LAUSR

Tumor-specific antigens can be targeted by T cells and cancer immunotherapies directed against these neoantigens have resulted in regression across several cancer types. Both missense mutations and gene fusions can serve as targets for cytotoxic T cells, but it is unknown whether one of these classes of mutations has unique parameters that make it more immunogenic. This study compares the immunogenicity of patient-specific missense mutations and a conserved gene fusion, ETV6/RUNX1, present in approximately 25% of pediatric B cell acute lymphoblastic leukemia cases to determine which is more effective at inducing CD8+ T cell responses. Utilizing patient HLA haplotype, mutation data obtained from whole-genome and mRNA sequencing, and the online prediction algorithm NetMHCcons, we identified several putative neoepitopes. To experimentally validate these predicted neoantigens, we performed peptide exchange assays with antigen presenting beads to evaluate the efficiency of peptide-HLA interactions and complex formation. To further investigate the immunogenicity of single nucleotide variants (SNVs) compared to the conserved gene fusion ETV6/RUNX1, we assessed peptide-HLA stability by measuring the dissociation of SNV or ETV6/RUNX1 peptides from the HLA binding groove and calculating the corresponding half-life. Higher affinity peptides would have a greater exchange rate than lower affinity peptides and would have a higher stability/half-life; these assays validated that ETV6/RUNX1 has a greater affinity/stability for HLA-A*02:01 compared to any of the patient-specific SNVs evaluated. To determine whether the higher affinity and stability of the fusion peptide was able to induce greater CD8+ T cell responses, we performed co-culture assays using peptide-pulsed artificial antigen presenting cells expressing HLA-A*02:01 and measured CD8+ T cell cytokine secretion and cytotoxicity. Interestingly, while both mutation types were able to activate CD8+ T cells, those targeting the fusion peptides displayed greater polyfunctionality. Additionally, we found a greater frequency of clonally expanded CD8+ T cells targeting fusion peptides using tetramer-binding assays. Overall, our findings suggest fusion neoantigens have greater immunogenicity than missense mutations and may provide better targets for TCR-T cell-based immunotherapies. Citation Format: Erin E. Cygan, Ravi K. Shah, Tanya Kozlik, Paul G. Thomas, Anthony E. Zamora. Conserved gene fusion ETV6/RUNX1 displays higher immunogenicity than patient-specific missense mutations within the context of HLA-A*02:01 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2992.