Antigenic Epitope Analysis Of Anti-bFGF And The Security Of VBP3 Poly Peptide Vaccine

Background & Aim:(1) Initially, three monoclonal antibody MabF7, MabF10, MabF12 of the neutralization activity had been screened in our lab, all of which can specificialy bind bFGF. Cell test showed that three monoclonal antibodies can inhibit vascular endothelial cell proliferation stimulated by bFGF, the growth of melanoma cells and induced apoptosis of melanoma cells in vitrto. And MabF7 showed significantly inhibit the growth of tumor in vivo. The antigen recognition epitope of antibody is much related to its affinity and pharmacokinetics. The research purpose is to determine antigene-epitope of the three rhbFGF Mabs.(2) In addition, the VBP3 complex peptide expressed by gene engineering in E.coli system was used to immunize C57BL/6J mouse, from which the high-titer anti-bFGF blood serum and anti-VEGF blood serum can be obtained. It was demonstrated that the VBP3 complexpeptide vaccine could effectively inhibit heterogeneous graft melanoma in vivo. In order to evaluate the security and stability of VBP3 complex peptide vaccine, we designed the local stimulation, systemic anaphylactic status and stability experiments.Methods:The analysis of antigene epitope of Mab:ELISA was used to determine the type of antigene epitope of three monoclonal antibodies; and the method of Affinity-Directed Mass Spectrometry was used to analyze antigenic epitope sequences of monoclonal antibodies. (a) For linear epitope:peptide fragments were obtained by the digestion of bFGF, and combined to monoclonal antibodies. The peptides non-binding with antibodies were removed, and the antibody binding peptides were filtrated for analysis. (b) For conformation epitope:the mixture of antigene and antibody was digested, and the non-binding peptides were removed. The antibody binding peptides were filtrated for analysis.The security evaluation of complex peptide vaccine VBP3:local stimulation reaction of subcutaneous injection of rabits and the systemic anaphylactic status of guinea pigs were observed by the injection of complex peptide vaccine VBP3.The stability evaluation of complex peptide vaccine VBP3:VBP3 complex peptides were kept for two month indifferent temperature conditions and then used to immunize Balb/c mice. The anti-bFGF antibodies titer from the mice serum was analyzed by ELISA.Results:The analysis of antigene epitope of Mab:MabF7 can recognize the continuous linear epitope of bFGF, MabF10 and MabF12 can recognize the conformation epitope of bFGF by the initial analysis of ELISA; By use of Affinity-Directed Mass Spectrometry analysis, the epitope sequence of MabF7 is located in 110-120, the epitope amino acids of MabF10 is located in five peptides of 53-60,34-44,99-108,111-119 and 136-145.The security evaluation of complex peptide vaccine VBP3:There were no local changes, like bleeding and edema in rabits, and A little inflammatory cell infiltration was observed in the injection part by histopathological examination, but there were no pathological changes, something like edema, thanatosis, edema, necrosis and so on. For the systemic anaphylactic status analysis, there were only three guinea pigs showed weak positive reaction and soon recovered, for the others, there were no hypersensitivity reaction emerged.The stability evaluation of complex peptide vaccine VBP3:After two month store, the biological activity of the complex peptide showed little decreasing in-20℃condition. But in 4℃and room temperature (25℃) condition, the biological activity of the complex peptide showed badly decreasing.Conclusions:The antigene epitope of MabF7 and MabF10 was recognized by the method of Affinity-Directed Mass Spectrometry, which demonstrated that antigene epitope of monoclonal antibody can be determined with the metod of Affinity-Directed Mass Spectrometry.According to the focal stimulation reaction of rabits and the systemic anaphylactic status of guinea pigs, The security evaluation revealed that the complex peptide vaccine VBP3 can be used safely.