Function-blocking Integrin αvβ6 Monoclonal Antibodies

Abstract

We have generated a panel of potent, selective monoclonal antibodies that bind human and mouse ␣ v ␤ 6 integrin with high affinity (up to 15 pM). A subset of these antibodies blocked the binding of ␣ v ␤ 6 to the transforming growth factor-␤1 latency-associated peptide with IC 50 values as low as 18 pM, and prevented the subsequent ␣ v ␤ 6 -mediated activation of transforming growth factor-␤1. The antibodies also inhibited ␣ v ␤ 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 ␣ v ␤ 6 -expressing cells. Despite containing an RGD sequence, 6.8G6 was specific for ␣ v ␤ 6 and showed no cross-reactivity with the RGD-binding integrins ␣ v ␤ 3 , ␣ v ␤ 8 , or ␣ IIb ␤ 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 ␣ v ␤ 6 , suggesting that they may bind overlapping epitopes. The "ligand-mimetic" antibodies are the first to be described for ␣ v ␤ 6 and resemble those described for ␣ IIb ␤ 3 . We also report for the first time the relative abilities of divalent cations to promote ␣ v ␤ 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 ␣ v ␤ 6 as well as the role of ␣ v ␤ 6 in vivo.

High expression of integrin β6 in association with the Rho–Rac pathway identifies a poor prognostic subgroup within HER2 amplified breast cancers

“…Primary blocking was done with 1% bovine serum albumin (BSA, Sigma, St. Loius, MO, USA) for 30 min at room temperature. Primary antibody for α v β 6, clone 6.2‐A1 (Biogen Inc., Cambridge, MA, USA) was used as previously described 26. The antibody was diluted to 0.17  μ g/mL and was applied for 1 h at room temperature.…”

αvβ6 Integrin Regulates Renal Fibrosis and Inflammation in Alport Mouse

“…23 Cells were harvested by trypsinization, washed in phosphate-buffered saline (PBS), and resuspended in flow cytometry (FC) buffer (1ϫ PBS, 2% FBS, 0.1% NaN 3 , 1 mmol/L CaCl 2 , and 1 mmol/L MgCl 2 ). Cells (0.2 ϫ 10 5 ) were incubated on ice for 1 hour in FC buffer containing purified primary antibodies in a total volume of 100 l. After incubation, cells were washed two times with ice-cold FC buffer and resuspended in 100 l of FC buffer containing 5 g/ml PE-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch) and incubated on ice for 30 minutes.…”

“…Primary antibody was diluted in PBS containing 0.1% bovine serum albumin, and tissues were incubated overnight at 4°C. For immunostaining ␤6 on mouse tissue, sections were incubated with a human/ mouse chimeric form of the anti-␣v␤6 mAb, 2A1, 23 and an anti-human biotinylated secondary antibody (PK-6103; Vector Laboratories). For immunostaining ␤6 on human tissue, sections were incubated with murine 2A1, 23 and an anti-mouse-biotinylated secondary antibody (PK-6102; Vector Laboratories).…”

“…For immunostaining ␤6 on mouse tissue, sections were incubated with a human/ mouse chimeric form of the anti-␣v␤6 mAb, 2A1, 23 and an anti-human biotinylated secondary antibody (PK-6103; Vector Laboratories). For immunostaining ␤6 on human tissue, sections were incubated with murine 2A1, 23 and an anti-mouse-biotinylated secondary antibody (PK-6102; Vector Laboratories). Avidin-biotin complex-horseradish peroxidase (Vector kit PK-6102) was applied to sections and incubated for 30 minutes at room temperature, and 3,3Ј-diaminobenzidine substrate was prepared as directed (SK-4100; Vector Laboratories) and applied to sections for 5 minutes at room temperature.…”

“…23 Human/mouse chimeric 2A1 and 3G9 cDNAs were generated from the respective parent hybridoma total RNAs with constant region primers CDL-739 for the heavy chain and CDL-738 for the light chain using the First Strand cDNA synthesis kit (Amersham/Pharmacia, Piscataway, NJ). The heavy and light chain variable regions were amplified by the polymerase chain reaction using the same 3Ј primers used for cDNA synthesis and pools of degenerate primers specific for most murine antibody gene signal sequences (sequences available on request) and Pfu DNA polymerase (Stratagene, La Jolla, CA).…”