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DIFFERENTIAL EXPRESSION OF HLA-A2 AND HLA-B7 PHOSPHORYLATED PEPTIDES ON CANCER AND NON-CANCER CELLS: NEW CANDIDATES FOR IMMUNOTHERAPY.
L.M. Hopkins PhD 1, J.M. Polefrone 1, A.L. Zarling PhD 2, A.M. Evans PhD 1, J. Shabanowitz PhD 1, D.F. Hunt PhD 1,3 and V.H. Engelhard PhD 2. 1 Dept of Chemistry, Univ of Virginia, Charlottesville, VA ; 2 Beirne B Carter Center & Dept of Microbiology, Univ of Virginia, Charlottesville, VA and 3 Dept of Pathology, Univ of Virginia, Charlottesville, VA .
Intracellular proteins are degraded into peptides by the ubiquitin-proteasome pathway, associated with MHC class I, and presented on the cell surface to the immune system. Since deregulated kinase activity is related to malignant transformation, MHC could present phosphorylated peptides unique to cancer. The purpose of this study is to compare phosphopeptides presented by control and cancer cells and identify those uniquely presented by cancer cells. MHC class I was immunoaffinity purified from EBV-B and cancer cell lines. Peptides were differentially labeled with stable isotopes and mixed. Phosphorylated peptides were enriched using IMAC. Peptides were gradient eluted into a Finnigan LTQ-FT or LTQ linear ion trap mass spectrometer adapted for electron transfer dissociation. HLA-A2 phosphopeptides have been detected on melanoma and ovarian cancer cells. Several phosphopeptides are unique to a specific cancer while others are common to both cancers. Source proteins for these phosphopeptides have been implicated in cancer or cell cycle deregulation. CD8+ T-cells have been generated against select peptides that lyse both peptide pulsed and cancer cells. From HLA-B7, several phosphorylated peptides have been identified. The binding motif for HLA-B7 is also a common kinase motif. As expected, the number of phosphopeptides presented by HLA-B7 exceeds that observed for HLA-A2. In summary, several phosphopeptides uniquely presented on cancer cells have been identified. Such peptides become candidates for immunotherapy or vaccines.