Enrichment and Identification of HLA-Associated Phosphopeptides for the Development of Novel Cancer Immunotherapeutics

Despite extensive research efforts, cancer remains the second leading cause of death in the United States. Current treatment options are often ineffective and result in severe side effects due to their inability to distinguish between healthy and cancerous cells. Alternatively, immunotherapy represents an ideal treatment option given the natural ability of the immune system to specifically recognize and clear malignant cells from the body. This is a result of the HLA class I processing pathway, a mechanism employed by the immune system to assess cellular health. This pathway involves the degradation of cytoplasmic proteins by the proteasome into peptides which are subsequently loaded onto HLA class I molecules. HLA:peptide complexes migrate to the cell surface for display to circulating cytotoxic T cells (CTLs) which can trigger the specific killing of cells presenting antigenic peptides. In cancer, cell signaling becomes largely dysregulated, leading to markedly increased, aberrant phosphorylation. Phosphopeptides arising from aberrantly phosphorylated proteins enter into the HLA class I processing pathway where circulating CTLs can recognize them as antigenic and mount a significant immune response against the malignant cells. Therefore, we aim to identify tumor-specific, HLA-associated phosphopeptides, specifically from hepatocellular carcinoma, for the development of novel immunotherapeutics which harness an individual’s own immune system to target and eliminate cancer.
In this dissertation, we first implement the use of STop And Go Extraction (STAGE) tips as an improved methodology for HLA-associated peptide sample desalting. Implementation of this robust and sensitive methodology allows for decreased sample losses, increased peptide recoveries, and the possibility of sample multiplexing. We then evaluate the effectiveness of alternative resins for use in immobilized metal affinity chromatography (IMAC) enrichment of phosphopeptides. By applying complementary IMAC enrichment techniques to a single hepatocellular carcinoma tumor tissue sample, we identify the largest number of phosphopeptides from a single sample in our laboratory to date. Additionally, we show that both enrichment techniques can be used to provide a more complete representation of the HLA-associated phosphopeptides present within tissue samples. We then apply our improved sample desalting methodology and complementary IMAC enrichment techniques to numerous healthy and cancerous liver tissues leading to the identification of a total of 459 HLA-associated phosphopeptides, many of which are tumor-specific. Finally, we show that these phosphopeptides are capable of eliciting an immune response in healthy donors and patients with chronic liver disease, suggesting that they are targets of cancer immune surveillance and therefore represent ideal candidates for use in the development of novel cancer immunotherapeutics.