Enrichment and Characterization of Post-Translationally Modified Peptides for the Development of Novel Cancer Immunotherapeutics

Phosphorylation and O-linked beta-N-acetylglucosamine (O-GlcNAc) are post-translational modifications (PTMs) that modulate cellular functions through extensive cross-talk with signaling cascades. As such, these PTMs are directly involved in the regulation of many cellular processes, and dysregulation of these pathways can result in malignancy. Changes in protein expression and/or metabolism that accompany cellular transformation can be conveyed to the immune system via the MHC class I processing pathway. This system involves intracellular proteins that get degraded to 8-10 amino acid peptides. These peptides are then loaded onto MHC-class I molecules and presented onto the cell surface. If the peptide is antigenic, circulating CD8+ T lymphocytes can bind to the MHC-peptide complex and stimulate an immune response. Since dysregulated cell signaling in malignant cells leads to aberrant enzymatic modification of proteins, we believe transformed cells will present post-translationally modified peptides unique to cancer. We aim to identify tumor-specific peptides on the cell surface that can stimulate immune action in order to develop specific cancer immunotherapeutics.
In this dissertation, we first identify HLA-associated tumor-specific phosphopeptides from the surface of esophageal tissues. We then demonstrate the ability of these peptides to generate a response in healthy individuals, suggesting that they may be excellent candidates for further study. We then go on to present the first HLA class I associated O-GlcNAcylated peptide antigens detected on the surface of tumor cells. Additionally, because these peptide antigens are estimated to be present at 0.1-1% of the total peptide pool (10-100 copies/cell), we develop an enrichment technique to help characterize the peptides. This technique allows us to sequence a total of 19 O-GlcNAcylated peptide antigens present at the attomole level. Finally, we show that healthy individuals have T-cell responses against the peptide antigens located in the central memory compartment, suggesting that the O-GlcNAcylated peptides may be targets of cancer immune surveillance and could be of importance in the design of cancer immunotherapies. Ideally, these peptide antigens will be utilized in the development of specific cancer immunotherapies such as adoptive T-cell therapy, T-cell receptor gene transfer, and/or immune-mobilizing monoclonal TCRs. Ultimately, we believe that post-translationally modified peptides are key to unlocking this potential and can be exploited in the development of cancer immunotherapies that will eradicate this devastating disease.