Immune Targeting of the Phosphoproteome in Lymphoma and Leukemia.

CD8⁺ T cells have been identified as potent effectors of the adaptive anti-tumor immune response. So far, only a small number of tumor antigens have been objectively linked to the oncogenic processes. Alteration in phosphorylation status of cellular proteins is a hallmark of malignant transformation and a proven important therapeutic target. Phosphorylated antigens thus represent attractive targets for immunotherapeutic development based on the hypothesis that immune escape through mutation may be more difficult without compromising the malignant phenotype. Using a mass spectrometry approach phosphopeptide display was analyzed in CLL, AML, ALL, hairy cell leukemia and mantle cell lymphoma samples and compared against normal tissue. 10⁹ or more primary tumor cells from HLA-A*0201⁺ or HLA-B*0702⁺ patients were lysed and peptides extracted from the surface HLA molecules and phosphopeptides isolated using in-line immobilized metal ion affinity chromatography and analyzed using tandem mass spectrometry using previously established methodology (Zarling A.L. et al PNAS 2006). 55 HLA-B*0702 phosphopeptides and 12 HLA-A*0201 phosphopeptides have been characterized. Many of these phosphopeptides are derived from phosphoproteins known to function in signaling cascades implicated in neoplastic transformation including MAP kinase pathway signaling proteins, c-Myc, NFAT and Bcl-11 with many representing novel phosphorylation sites. Peptide binding assays reveal IC₅₀ binding affinities ranging between 4 and 300nM within the immunogenic range for the subset of phosphopeptides investigated. To investigated the mechanism of binding X-ray crystallography was performed in combination of peptide binding assays. Six HLA-phosphopeptide structures were resolved and demonstrate upward facing phosphate moiety directly interacting with HLA alpha helix residues yet with potential for direct recognition by the TCR. Peptide binding data support contributions from phosphate group in binding to the HLA in some but not all phosphopeptides tested and these data support a novel non-canonical binding mechanism for phosphopeptides complexed with HLA-A*0201. We have used mice transgenic for HLA-A*0201 and HLA-B*0702 to demonstrate in vivo immunogenicity of a subset of these peptides in a dendritic cell-based vaccination model. T cells generated differentially recognized phosphopeptides over non-phosphopeptide counterparts. Phosphopeptide-specific primary T cell lines were also generated ex-vivo from healthy lab donors which bound HLA-phosphopeptide tetramers and recognized HLA-matched primary tumor samples. In summary this work characterizes phosphopeptides that are differentially presented on primary leukemia and lymphoma samples by class I MHC molecules. These post-translationally modified peptide antigens represent distinct antigenic determinants which may overcome barriers of immune tolerance and autoimmunity inherent with other tumor antigens. These phosphopeptides therefore represent attractive novel candidates for future cancer immunotherapy.