Identification of highly selective MMP‐14 inhibitory Fabs by deep sequencing

Abstract

Matrix metalloproteinase (MMP)-14 is an important target for cancer treatment due to its critical roles in tumor invasion and metastasis. Previous failures of all compound-based broad-spectrum MMP inhibitors in clinical trials suggest that selectivity is the key for a successful therapy. With inherent high specificity, monoclonal antibodies (mAbs) therefore arise as attractive inhibitors able to target the particular MMP of interest. As a routine screening method, enzyme-linked immunosorbent assays (ELISA) have been applied to panned phage libraries for the isolation of mAbs inhibiting MMP-14. However, because of suboptimal growth conditions and insufficient antibody expression associated with monoclonal ELISA, a considerable number of potentially inhibitory clones might not be identified. Taking advantage of next-generation sequencing (NGS), we monitored enrichment profiles of millions of antibody clones along three rounds of phage panning, and identified 20 Fab inhibitors of MMP-14 with inhibition IC50 values of 10–4000 nM. Among these inhibitory Fabs, 15 were not found by monoclonal phage ELISA. Particularly, Fab R2C7 exhibited an inhibition potency of 100 nM with an excellent selectivity to MMP-14 over MMP-9. Inhibition kinetics and epitope mapping suggested that as a competitive inhibitor, R2C7 directly bound to the vicinity of the MMP-14 catalytic site. This study demonstrates that deep sequencing is a powerful tool to facilitate the systematic discovery of mAbs with protease inhibition functions.

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.…”

Efficient Antibody Assembly in E. coli Periplasm by Disulfide Bond Folding Factor Co-expression and Culture Optimization

“…Furthermore, the expression level at 30 °C was two-fold higher than that at 37 °C. In addition, there was a band with an apparent MW of ~35 kDa in the 37 °C culture sample (arrow in Fig 5B), likely due to a low degree of cleavage at the long CDR-H3 region, observed phenomena for certain protease inhibitory antibodies [24, 25]. But the level of truncated Fab was reduced when cultured at 30 °C.…”

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.…”