Reducing proteolytic liability of a MMP-14 inhibitory antibody by site-saturation mutagenesis.

ABSTRACT: 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 potency K_I of 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.

SUBMITTER: Lee KB 

PROVIDER: S-EPMC6371627 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature