Immunological targeting of the cancer phosphoproteome


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Mark Cobbold2, Andrew Norris4, Fiyas Mohammed1, Joy M. Polefrome4, Jie Qian4, Andrea M. De Santis4, Angela L. Zarling3, Jefferey Shabanowitz4, Benjamin E. Willcox1, Michael E. Williams5, Donald F. Hunt4, Victor H. Engelhard3

1CR-UK Institute for Cancer Studies, University of Birmingham, Birmingham, UK, 2Department of Clinical Immunology, University of Birmingham, Birmingham, UK, 3Beirne Carter Center for Immunology Research, University of Virginia, Charlottesville, VA, USA, 4Department of Chemistry, University of Virginia, Charlottesville, VA, USA, 5Division of Hematology/Oncology, University of Virginia School of Medicine, Charlottesville, VA, USA

Abstract

Immunological targeting of the cancer phosphoproteome

Background

CD8+ T cells have been identified as potent effectors of the adaptive anti-tumour immune response. So far, only a small number of tumour 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. A novel immunological approach to target the altered cancer phosphoproteome is described.

Method

Using a mass spectrometry approach phosphopeptide display was analyzed in primary haematolymphoid tumour samples and compared against normal tissue.

109 or more primary tumour cells were lysed and Class I MHC molecules extracted. MHC-bound phosphopeptides were isolated and analyzed using tandem mass spectrometry. X-ray crystallography was performed on MHC-phosphopeptide complexes. Phosphopeptide-specific T cells were generated from lab donors using a dendritic cell stimulation.

Results

88 phosphopeptides were characterized with many derived from phosphoproteins known to function in signaling cascades implicated in neoplastic transformation. Six HLA-phosphopeptide structures were resolved and demonstrate upward facing phosphate moiety directly interacting with MHC alpha helix residues yet with potential for direct recognition by the TCR. Phosphopeptide-specific primary T cell lines were also generated ex-vivo from healthy lab donors which bound HLA-phosphopeptide tetramers and recognized and killed primary tumour samples. Xenograft studies demonstrated delayed outgrowth of human lymphoma cell lines in NOD/SCID mice receiving phosphopeptide-specific T cells.

Discussion

In summary post-translationally modified peptide antigens represent distinct antigenic determinants which may overcome barriers of immune tolerance inherent with other tumour antigens. These phosphopeptides therefore represent attractive novel candidates for future cancer immunotherapy.