Abstract PR011: Immunogenic landscape and therapeutic targeting of mutant NRAS ”public” neoantigens
Authors: Etxeberria Inaki , Perera Gihan , Lyudovyk Olga , Chandran Smita , Banks Lauren , Gormally Michael , Postow Michael , Merghoub Taha , Wolchok Jedd , Greenbaum Benjamin , Baker Brian , Klebanoff Christopher 
Published: February 2025

Abstract
Neoantigens (NeoAgs) are a critical class of human cancer rejection antigens. The vast majority arise from random passenger mutations unique to a single individual, which limits the systematic study of the molecular basis of NeoAg immunogenicity in patients. In contrast, “public” NeoAg are clonally conserved epitopes derived from recurrently mutated driver genes that are shared among patients. In this study, we developed of a unique research platform to perform a comprehensive structural, biophysical, genetic, and immunogenic analysis of a family of shared NeoAgs derived from mutant NRAS Q61, the second most prevalent RAS mutation. We combined a mass-spectrometry screen, x-ray crystallography, dextramer-based T cell detection, single cell TCRa/b-sequencing, and functional immune validation assays to determine the immunogenicity of NRAS(Q61) epitopes. From our mass-spectrometry screen, we discovered that neoepitopes derived from the three most common NRAS(Q61) hotspot substitutions (R, K, and L) are naturally processed and presented in HLA-A*01, thereby generating a family of public NeoAgs. By resolving the x-ray structures of the mutant pep/HLA complexes and their wild-type counterpart, we established that each hotspot substitution “untethers” a TCR contact residue from the side wall of HLA, increasing solvent exposure of the mutated residue and facilitating immune recognition. We subsequently performed immune monitoring of peripheral blood and tumor samples from n=47 HLA-A*01+ patients with an NRAS(Q61) mutated cancer, the largest biorepository from patients who express an identical NeoAg ever assembled. Detectable NRAS public NeoAg-specific T cell responses were observed across all hotspot substitutions; however, the proportion of patients who developed a response correlated with the relative hydrophobicity of the mutant sidechains (L&gt;R&gt;K). In addition, we found that exposure to immune checkpoint blockade, but not tumor mutational burden, was significantly correlated with the detection and clonal dynamics of CD8+ T cells reactive to NRAS NeoAgs. Using single-cell sequencing, we retrieved, functionally validated, and characterized a panel of n=30 TCRs from patient samples that specifically confer recognition to cancer cells expressing NRAS Q61 public NeoAgs. Each of these TCRs display high functional-avidity and function in a CD8 co-receptor independent manner. Moreover, a subset of TCRs demonstrate therapeutic “cross-protection” in vitro and in vivo towards multiple NRAS Q61 mutated variants, allowing a single receptor to provide therapeutic coverage for &gt;90% of NRAS mutations. Importantly, cross-protective TCRs were not more likely to be cross-reactive to normal human proteins compared with TCRs that recognize a single NRAS hotspot substitution. Together, these findings reveal that NRAS mutations give rise to immunogenic public NeoAgs that can be studied across patients. Further, our data helps establish generalizable principles regarding NeoAg immunogenicity while also providing a therapeutically actionable target for a TCR-based approach.
Citation Format: Inaki Etxeberria, Gihan Perera, Olga Lyudovyk, Smita Chandran, Lauren Banks, Michael Gormally, Michael Postow, Taha Merghoub, Jedd Wolchok, Benjamin Greenbaum, Brian Baker, Christopher Klebanoff. Immunogenic landscape and therapeutic targeting of mutant NRAS ”public” neoantigens [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr PR011.