Characterization of the neutralizing activity of three anti-human TNF monoclonal antibodies and prediction of their TNF epitopes by molecular modeling and mutant protein approach.

The neutralizing activity of three anti-human TNF monoclonal antibodies, designated D2, E6, and F6 were investigated by three experimental systems. The results from the systems showed that all the three mAbs could neutralize TNF-mediated cytotoxicity in L929 cells, TNF-induced NF-kappaB activation in ECV304 cells, and TNF-upregulated ICAM-1 surface expression on ECV304 cells in dose-dependent manners. D2 had the highest neutralizing activity of the three mAbs, and F6 had higher level of neutralizing activity than E6. We also cloned the VH and VL cDNAs and obtained their cDNA sequences. The sequences were used in molecular modeling to establish the complex structures of TNF with variable regions of the three mAbs, respectively. In the structures, the TNF epitopes of D2, E6, and F6 were predicted at amino acids of (A109, A111-A112, C19, C21-C29, C44-C46, C66-C75, C77, C79, C90, C101, C103, C105, C114, C134-C148), (C18-C19, C21-C30, C32, C37, C43-C47, C67-C75, C83, C105-C106, C131, C135-C141), and (C21-C32, C45-C47, C65, C67-C72, C74, C81, C83, C90-C95, C105-C113, C133-C147), respectively, and the affinities of D2, E6, and F6 to TNF were predicted as -252.69, -232.83, and -299.92 kcal, respectively. Moreover, we proved the binding ability of F6 to the epitopes of amino acids of 141-146 in TNF molecule was better than that of E6, and that of D2 was the best of the three mAbs by Western blot and ELISA, in which the mutant TNF deleted the amino acids of 141-146 in TNF molecule was employed. These results make a basic foundation for selecting candidate mAbs for various purposes, such as construction of chimeric or humanized mAbs for therapeutic purpose, establishment of ELISA kits for determination of TNF, and production of affinity columns to purify TNF.