Function-blocking integrin alphavbeta6 monoclonal antibodies: distinct ligand-mimetic and nonligand-mimetic classes

J Biol Chem. 2004 Apr 23;279(17):17875-87. doi: 10.1074/jbc.M312103200. Epub 2004 Feb 11.

Abstract

We have generated a panel of potent, selective monoclonal antibodies that bind human and mouse alpha(v)beta(6) integrin with high affinity (up to 15 pm). A subset of these antibodies blocked the binding of alpha(v)beta(6) to the transforming growth factor-beta1 latency-associated peptide with IC(50) values as low as 18 pm, and prevented the subsequent alpha(v)beta(6)-mediated activation of transforming growth factor-beta1. The antibodies also inhibited alpha(v)beta(6) binding to fibronectin. The blocking antibodies form two biochemical classes. One class, exemplified by the ligand-mimetic antibody 6.8G6, bound to the integrin in a divalent cation-dependent manner, contained an RGD motif or a related sequence in CDR3 of the heavy chain, was blocked by RGD-containing peptides, and was internalized by alpha(v)beta(6)-expressing cells. Despite containing an RGD sequence, 6.8G6 was specific for alpha(v)beta(6) and showed no cross-reactivity with the RGD-binding integrins alpha(v)beta(3), alpha(v)beta(8),or alpha(IIb)beta(3). The nonligand-mimetic blocking antibodies, exemplified by 6.3G9, were cation-independent, were not blocked by RGD-containing peptides, were not internalized, and did not contain RGD or related sequences. These two classes of antibody were unable to bind simultaneously to alpha(v)beta(6), suggesting that they may bind overlapping epitopes. The "ligand-mimetic" antibodies are the first to be described for alpha(v)beta(6) and resemble those described for alpha(IIb)beta(3). We also report for the first time the relative abilities of divalent cations to promote alpha(v)beta(6) binding to latency-associated peptide and to the ligand-mimetic antibodies. These antibodies should provide valuable tools to study the ligand-receptor interactions of alpha(v)beta(6) as well as the role of alpha(v)beta(6) in vivo.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal / metabolism
  • Antigens, Neoplasm / chemistry*
  • Antigens, Neoplasm / immunology
  • Binding, Competitive
  • Cations
  • Cell Adhesion
  • DNA, Complementary / metabolism
  • Dose-Response Relationship, Drug
  • Epitopes / chemistry
  • Fibronectins / chemistry
  • Fibronectins / metabolism
  • Flow Cytometry
  • Humans
  • Immunoassay
  • Inhibitory Concentration 50
  • Integrins / chemistry*
  • Integrins / immunology
  • Kinetics
  • Ligands
  • Mice
  • Mice, Transgenic
  • Molecular Sequence Data
  • NIH 3T3 Cells
  • Oligopeptides / chemistry
  • Peptides / chemistry
  • Platelet Aggregation
  • Protein Binding
  • Recombinant Proteins / chemistry
  • Sequence Homology, Amino Acid
  • Transfection
  • Transforming Growth Factor beta / metabolism
  • Transforming Growth Factor beta1

Substances

  • Antibodies, Monoclonal
  • Antigens, Neoplasm
  • Cations
  • DNA, Complementary
  • Epitopes
  • Fibronectins
  • Integrins
  • Ligands
  • Oligopeptides
  • Peptides
  • Recombinant Proteins
  • TGFB1 protein, human
  • Tgfb1 protein, mouse
  • Transforming Growth Factor beta
  • Transforming Growth Factor beta1
  • integrin alphavbeta6
  • arginyl-glycyl-aspartic acid