.12 H23 Monoclonal Antibody, Chimeric Antibody Biological Activity Analysis Of Its Analog Epitope Screening

Epidermal Growth Factor Receptor (EGFR) has been implicated in in cancer initiation and development and it has been an important therapeutic target. Mouse monoclonal antibody 12H23 which can specifically recognize overexpressed EGFR and EGFR variantⅢ(EGFRvⅢ) showed its inhibitory in EGFRvⅢ-positive HCC. Chimeric antibody CH12 derived from 12H23 showed almost the same affinity to EGFRvⅢas 12H23. In this study, we would further study the biological activities of CH12 antibody.However, passive antibody immunization had serious practical limitations, like production obstacle, expensiveness, poor efficacy, limiting side effects due to passive application of the murine monoclonal antibodies. To overcome this limitation, we generated epitope mimics (mimotope) that bind to 12H23 and tested whether the peptide mimics could induce the production of similar antibodies when the isolated peptides were used to immunize the Balb/c mice.Section one Biological activities of a chimeric antibody derived from monoclonal antibody 12H23[Objective]:To examine the biological activities of chimeric antibody (CH12) derived from mouse monoclonal antibody 12H23. [Methods]:CH12 antibody was labeled with 125I. The binding affinity and biodistribution of 125I-CH12 were then performed. Antibody dependent cellular cytotoxity (ADCC) were applied to evaluate the killing effect of 12H23 to Huh7-EGFRvⅢand U87-EGFRvⅢcells. In vivo antitumor effect of CH12 antibody was also tested on nude mice bearing hepatocellular carcinoma xenografts. [Result]:CH12 has a binding affinity of 1.346 nm to Huh7-EGFRvⅢcells. CH12 can be accumulated to Huh7-EGFRvⅢxenografts after tail-vein injection into the tumor-bearing mice. In ADCC assay, 12H23 showed the killing effect on Huh7-EGFRvⅢand U87-EGFRvⅢcells but not on Huh-7 and U-87 MG cells. Importantly, CH12 antibody showed more growth inhibition effect on the growth of Huh7-EGFRvⅢ(bearing exogenous EGFRvⅢexpression) and SMMC-7721 (bearing endogenous EGFRvⅢexpression) xenograft in vivo when compared to C225 (cetuximab) antibody. [Conclusion]:Our data indicate that the chimeric antibody of 12H23 (CH12) is a promising therapeutic agent for the treatment of EGFRvⅢ-positive hepatocellular carcinoma (HCC).Section two Biopanning of mimotopes of 12H23 monoclonal antibody[Objective]:To identify the mimotopes of 12H23 (or CH12) monoclonal antibody.[Methods]:PH.D-12 phage display peptide library was applied to isolate the mimic peptides which could bind to the 12H23 monclonal antibody. We performed the Specificity ELISA and phage competitive binding assay to identify the affinity of the mimotopes binding to the 12H23 and ch806 (which also recognizes the same epitope of 12H23). [Result]:Two phages displaying peptides (WHTEILKSYPHE and LPAFFVTNQTQD) that can bind specifically to 12H23 and ch806 were isolated. The binding of the two phage peptides to 12H23 and ch806 can be competed by peptide or recombinant proteins bearing the natural epitope of 12H23 antibody. [Conclusion]: Our data indicate that we have successfully isolated two mimotopes of 12H23.Section three Immune activities of 12H23 mimotopes[Objective]:To analyze the immune activities of the 12H23 mimotopes.[Methods]:The isolated mimotopes was linked to KLH and the resulted conjugates were used to intraperitoneally immunize BALB/c mice. The sera titer was determined by ELISA. Western Blot Assays and Fluorescent immunostaining were performed to further demonstrate whether the induced antibodies could bind to EGFR overexpressed in A431 and EGFRvⅢexpressed in Huh7-EGFRvⅢcells. Antibody dependent cellular cytotoxicity assay (ADCC) was done to assess whether the antibodies induced by the mimotopes can exhibit specific lysis on Huh7-EGFRvⅢ, A431 and Huh-7 cells. [Result]:Sera from the mice immunized with the isolated mimotopes not only recognize the recombinant or synthetic 806 eptitope, but can also recognize EGFR that is overexpressed in A431 cells and EGFRvⅢexpressed in Huh7-EGFRvⅢcells, whereas sera from mice immunized with the control peptide-KLH and carrier KLH alone failed to show a similar reactivity. Furthermore, in antibody dependent cellular cytotoxicity assay (ADCC), the mimotope-induced antibodies specifically lysed Huh-7-EGFRvⅢcells but not on Huh-7 and A431 cells.[Conclusion]:Our data indicate that the isolated mimotopes reported here may potentially be used as new alternative agents for treating cancer with EGFRvⅢexpression.