Anti-TNF antibody has been an effective therapeutic strategy for the diseases related to aberrant production of
TNF-alpha, such as
rheumatoid arthritis (RA) and
Crohn's disease. The limitations of large molecule inhibitors in the
therapy of these diseases prompted the search for other potent novel
TNF-alpha antagonists. Antagonistic
peptides, derived directly or designed rationally from
complementarity-determining regions (CDRs) of
neutralizing antibodies against
TNF-alpha, have been demonstrated for their ability of inhibiting
TNF-alpha. However, their activity is very low. In this study, to increase the affinity and bioactivity, human antibody variable region was used as scaffold to display antagonistic
peptides, which were designed on the interaction between
TNF-alpha and its neutralizing
monoclonal antibody (mAb Z12). Based on the previously designed domain antibody (framework V(H)5), framework V(kappa)1 was used as light chain scaffold. On the basis of computer-guided molecular design method, a novel human scFv fragment (named as TSA1) was designed. Theoretical analysis showed that TSA1 could bind to
TNF-alpha with more hydrogen bonds and lower binding free energy than the designed domain antibody. The biological experiments demonstrated that TSA1 could directly bind with
TNF-alpha, competitively inhibit the binding of mAb Z12 to
TNF-alpha and block the binding of
TNF-alpha to
TNFR I and TNFR II. TSA1 could also inhibit TNF-induced cytotoxicity on L929 cells and TNF-mediated
NF-kappaB activation on HEK-293T cells. The bioactivity of TSA1 was significantly increased over the domain antibody. This study indicated that the framework of antibody variable region could serve as an ideal scaffold for displaying the
peptides and provides a novel strategy to design
TNF-alpha inhibitors with the ability to block the deleterious biological effects of
TNF-alpha.