Identification of post-translationally modified antigens using genetic code expansion

PROJECT SUMMARY Post-translational modifications (PTMs) are chemical modifications of amino acid side chains in proteins. PTMs are indispensable for biological processes and are intimately involved in numerous diseases. Post-translational modifications of proteins lead to alteration of their biochemical properties and are a crucial component of many cellular processes. Interestingly, PTMs also alter the immunogenicity of proteins by positively or negatively influencing recognition by antibodies or T cells. In particular, PTM epitopes have been shown to alter presentation of epitopes on MHC/HLA molecules and their recognition by T cells. PTMs such as deamidations, Hybrid Insulin Peptide (HIP) formation, citrullination, and phosphorylation have been known to create immunogenic epitopes across many diseases including Rheumatoid Arthritis, Type 1 Diabetes, and cancer. Despite their recognized importance, there is a lack of robust, scalable, and high-throughput approaches to interrogate PTMs as T cell antigens. We have previously developed a cell-based T cell antigen discovery approach called SABRs that can present >10,000 epitopes to T cells and identify their cognate epitopes. Using SABR libraries, we have previously identified novel HIPs as autoantigens in Type 1 Diabetes and personalized neoepitopes in melanoma. Here, we propose to build upon this platform for the identification of PTM epitopes. We will employ genetic code expansion to use orthogonal tRNA/tRNA synthetases to site-specifically incorporate PTMs in genetically-encoded SABR libraries. Our preliminary studies have demonstrated the feasibility of this approach in incorporating modified lysine and citrulline into epitopes. We aim to: 1) extend this platform to incorporate phosphorylated serine, acetylated lysine, and trimethylated lysine into SABRs; and 2) use this approach to identify citrullinated autoantigens in Type 1 Diabetes. Successful completion of this project will establish a robust, scalable, and versatile platform capable of presenting thousands of epitopes with precisely defined PTMs. These studies will pave the way for interrogating PTM epitopes at unprecedented scale, will lead to identification of neoepitopes across multiple diseases including autoimmune disorders and cancers, and enhance our mechanistic understanding of the immunological consequences of PTMs.