Development and characterisation of a novel monoclonal antibody against overexpressed integrin alpha6beta4 on human pancreatic cells

Despite major advances in the diagnosis and treatment of patients with cancer, pancreatic cancer remains one of the most aggressive and fatal cancer types. There is an urgent need for the discovery of additional therapeutic targets and the development of more effective therapeutics. Monoclonal antibody (mAb) technology is an excellent tool for the discovery of overexpressed cell surface tumour antigens, and antibodies generated may have significant diagnostic and therapeutic value. Here, we report the generation and characterisation of a novel antibody KU44.71 against the human ductal pancreatic adenocarcinoma metastasised to the liver cell line CFPAC-1, using hybridoma technology. Following screening, mAb KU44.71 which was directed against overexpressed cell surface antigens on pancreatic cancer cells was selected and purified by affinity chromatography. Further characterisation of the mAb was performed by ELISA, flow cytometry, cell proliferation assay, internalisation studies, immunoprecipitation and mass spectrometry, western blotting, and immunohistochemistry. We discovered that KU44.71 recognises the external domain of integrin alpha6beta4 that is overexpressed by varying amounts in pancreatic cancer cell lines, derived from different sources including primary pancreatic cancer, liver metastasis, ascites and lymph node metastasis. Treatment with the naked antibody did not affect growth of alpha6beta4 expressing pancreatic cancer cells. In contrast, exposure to anti-alpha6beta4 KU44.71 induced internalisation of integrin alpha6beta4 in pancreatic cancer cells. Furthermore, KU44.71 was found to be effective in detecting alpha6beta4 expression by western blot and immunohistochemistry. Our results suggest that mAb KU44.71 is an useful tool for studying the role of alpha6beta43 in the complex biology of human cancer, and for investigating the relative expression, prognostic significance, and predictive value of integrin alpha6beta43 in patients with pancreatic cancer. Further studies are warranted to elucidate the therapeutic potential of this novel mAb including its humanised or drug conjugated versions in patients with alpha6beta43 overexpressing pancreatic and other type of cancer.