Abstract 5640: Mono-allelic immunopeptidomics data from 109 MHC-I alleles reveals variability in binding preferences and improves neoantigen prediction algorithm

Abstract Neoantigen-based biomarkers are a promising approach for stratifying patient response to immunotherapy; however, current neoantigen prediction methods are not accurate enough to optimize these biomarkers. Sequence variability in the major histocompatibility complex (MHC) leads to the presentation of diverse neoantigens to T cells, and accurately representing this diversity in neoantigen prediction is critical for improvement. Previously, we published data from 25 mono-allelic cell lines and built an associated MHC class I, pan-allelic neoantigen prediction algorithm (SHERPATM). Here, we profile an additional 84 MHC alleles including 37 that have never previously been profiled with mono-allelic immunopeptidomics, explore the impact of MHC variability on peptide binding and improve neoantigen prediction of the SHERPA algorithm. To generate the data, we stably and transiently transfected 109 different MHC alleles (43 HLA-A, 56 -B and 10 -C alleles) into independent K562 HLA-null cell lines, immunoprecipitated intact MHC complexes using a W6/32 antibody and profiled the bound peptides using LC/MS-MS. We recovered a median of 1430 peptides per allele, with yields from the transient transfections being consistently higher than the stable transfections. Nearly all alleles have a strong anchor residue in the ninth position, but the positions of the secondary anchor residue vary by gene. HLA-B showed a stronger preference for the second position while HLA-A exhibited more variability across the first, second and third positions. In addition to the 109 mono-allelic cell lines, SHERPA increases generalizability by systematically integrating an additional 104 mono-allelic and 384 multi-allelic samples with publicly available immunopeptidomics data. The 186 alleles in the resulting training dataset have an average allelic coverage of 98% across 18 different US-based ethnicities. We evaluated our updated performance on 10% held-out mono-allelic test data from multiple cell line sources. The positive predictive value (PPV) of SHERPA was markedly higher than either NetMHCPan 4.1 or MHCFlurry-2.0 (1.45 and 1.58-fold increase, respectively), with further gains when only the 37 previously unprofiled alleles were considered (1.51 and 1.79-fold increase, respectively). Furthermore, the SHERPA model was able to detect 1.38-fold more immunogenic epitopes than either other method. Finally, we performed predictions with SHERPA across millions of synthetic binding pockets and peptides to elucidate the impact of MHC variability on peptide diversity. We found a strong correlation between binding pocket positions that highly influence peptide binding and those that are evolutionarily divergent. In conclusion, we profiled 109 mono-allelic cell lines, showed key trends in MHC-associated peptides and improved the SHERPA neoantigen prediction model. Citation Format: Rachel Marty Pyke, Steven Dea, Hima Anbunathan, Charles W. Abbott, Neeraja Ravi, Jason Harris, Gabor Bartha, Sejal Desai, Rena McClory, John West, Michael P. Snyder, Richard O. Chen, Sean Michael Boyle. Mono-allelic immunopeptidomics data from 109 MHC-I alleles reveals variability in binding preferences and improves neoantigen prediction algorithm [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5640.