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Playing pivotal roles in tumor growth and metastasis, matrix metalloproteinase‐14 (MMP‐14) is an important cancer target. Potent inhibitory Fab 3A2 with therapy‐desired high selectivity has been isolated from a synthetic antibody library carrying long CDR‐H3s. However, like many standard mechanism protease inhibitors, Fab 3A2 can be cleaved by high concentrations of MMP‐14 after extended incubation at acidic pH. Edman sequencing of generated 3A2 fragments indicated that cleavage occurred within its CDR‐H3 between residues N100h (P1) and L100i (P1’). To improve proteolytic stability of 3A2, three positions adjacent to its cleavage site (P1, P1’, and P3’) were subjected to site‐saturation mutagenesis (SSM). Mutations at P1’ (L100i) resulted in loss of inhibition function, while screening of 3A2 Fab mutants at P1 (N100h) or P3’ (A100k) positions identified four clones exhibiting improvements in both stability and inhibition potency. The majority of these mutants with improved stability were substitutions to either hydrophobic (Lue, Trp) or basic residues (Arg, Lys, His). Combinations of these beneficial mutations resulted in a double mutant N100hR/A100kR, which prolonged half‐life twofold with an inhibition potencyK_Iof 6.6 nM. Enzyme kinetics and competitive ELISA suggested that N100hR/A100kR was a competitive inhibitor overlapping its binding epitope with that of nTIMP‐2. This study demonstrated that site‐directed mutagenesis at or near the cleavage position reduced proteolytic liability of standard mechanism protease inhibitors especially inhibitory antibodies.

Matrix metalloproteinase‐12 (MMP‐12), also known as macrophage elastase, is a potent inflammatory mediator and therefore an important pharmacological target. Clinical trial failures of broad‐spectrum compound MMP inhibitors suggested that specificity is the key for a successful therapy. To provide the required selectivity, monoclonal antibody (mAb)‐based inhibitors are on the rise. However, poor production of active recombinant human MMP‐12 catalytic domain (cdMMP‐12) presented a technical hurdle for its inhibitory mAb development. We hypothesized that this problem could be solved by designing an expression‐optimized cdMMP‐12 mutant without structural disruptions at its reaction cleft and surrounding area, and thus isolated active‐site inhibitory mAbs could maintain their binding and inhibition functions toward wild‐type MMP‐12. We combined three advances in the field—PROSS algorithm for cdMMP‐12 mutant design, convex paratope antibody library construction, and functional selection for inhibitory mAbs. As a result, isolated Fab inhibitors showed nanomolar affinity and potency toward cdMMP‐12 with high selectivity and high proteolytic stability. Particularly, Fab LH11 targeted the reaction cleft of wild‐type cdMMP‐12 with 75 nM bindingK_Dand 23 nM inhibition IC₅₀. We expect that our methods can promote the development of mAbs inhibiting important proteases, many of which are recalcitrant to functional production.