Generation of inhibitory monoclonal antibodies targeting matrix metalloproteinase-14 by motif grafting and CDR optimization

Abstract

Matrix metalloproteinase-14 (MMP-14) plays important roles in cancer metastasis, and the failures of broad-spectrum MMP compound inhibitors in clinical trials suggested selectivity is critical. By grafting an MMP-14 specific inhibition motif into complementarity determining region (CDR)-H3 of antibody scaffolds and optimizing other CDRs and the sequences that flank CDR-H3, we isolated a Fab 1F8 showing a binding affinity of 8.3 nM with >1000-fold enhancement on inhibition potency compared to the peptide inhibitor. Yeast surface display and fluorescence-activated cell sorting results indicated that 1F8 was highly selective to MMP-14 and competed with TIMP-2 on binding to the catalytic domain of MMP-14. Converting a low-affinity peptide inhibitor into a high potency antibody, the described methods can be used to develop other inhibitory antibodies of therapeutic significance.

Epitope‐specific affinity maturation improved stability of potent protease inhibitory antibodies

“…However, the high similarity of protein folding and catalytic chemistry among MMP family members presents a daunting challenge for the generation of highly selective compound inhibitors (Turk, ). Our studies (Lopez, Nam, Kaihara, Mustafa & Ge, ; Nam, Fang, Rodriguez, Lopez, & Ge, ; Nam, Rodriguez, Remacle, Strongin, & Ge, ), among others (Appleby et al, ; Devy et al, ; Ling et al, ; Udi et al, ), demonstrated the feasibility that antibody‐based inhibitors could exhibit the desired high selectivity. Particularly, Fab3A2 with 4.8 nM affinity, 9.7 nM potency, and high selectivity toward MMP‐14 was isolated from a library containing ultralong CDR H3s (Nam et al, ).…”

“…Our studies (Lopez, Nam, Kaihara, Mustafa & Ge, 2017;Nam, Fang, Rodriguez, Lopez, & Ge, 2017;Nam, Rodriguez, Remacle, Strongin, & Ge, 2016), among others (Appleby et al, 2017;Devy et al, 2009;Ling et al, 2017;Udi et al, 2015), demonstrated the feasibility that antibody-based inhibitors could exhibit the desired high selectivity.…”

“…It is possible that through conventional affinity maturation, the epitope can migrate to a region, which interferes less with the catalytic pocket, resulting in reduced inhibition potency. By conjugating MMP‐14 and its native inhibitor TIMP‐2 with different fluorescent dyes, we have demonstrated that dual color FACS can distinguish inhibitory clones from noninhibitory clones (Nam et al, ). This study further develops this method to improve the proteolytic stability of 3A2, while avoiding unwanted epitope drift and retaining inhibition potency.…”

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

“…active‐site zinc‐chelating hydroxamates, have been tested in clinical trials but all failed due to low overall efficacy and severe side effects such as musculoskeletal pain and inflammation caused by poor selectivity . Compared to small molecule inhibitors, monoclonal antibodies (mAbs) usually render exclusive specificity, and have emerged as a promising alternative for MMP inhibition . In our previous study, a panel of Fabs inhibiting MMP‐14 was isolated from a phage displayed synthetic human antibody library carrying long CDR‐H3s .…”

“…17 Compared to small molecule inhibitors, monoclonal antibodies (mAbs) usually render exclusive specificity, and have emerged as a promising alternative for MMP inhibition. [19][20][21][22][23][24][25][26] In our previous study, a panel of Fabs inhibiting MMP-14 was isolated from a phage displayed synthetic human antibody library carrying long CDR-H3s. 27 Particularly, Fab 3A2 exhibited nM potency competitive inhibition toward MMP-14 with no reactivity with MMP-2 or -9.…”