Construction And Characterization Of A Novel Pro-Antibody Against Epidermal Growth Factor Receptor

Objective:Panitumumab, as a commercially available antibody, is an effective anti-tumor therapeutic against epidermal growth factor receptor (EGFR), although it exerts weak antibody-dependent cell-mediated cytotoxicity (ADCC) activity owing to its IgG2 nature. Therefore we engineered panitumumab by grafting its variable region into IgG1 backbone. The newly generated antibody was denoted as Pan. To reduce on-target toxicities associated with EGFR inhibitors and improve the safety profile of Pan, we then constructed pro-antibody termed Pan-P, which could be activated by urokinase-type plasminogen activator (uPA), based on Pan by utilizing ProbodyTM technology. In conclusion, we report here the generation and characterization of Pan-P with enhanced anti-tumor activity and selectively activated potency that was developed for improving the therapeutic index.Methods:In the present study, we firstly developed a novel anti-EGFR antibody, termed Pan, by grafting the variable region of panitumumab into Fc domain of IgG1 subclass. Then Pan was produced in CHO cell line. After purified with affinity-chromatography, Pan was characterized as follows. First of all, SDS-PAGE was utilized to evaluate the purity and molecular weight. Then affinity and antigen-binding ability were determined by Biacore and ELISA. CCK-8 assay was utilized to determine the inhibitory activity of Pan on proliferation of A431 cells. Furthermore, N-glycosylation species percentage of Pan was determined by LC-MS and ADCC activity was evaluated by ADCC-reporter bioassay. At last, in vivo therapeutic effect of Pan was tested in A431 tumor-bearing nude mice.Next, we engineered Pan-P that may enhance target specificity. The molecular modification included added blocking peptide, linker peptide and substrate peptide for uPA at N-terminal of light chain of Pan. The engineered pro-antibody, Pan-P was characterized as follows. Firstly, SDS-PAGE and LC-MS were applied to evaluate the uPA-mediated cleavage process on Pan-P in vitro. Biacore, ELISA and FACS assays were then performed to analyze that Pan-P could be activated selectively by uPA. Moreover, growth-inhibitory activity of activated Pan-P was determined on both A431 and DiFi cells by CCK-8 assay. Then in situ activation of Pan-P was tested in frozen tissue samples from human colorectal cancer (CRC) patients. Meanwhile, in vivo activation of Pan-P was evaluated in BALB/c nude mice bearing A431 cells. In the end, in vivo anti-tumor activity of Pan-P was studied in mice xenografted with A431 cells.Results:We obtained Pan and analyzed its purity was above 99%. It has been determined that the affinity of Pan was approximately 7.4×10-10M that is similar to that of panitumumab. Furthermore, Pan and panitumumab were equally effective in vitro, showing significant inhibition ability on proliferation of A431 cells. Especially, the afucosylation percentage of Pan was approximately 33%, which may contribute to inducing ADCC activity. Notably, Pan prevented and inhibited tumor progression more effectively than panitumumab in both prophylactic and established tumor models. Pan-P, which was derived from Pan by using previously reported ProbodyTM techniques, could be activated selectively by uPA in vitro and in vivo. In SDS-PAGE and LC-MS assay, the site-specific cleavage of Pan-P by uPA was verified. Further, results from Biacore, ELISA and CCK-8 assays showed that Pan-P exhibited fully functional activity to a similar extent as Pan when it was selectively activated by uPA. More importantly, targeted activation and localization was observed in tumor samples from colorectal cancer (CRC) patients and tumor-bearing nude mice. Moreover, Pan-P also exhibited effective in vivo anti-tumor potency similar to Pan.Conclusion:Taken together, the data shown here evidence the enhanced anti-rumor activity and target selectivity of Pan-P which is derived from panitumumab and engineered by utilizing ProbodyTM technology. Therefore, Pan-P has the potential to provide an improved clinical benefit/risk ratio in the treatment of colorectal cancer in future.