title: 7B7: a novel antibody directed against the Ku70/Ku80 heterodimer blocks invasion in pancreatic and lung cancer cells.
abstract: Development of more effective therapeutic strategies for cancers of high unmet need requires the continued discovery of disease-specific protein targets for therapeutic antibody targeting. In order to identify novel proteins associated with cancer cell invasion/metastasis, we present here an alternative to antibody targeting of cell surface proteins with an established role in invasion; our functional antibody screening approach involves the isolation and selection of MAbs that are primarily screened for their ability to inhibit tumour invasion. A clonal population of the Mia PaCa-2, a pancreatic ductal adenocarcinoma (PDAC) cell line, which displays a highly invasive phenotype, was used to generate MAbs with the objective of identifying membrane targets directly involved in cancer invasion. Selected MAb 7B7 can significantly reduce invasion in a dose-responsive manner in Mia PaCa-2 clone 3 and DLKP-M squamous lung carcinoma cells. Using immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis, the target antigen of anti-invasive antibody, 7B7, was determined to be the heterodimeric Ku antigen, Ku70/80, a core protein composed of the Ku70 and Ku80 subunits which is involved in non-homologous end-joining (NHEJ) DNA repair. RNA interference-mediated knockdown of Ku70 and Ku80 resulted in a marked decrease in the invasive capacity of Mia PaCa-2 clone 3 and DLKP-M cells, indicating that Ku70/Ku80 is functionally involved in pancreatic and lung cancer invasion. Immunohistochemical analysis demonstrated Ku70/Ku80 immunoreactivity in 37 PDAC tumours, indicating that this heterodimer is highly expressed in this aggressive cancer type. This study demonstrates that a functional MAb screening approach coupled with immunoprecipitation/proteomic analyses can be successfully applied to identify functional anti-invasive MAbs and potential novel targets for therapeutic antibody targeting.
authors.affiliation: National Institute for Cellular Biotechnology (NICB), Dublin City University, Glasnevin, Dublin 9, Ireland.
authors.forename: Dermot
authors.initials: D
authors.lastname: O'Sullivan
authors.forename: Michael
authors.initials: M
authors.lastname: Henry
authors.forename: Helena
authors.initials: H
authors.lastname: Joyce
authors.forename: Naomi
authors.initials: N
authors.lastname: Walsh
authors.forename: Edel
authors.initials: E
authors.lastname: Mc Auley
authors.forename: Paul
authors.initials: P
authors.lastname: Dowling
authors.forename: Niall
authors.initials: N
authors.lastname: Swan
authors.forename: Michael
authors.initials: M
authors.lastname: Moriarty
authors.forename: Paul
authors.initials: P
authors.lastname: Barnham
authors.forename: Martin
authors.initials: M
authors.lastname: Clynes
authors.forename: Annemarie
authors.initials: A
authors.lastname: Larkin
mesh_terms: D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D034961:Antigens, Nuclear / Q000737:chemistry / Q000276:immunology*; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007167:Immunotherapy / Q000379:methods; D000072200:Ku Autoantigen; D008175:Lung Neoplasms / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D009361:Neoplasm Invasiveness / Q000276:immunology; D010190:Pancreatic Neoplasms / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D055503:Protein Multimerization / Q000276:immunology; D040901:Proteomics; D034622:RNA Interference
chemical_list: D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen
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