the accuracy of ERV detection and quantification, we utilized a comprehensive ERV database developed by Dr. Bradley Bernstein’s laboratory and performed stranded RNA sequencing to enable precise transcript identification. In parallel, targeted long-read DNA sequencing was used to resolve locus-specific ERV sequence variants, which are critical for accurate open reading frame (ORF) prediction and for constructing customized databases for mass spectrometry (MS) analysis. To identify peptides presented on ccRCC cells, we performed HLA immunoprecipitation using both endogenous HLA and engineered cell lines expressing tagged alleles, followed by immunopeptidomic profiling. Specifically, ccRCC cell lines were engineered to overexpress tagged common HLA alleles such as HLA-A*02:01, HLA-A*03:01 and HLA-B*07:02 to enable detailed characterization of HIF-regulated ERV-derived peptides presented by these alleles. Candidate ERV-derived peptides were prioritized based on known immunogenicity and recurrence across datasets. To evaluate T cell recognition, we performed ex vivo priming and expansion of peripheral blood mononuclear cells (PBMCs) from healthy donors, followed by single cell TCR sequencing, which enabled identification and characterization of T cell receptors (TCRs) specific for ERV-derived antigens. Results Integration of the updated ERV database with stranded RNA-seq substantially improved detection sensitivity, increasing the number of identified HIF-regulated ERVs from approximately 100 to nearly 300. Targeted long-read sequencing revealed numerous sequence variations at ERV loci relative to the reference genome, allowing more accurate ORF prediction and incorporation of sample-specific sequences into MS search databases. Immunopeptidomic profiling of engineered ccRCC cell lines expressing tagged common HLA alleles enabled the generation of a reference library of ERV-derived peptides presented across multiple prevalent HLA types. Importantly, we identified dozens of TCRs capable of recognizing these ERV-derived peptides, demonstrating their immunogenicity and reinforcing their relevance as tumor-specific targets. Conclusions These findings demonstrate that HIF2 drives the expression of tumor-specific ERVs in ccRCC that are actively translated and presented on the cell surface via HLA molecules across multiple common alleles. The ability of T cells to recognize these ERV-derived peptides highlights their promise as novel targets for immunotherapy. Collectively, this work provides a foundation for the development of ERV-targeted therapeutic strategies, including TCR-based therapies and cancer vaccines, to improve outcomes for patients with ccRCC.