This inorganic complex was used in a mouse immunization scheme followed by hybridoma generation and screening for antibodies directed against both the zinc tripod hapten and the metalloprotease MMP-9. Two clones, SDS3 and SDS4, were selected for detailed biophysical investigation. The antibodies bound MMP-9 at nanomolar affinity and displayed double-digit nanomolar inhibition of the catalytic activity of the enzyme. The antibodies were also found to be nonreactive against a range of metal-containing enzymes, such as carbonic anhydrase and alcohol dehydrogenase, as well as against the related MMP-1, MMP-7, and MMP-12.
An X-ray crystal structure revealed that the new antibodies possess a unique wide concave-shaped cleft with protruding light- and heavy-chain complementarity-determining regions. One of the new antibodies, SDS3, was evaluated for therapeutic utility in a model for Crohn's disease and related ulcerative colitis syndromes by testing the effect of the antibody on dextran sulfate (DSS)–induced colitis in mice (Figure 2).
While the untreated mice presented severe colon damage 13 days after DSS insult, the SDS3-treated animals had limited inflammation and a well-preserved mucosal architecture of the colon. Treatment with nonspecific antibody failed to produce a protective effect. This study represents a key milestone not only for the field of protein engineering in general, by showing that an antibody possessing a unique antigen binding architecture can be generated using metal complex mimicry approach, but also because it demonstrates that antibodies generated through this approach can in fact be useful in treating a debilitating disease.