7B7: a novel antibody directed against the Ku70/Ku80\ud heterodimer blocks invasion in pancreatic and lung cancer cells

摘要

Development of more effective therapeutic strategies\udfor cancers of high unmet need requires the continued\uddiscovery of disease-specific protein targets for therapeutic\udantibody targeting. In order to identify novel proteins associated\udwith cancer cell invasion/metastasis, we present here an\udalternative to antibody targeting of cell surface proteins with\udan established role in invasion; our functional antibody\udscreening approach involves the isolation and selection of\udMAbs that are primarily screened for their ability to inhibit\udtumour invasion. A clonal population of the Mia PaCa-2, a\udpancreatic ductal adenocarcinoma (PDAC) cell line, which displays a highly invasive phenotype, was used to generate\udMAbs with the objective of identifying membrane targets\uddirectly involved in cancer invasion. Selected MAb 7B7 can\udsignificantly reduce invasion in a dose-responsive manner in\udMia PaCa-2 clone 3 and DLKP-M squamous lung carcinoma\udcells. Using immunoprecipitation and liquid chromatographytandem\udmass spectrometry (LC-MS-MS) analysis, the target\udantigen of anti-invasive antibody, 7B7, was determined to be\udthe heterodimeric Ku antigen, Ku70/80, a core protein composed\udof the Ku70 and Ku80 subunits which is involved in\udnon-homologous end-joining (NHEJ) DNA repair. RNA\udinterference-mediated knockdown of Ku70 and Ku80 resulted\udin a marked decrease in the invasive capacity of Mia PaCa-2\udclone 3 and DLKP-M cells, indicating that Ku70/Ku80 is\udfunctionally involved in pancreatic and lung cancer invasion.\udImmunohistochemical analysis demonstrated Ku70/Ku80 immunoreactivity\udin 37 PDAC tumours, indicating that this\udheterodimer is highly expressed in this aggressive cancer type.\udThis study demonstrates that a functional MAb screening\udapproach coupled with immunoprecipitation/proteomic analyses\udcan be successfully applied to identify functional antiinvasive\udMAbs and potential novel targets for therapeutic\udantibody targeting.