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June 22, 2012

Sculpting the Right Epitope

Researchers discuss a new form of antibody-based therapy utilizing binding mechanisms that mimic TIMPs.

Sculpting the Right Epitope

Antibodies generated through the metal complex mimicry approach were shown to be effective in treating Crohn disease in mice. [© Markus Schnatmann - Fotolia.com]

  • ASSAY & Drug Development Technologies offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents one article that was analyzed in the "Literature Search and Review" column, a paper published in Nature Medicine titled “Antibodies targeting the catalytic zinc complex of activated matrix metalloproteinases show therapeutic potential.” Authors of the paper are Sela-Passwell N, Kikkeri R, Dym O, Rozenberg H, Margalit R, Arad-Yellin R, Eisenstein M, Brenner O, Shoham T, Danon T, Shanzer A, and Sagi I.

  • Abstract from Nature Medicine

    Endogenous tissue inhibitors of metalloproteinases (TIMPs) have key roles in regulating physiological and pathological cellular processes. Imitating the inhibitory molecular mechanisms of TIMPs while increasing selectivity has been a challenging but desired approach for antibody-based therapy. TIMPs use hybrid protein-protein interactions to form an energetic bond with the catalytic metal ion, as well as with enzyme surface residues. We used an innovative immunization strategy that exploits aspects of molecular mimicry to produce inhibitory antibodies that show TIMP like binding mechanisms toward the activated forms of gelatinases (matrix metalloproteinases 2 and 9). Specifically, we immunized mice with a synthetic molecule that mimics the conserved structure of the metalloenzyme catalytic zinc histidine complex residing within the enzyme active site. This immunization procedure yielded selective function-blocking monoclonal antibodies directed against the catalytic zinc-protein complex and enzyme surface conformational epitopes of endogenous gelatinases. The therapeutic potential of these antibodies has been demonstrated with relevant mouse models of inflammatory bowel disease. Here we propose a general experimental strategy for generating inhibitory antibodies that effectively target the in vivo activity of dysregulated metalloproteinases by mimicking the mechanism employed by TIMPs.

  • Commentary

    Targeting metalloproteases in a tissue-specific manner has been challenging due to difficulties associated with dialing in selectivity based on active enzyme conformations involving the catalytic metal center. Such catalytic metal-protein clefts have been deemed nonimmunogenic due to the poor epitope presentation and instability during B-cell surface display, thus precluding generation of highly specific antibodies. Here, a mimicry-based approach was adopted in which the catalytic zinc-containing center consisting of the zinc metal typically liganded by three histidines was mimicked by a zinc ion coordinated by a tridentate ligand incorporating three imidazoles (Figure 1).

  • Click Image To Enlarge +
    Figure 1

    Figure 1. Mimicry of the zinc-protein motif of MMPs. Based on the resolved structure of the conserved HEXXHXXGXXH zinc-binding motif (where X is any amino acid) in MMPs, a symmetrical tripodal tris-imidazol–zinc complex (Zn-tripod; ZnC36H59N11O8) was designed and synthesized as a mimicry complex of the natural tetrahedral zinc-protein motif in MMPs. (a) Left, MMP-9 catalytic domain shown in a secondary structure representation with a semitransparent surface. Right, a close-up view of the catalytic metalloprotein site. The three histidine side chains and a water molecule (not shown) bind the zinc ion (orange sphere) in a tetrahedral conformation. (b) Chemical structure of Zn-tripod in which three imidazoles and a water molecule (not shown) bind the zinc ion (sphere) in a tetrahedral conformation, thus structurally and chemically mimicking the zinc–protein interactions in MMPs.

  • Click Image To Enlarge +
    Figure 2. Prophylactic and therapeutic treatment with SDS3 protects C57BL/6J mice against DSS-induced colitis. (a–c) Clinical colitis severity, as monitored by body weight loss (a), colon length (b) and diarrhea on day 9 (left bar) and on day 14 (right bar) after DSS induction (c). (d) Representative photomicrographs of colons following prophylactic (top) and therapeutic (bottom) treatment with antibodies (control IgG [left] and SDS3 [right]). H&E stain; magnification: × 10. (e) Colonic IL-6 cytokine analysis 9 d after DSS induction. *P < 0.05 compared with mice treated with nonspecific mouse immunoglobulin control (IgG) and mice treated with vehicle (PBS). n = 10 mice per group in all panels except (e); n = 6 for IL-6 analysis. Results are expressed as mean ± s.e.m.

    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.

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