Abstract B28: MS-based HLA peptide discovery: Tumor neoantigens and biotherapeutic T-cell epitopes
Authors: Pisano Michael , Rizzo Paul Del , Mobley James , Houssain Kamal , Ho Bill , Jones Richard , Allen David , Amunugama Ravi , Ford Michael 
Published: June 2021

Abstract
To enable the molecular-level characterization of MHC- associated peptides for neoantigen discovery and identification of antigenic peptides on biotherapeutics, we have developed optimized workflows that allow the identification of MHC- presented epitopes. MHC Class I and II function to display peptide fragments of processed self and foreign proteins (antigens) on the cell surface for inspection by T cells (CD8+ cytotoxic T cells for Class I, and CD4+ helper T cells for Class II). Recognition of foreign antigens by T cell receptors triggers an immediate T-cell activation and expansion, resulting in the immune response. Characterizing these antigens is paramount to understanding the immunogenicity of proteins (e.g., biotherapeutic proteins) and generating tools for targeted cell destruction (e.g., cancer immunotherapy). Tumor neoantigens result from somatic mutations during oncogenesis and have immense immunotherapeutic value. Genomic sequencing of cancer tumor tissues combined with bioinformatics has enabled the identification of tumor specific mutations and in silico prediction of MHC-associated neoantigenic peptides. Often the results of the prediction algorithms are discordant with actual binding information. Neoantigen prediction by gene sequencing and in silico approaches can be complemented and further validated by empirical MHC peptide sequencing. Of interest to BioPharma is that biotherapeutics have the potential to exhibit immunogenicity, resulting in the expression of antidrug antibodies, which reduce drug load and effectiveness. Even though most protein therapeutics are humanized, single-nucleotide polymorphisms in endogenous related proteins can be the source of the immunogenic response. Thus, it is imperative to identify the biotherapeutic derived epitopes presented by MHC Class II to T helper cells with the goal of modifying the drug to remove the antigenic peptides without reducing efficacy. It also can lead to a personalized approach with multiple versions of the same drug mapped to the appropriate patient haplotype to insure efficacy without immunogenic response. The molecular-level characterization of peptides associated with MHC Class I and II requires MHC enrichment by immunoprecipitation, an unbiased peptide elution, and analysis by mass spectrometry. Here we present case studies of our recent work applying workflows for the analysis of peptides associated with Class I and Class II MHC molecules. Using human cell lines (HCT116 and Colo205), we present optimized methods for cell treatment/growth and lysis conditions, MHC immunoaffinity purification, peptide enrichment, mass spectrometry and data processing for sensitive and specific analysis of MHC-associated peptides. These assays have utility to improve prediction of neoantigen identification and mitigate risk of biotherapeutic immunogenicity.
Citation Format: Michael Pisano, Paul Del Rizzo, James Mobley, Kamal Houssain, Bill Ho, Richard Jones, David Allen, Ravi Amunugama, Michael Ford. MS-based HLA peptide discovery: Tumor neoantigens and biotherapeutic T-cell epitopes [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B28.