Research Of Epitope And Humanization Of A Mouse Monoclonal Antibody (Z12) Neutralizing Human TNF-α

The innovation of the dissertation is to construct a new method, so as to make the experimental work based on the computer model and proceed under its estimated results. As a powerful tool, the method can be applied in the analysis of structure-function relationships of proteins (for example, humanization of murine antibodies) and the rational design of drug (for example, epitope mapping of antibodies).Human Tumor necrosis factor-α (hTNF-α) whose crystal structure had been published was the target antigen of our research. hTNF-α plays a key role in the pathogenesis of many diseases, therefore, increased level of human TNF-α (hTNF-α), became a useful target of therapy for several autoimmune diseases. Anti-hTNF-α monoclonal antibody (mAb), as an agent blocking hTNF-α avtivity, has been used for therapy of rheumatoid arthritis, septic shock, inflammatory bowel disease and so on, displaying a nice development prospect. A murine monoclonal antibody against hTNF-α, product by us named Z12, was of theory and practice research interest.Epitope mapping and humanization are two main facts in research of antibodies, and computer model contributes a lot to the work above. Firstly, the genes of Z12 variable regions were cloned, and the three-dimensional (3D) structure of Fv and the complex model of antibody acted with antigen were modeled by means of homology modeling and molecular docking. The antigenic epitope recognized by Z12 mAb was estimated by the complex model. It was confirmed through experiments to locate the epitope region subsequently that hTNF-α141-146 was the main part combined with Z12 mAb. The rationality of computer model above and estimated data deduced from it, were then validated. Design of humanized antibody for Z12 based on the computer model approved credible, involved the replacement of Ser88 with Ala in heavy chain variable region(VH) and Glu17 and LyslS with Asp and Arg, respectively in light chainvariable region(VL). Finally, the mutant genes were cloned and expressed. As the results shown by its activity research, the humanized version retained the specific binding affinity for the original mouse antibody Z12. Two aspects were mainly researched in the dissertation. One was to identify the antigenic epitope recognized by Z12 mAb, and the other was to make Z12 mAb humanized. A native inventing patent was applied for.The genes of Z12 mAb variable regions (VH and VL) obtained by our lab, have our own intellectual property right. The nucleotide sequence of VH and VL and the antigenic epitope and humanization method are different from those anti-hTNF- antibodies published.