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

Abstract

Targeting effectual epitopes is essential for therapeutic antibodies to accomplish their desired biological functions. This study developed a competitive dual color fluorescence-activated cell sorting (FACS) to maturate a matrix metalloprotease 14 (MMP-14) inhibitory antibody. Epitope-specific screening was achieved by selection on MMP-14 during competition with N-terminal domain of tissue inhibitor of metalloproteinase-2 (TIMP-2) (nTIMP-2), a native inhibitor of MMP-14 binding strongly to its catalytic cleft. 3A2 variants with high potency, selectivity, and improved affinity and proteolytic stability were isolated from a random mutagenesis library. Binding kinetics indicated that the affinity improvements were mainly from slower dissociation rates. In vitro degradation tests suggested the isolated variants had half lives 6-11-fold longer than the wt. Inhibition kinetics suggested they were competitive inhibitors which showed excellent selectivity toward MMP-14 over highly homologous MMP-9. Alanine scanning revealed that they bound to the vicinity of MMP-14 catalytic cleft especially residues F204 and F260, suggesting that the desired epitope was maintained during maturation. When converted to immunoglobulin G, B3 showed 5.0 nM binding affinity and 6.5 nM inhibition potency with in vivo half-life of 4.6 days in mice. In addition to protease inhibitory antibodies, the competitive FACS described here can be applied for discovery and engineering biosimilars, and in general for other circumstances where epitope-specific modulation is needed.

Functional selection of protease inhibitory antibodies

“…In summary, this study described a high-throughput method for selecting protease inhibitory mAbs. Compared with recent technology developments such as epitope synthetic mimicry (37), convex paratope design (21), competitive phage elution (16), cytoplasmic genetic selection (38), and epitope-specific fluorescenceactivated cell sorter (39), this method directly relies on functional inhibition and offers the following advantages: 1) an exceptionally high successful rate as the ratio of inhibitors over binders (SI Appendix, Table S1); 2) exclusive selectivity against proteases of the same family ( Fig. 2 and SI Appendix, Fig.…”

Inhibitory Antibodies Designed for Matrix Metalloproteinase Modulation

“…It was suggested that Fab 3A2 targets the S1’ pocket within MMP-14 and competes with TIMP-2 and the substrate without reaching out to the catalytic zinc(II) ion [32,108]. As Fab 3A2 can be cleaved by high concentrations of MMP-14, mutations were introduced that vary in the positions adjacent to the cleavage site, which led to more stable mutants with prolonged half-life and high potency [109,110]. In a syngeneic mouse breast cancer model with fast and spontaneous metastasis, IgG 3A2 displayed significant impact on tumor growth as well as metastatic spread and proved its therapeutic potential [25].…”

Selectivity Conversion of Protease Inhibitory Antibodies

“…Our studies [ 19–21 ], consistent with others [ 22–31 ], have demonstrated the feasibility of monoclonal antibodies (mAbs) to act as highly potent and highly selective inhibitors of secreted or cell surface proteases. However, even with recent technology advancements such as epitope synthetic mimicry [ 26 ], competitive phage elution [ 25 ], convex paratope design [ 19 ], and epitope-specific FACS [ 32 ], the discovery of protease inhibitory mAbs still presents a challenge in general. This is largely because the isolated antibodies need to not only specifically bind, but also efficiently inhibit the protease of interest.…”