Preliminary Study Of The Antigenic Epitope Of Anti-human CD40 Monoclonal Antibody (5C11)

This issue was based on the human CD40 antagonist mouse monoclonal antibody (5C11) by us. The main binding sites between antigen CD40 and antibody 5C11 were obtained by the means of Computer Aided Molecular Design (CAMD) and protein spatial structure simulation technologies. Then we could infer the antigenic epitope of antibody. We used experimental methods in biology such as site-directed mutagenesis and the construction of mutational transgene cell lines to prove the reliability of computer modeling results and then to confirm the epitope. This had theoretical and potential clinical significance to research humanized antibody of CD40.Objective: Identify the antigenic epitope of anti-CD40 monoclonal antibody (5C11) preliminarily by means of computer modeling and exprimental methods.Methods: Model the structures of antigen, antibody and complex respectively by using InsightⅡ. Then calculate and infer the antigen epitope of antibody. The target gene encoding full length human CD40 (wtCD40) and two site-directed mutational gene of CD40 (70muCD40 and 114muCD40) were cloned by RT-PCR and then constructed to be the recombinant vectors pIRES2-EGFP/wtCD40, pIRES2-EGFP/70muCD40 and pIRES2-EGFP/114muCD40. The recombinants were transfected into HEK293 cells with LipofectamineTM 2000 Transfection Reagent (HEK293/wtCD40, HEK293/70muCD40 and HEK293/114muCD40). The recombinant vectors were detected by RT-PCR and flow cytometry. The binding abilities among three transgene cells with 5C11 were compared by flow cytometry and Western Blot.Results: (1) We obtained the optimized complex model of 5C11 Fv fragment interacting with CD40 constructed using molecular docking. (2) The results of enzyme digestion of recombinant vectors and DNA sequencing showed the three genes wtCD40, 70muCD40 and 114muCD40 were inserted correctly into plasmid pIRES2-EGFP, the three recombinant vectors were constructed successfully. (3) RT-PCR and flow cytometry revealed that HEK293 cells have been transfected with three recombinant vectors. Comparing the mean fluorescence intensity between site-directed mutational transgene cells and wtCD40 transgene cell with 5C11, the former is lower. The result of Western blot showed that 5C11 only recogonized HEK293/wtCD40 but not HEK293/70muCD40 or HEK293/114muCD40.Conclusion: We infered that the antigenic epitope of 5C11 was 15-17, 23, 43-56, 68-80, 85, 87, 100-115; HEK293/wtCD40, HEK293/70muCD40 and HEK293/114muCD40 transgene cells were constructed successfully. NO.70 threonine and NO.114 glutamic acid of human CD40 were comfirmed as the recogonized epitope of 5C11. At the same time, we proved the reliability of computer modeling results. This will lay the foundation for the study of humanized 5C11 antibody by CAMD and experimental research in the future.