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

Biotechnol Bioeng. 2018 Nov;115(11):2673-2682. doi: 10.1002/bit.26814. Epub 2018 Sep 15.

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.

Keywords: FACS; MMP; epitope specificity; inhibitory antibody; proteolytic stability.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Antibodies / immunology
  • Antibodies / isolation & purification*
  • Antibody Affinity*
  • Binding Sites
  • Drug Evaluation, Preclinical / methods*
  • Epitopes / immunology*
  • Flow Cytometry / methods
  • Half-Life
  • Immunologic Factors / immunology
  • Immunologic Factors / isolation & purification*
  • Kinetics
  • Matrix Metalloproteinase 14 / immunology*
  • Matrix Metalloproteinase 14 / metabolism
  • Matrix Metalloproteinase Inhibitors / isolation & purification*
  • Mice
  • Mutagenesis
  • Protein Binding

Substances

  • Antibodies
  • Epitopes
  • Immunologic Factors
  • Matrix Metalloproteinase Inhibitors
  • Matrix Metalloproteinase 14