Active-site MMP-selective antibody inhibitors discovered from convex paratope synthetic libraries

Abstract

Proteases are frequent pharmacological targets, and their inhibitors are valuable drugs in multiple pathologies. The catalytic mechanism and the active-site fold, however, are largely conserved among the protease classes, making the development of the selective inhibitors exceedingly challenging. In our departure from the conventional strategies, we reviewed the structure of known camelid inhibitory antibodies, which block enzyme activities via their unusually long, convex-shaped paratopes. We synthesized the human Fab antibody library (over 1.25 × 10 9 individual variants) that carried the extended, 23-to 27-residue, complementarity-determining region (CDR)-H3 segments. As a proof of principle, we used the catalytic domain of matrix metalloproteinase-14 (MMP-14), a promalignant protease and a drug target in cancer, as bait. In our screens, we identified 20 binders, of which 14 performed as potent and selective inhibitors of MMP-14 rather than as broad-specificity antagonists. Specifically, Fab 3A2 bound to MMP-14 in the vicinity of the active pocket with a high 4.8 nM affinity and was similarly efficient (9.7 nM) in inhibiting the protease cleavage activity. We suggest that the convex paratope antibody libraries described here could be readily generalized to facilitate the design of the antibody inhibitors to many additional enzymes. family members control various physiological and pathological processes. Multiple diseases are associated with altered MMP expression and aberrant proteolysis, including cancer (1), wound healing (2), inflammatory diseases (3, 4), neurological pain (5, 6), and hypertension (7). There is consensus among researchers that the individual MMPs are promising drug targets in diversified pathologies and that inhibitor specificity is required for selective and successful MMP therapies (8-10).However, achieving target specificity and selectivity in small-molecule MMP inhibitors is remarkably challenging (11,12). Because the catalytic mechanism and catalytic domain fold are conserved among the MMP/ADAM (a disintegrin and metalloproteinase)/ ADAMTS (ADAM with thrombospondin motifs) superfamily members, the available small-molecule inhibitors (most frequently, active-site zinc-chelating hydroxamates) target multiple proteinases, resulting in off-target side effects (8,(12)(13)(14). This aspect is problematic, given that some MMPs (e.g., MMP-14) are always protumorigenic, whereas some other MMPs are antitumorigenic in certain cancer microenvironments (15, 16). As a result, broad-spectrum hydroxamates failed in cancer clinical trials due to their low overall efficacy and side effects (13). Alternatively, antibody-based MMP inhibitors are emerging as both research tools and potential therapeutic agents (10, 17-21) because of (i) high affinity and specificity due to the large antigen-antibody interaction area and multiple complementarity-determining regions (CDRs), (ii) long half-life and well-defined action mechanisms, (iii) low immunogenicity and toxicity, and (iv) multiple MMPs potentially targe...

Antigen recognition by single-domain antibodies: structural latitudes and constraints

“…105 By contrast, sdAb paratopes can clearly adopt both flat 106,107 and convex 11 topologies, although possibly only inefficiently adopt concave ones. The CDR1 and CDR2 loops of V H Hs depart from the typical canonical structures of conventional antibodies (Figure 2A), potentially through somatic mutation since germline human V H and camelid V H H repertoires appear to have similar canonical structures.…”

Generation of Highly Selective MMP Antibody Inhibitors

“…Scheme that convex antibody paratope formed by an extended CDR-H3 mediates enzyme inhibition (reprinted from Ref [24]).…”

“…Typical results of MMP inhibitory Fab characterizations (reprinted from Ref [24]). (A) Binding affinity and inhibition potency measured by ELISA and FRET assays.…”

“…Here we describe the design and construction of human Fab libraries in which the long, convex-shaped, camelid-like paratopes are incorporated into the human antibody scaffold [23, 24] (Fig. 1).…”

Engineering a potent inhibitor of matriptase from the natural hepatocyte growth factor activator inhibitor type-1 (HAI-1) protein

“…2C), which was 20-fold weaker than KD1. Notably, inhibitors that demonstrate protease binding do not always demonstrate protease inhibition of the target active site (47,48). Thus, proteins were tested for functional matriptase inhibition by incubating an increasing number of yeast cells displaying KD2/1 or wild-type KD1 or KD2 with soluble matriptase and substrate.…”