Major Histocompatibility Complex (MHC) Class I-associated phosphopeptides as potential targets for immunotherapy in hepatocellular carcinoma (HCC)

Despite the high incidence of HCC, being the fifth most common cancer worldwide, current therapeutic options remain limited for advanced HCC. There is, however, first evidence of clinical improvement after checkpoint blockade and it has also been shown that CD8+ T-cell responses targeting tumor antigens beneficially impact the survival of HCC patients. Immunotherapy therefore seems to be a promising approach for HCC treatment. The identification of tumor-specific antigens provides the basis for the development of an efficient targeted immunotherapy. Of note, until today only few tumor-specific antigens for HCC were identified. Promising proteins that harbor tumor antigens are so called phosphoproteins. This is because phosphoproteins are abundantly integrated in most signaling pathways and dysregulation of signaling pathways including aberrant and increased phosphorylation of proteins represents one hallmark of cancer. These emerging phosphoproteins can be degraded and the derived peptide fragments are presented via MHC-class I molecules on the cell surface of altered cells leading to T-cell recognition. We therefore claim that MHC-class I-associated phosphopeptides are attractive new tumor antigens for CD8+ T-cell-based immunotherapy of HCC.

In order to identify tumor-associated phosphopeptides, to understand their significance for tumor immunity and to evaluate whether they could serve as potential tumor-specific antigens in HCC, HCC and adjacent liver tissues were lysed and MHC-class I peptide complexes were affinity-isolated. Subsequently, phosphopeptides were enriched and sequenced, using mass spectrometry. Interestingly, most of the identified phosphopeptides were displayed by Human Leukocyte Antigen (HLA)-B7 or HLA-B27 molecules and nearly 100 HLA-B7-associated phosphopeptides were identified. HLA-class I-presented phosphopeptides were found predominantly on cirrhotic liver tissue and HCC when compared to healthy tissue. Notably, many of the underlying proteins play an important role in tumor progression or survival, making them especially interesting for immunotherapeutic strategies.

Based on their potential involvement in tumor progression, 21 phosphopeptides were further selected for immunological testing. Peripheral blood lymphocytes from healthy individuals, patients with chronic liver diseases or HCC patients were isolated and stimulated with the phosphopeptides for 7 days followed by intracellular cytokine staining. CD8+ T cell responses against this novel class of tumor antigens were comparable in quantity and quality to those seen against viral epitopes. Phosphopeptide-specific CD8+ T-cell responses were found in patients with chronic liver disease and liver cirrhosis. Our results therefore suggest that MHC-class I-presented phosphopeptides may be the target of cancer immune surveillance in liver disease and therefore represent an attractive target for future cancer immunotherapies of HCC.