Development of monoclonal antibodies for the identification of novel invasion associated targets in human cancer.

Monoclonal antibodies (MAbs) have emerged as an important therapeutic modality for the treatment of cancer, due to their high specificity, low toxicity, and the ability to activate components of the immune system. The research carried out in this thesis aims to identify novel antigens associated with cancer invasion, that could form the basis of anti-invasive therapeutic targets, through the generation of MAbs directed against the highly invasive MiaPaCa-2 clone 3 pancreatic cell line, and the MDA-MB-435-SF breast cancer cell line. Two MAbs were identified that could successfully block cancer invasion in vitro. MAb 7B7 G5 (2) significantly reduced invasion in the MiaPaCa-2 clone 3 pancreatic cell line; the SKBR-3 and MDA-MB-231 breast cancer cell lines; the DLKP-M and H1299 lung cancer cell lines; the SNB-19 glioma cell line and the HCT-116 colon cancer cell line. Inhibition of invasion was also observed in the Lox IMVI melanoma cell line, but not significantly so. This MAb also significantly decreased cell motility in the MiaPaCa-2 clone 3 cell line. MAb 9E1 24 (6) significantly decreased cell invasion in the MiaPaCa-2 clone 3, MDA-MB-231, DLKP-I, DLKP-M, H1299, C/68 and Lox IMVI cell lines. Invasion was also inhibited in the SKBR-3 cell line, but not significantly so. Surprisingly, invasion was increased in the HCT-116 colon cancer cell line, following incubation with this MAb. Other invasion-related processes were also decreased following incubation with the MAb 9E1 24 (6) and 7B7 G5 (2); MiaPaCa-2 clone 3 adhesion to fibronectin, and MMP-9 activity in the MDA-MB-231 breast cancer cell line. Immunohistochemical analysis of 9E1 24 (6) revealed that its target antigen is expressed to varying degrees in a wide range of tumour types (colon adenocarcinoma, pancreatic, breast, B-Cell lymphoma, Retinoblastoma and Glioma). Weaker staining was observed in normal colon, liver and prostate tissues. MAb 9E1 24 (6) was shown to react with a 75kDa protein band on Western blot analysis. Immunoprecipitation studies, followed by LC-MS/MS analysis, revealed that its target antigen was Annexin A6, a 75kDa cellular calcium and phospholipid binding protein. This was further corroborated by decreased expression of the reactive 9E1 24 (6) band in Annexin A6-silenced cells. siRNA silencing of Annexin A6 significantly reduced invasion in the MiaPaCa-2 clone 3 and DLKP-M cell lines, suggesting a role for this protein in the invasion process. A cross-linked immunoprecipitation approach with MAb 7B7 G5(2) revealed two bands at approximately 70 and 80kDa. LC-MS/MS analysis identified these as Ku70 and Ku80 respectively, two subunits of the Ku heterodimer, which is involved in DNA double strand break repair. siRNA silencing of these two subunits in the MiaPaCa-2 clone 3 and DLKP-M cell lines significantly reduced levels of invasion and motility, indicating that they play a role in both processes. Immunofluorescence analysis on Ku70 and Ku80 silenced MiaPaCa-2 clone 3 cells revealed a significant decrease in MAb 7B7 G5 (2) reactivity on Ku80, but not Ku70, silenced cells, suggesting that the Ku80 subunit is the main target antigen for MAb 7B7 G5 (2). The research presented in this thesis is proof of principle of how MAbs can be successfully generated that specifically target invasion-related proteins, and can block cancer invasion in vitro. The identified proteins may have the potential to become useful therapeutic targets for the treatment of invasive cancers, and could lead to the development of new drugs that specifically target metastatic cancer cells