Atomic Basis for the Species-specific Inhibition of αV Integrins by Monoclonal Antibody 17E6 Is Revealed by the Crystal Structure of αVβ3 Ectodomain-17E6 Fab Complex

Bhuvaneshwari Mahalingam,Johannes F Van Agthoven,Jian-Ping Xiong,José Luis Alonso,Brian D Adair,Xianliang Rui,Saurabh Anand,Mehrdad Mehrbod,Mohammad R.K Mofrad,Christa Burger,Simon L Goodman,M Amin Arnaout

doi:10.1074/jbc.m113.546929

Abstract

The function-blocking, non-RGD-containing, and primate-specific mouse monoclonal antibody 17E6 binds the αV subfamily of integrins. 17E6 is currently in phase II clinical trials for treating cancer. To elucidate the structural basis of recognition and the molecular mechanism of inhibition, we crystallized αVβ3 ectodomain in complex with the Fab fragment of 17E6. Protein crystals grew in presence of the activating cation Mn(2+). The integrin in the complex and in solution assumed the genuflected conformation. 17E6 Fab bound exclusively to the Propeller domain of the αV subunit. At the core of αV-Fab interface were interactions involving Propeller residues Lys-203 and Gln-145, with the latter accounting for primate specificity. The Propeller residue Asp-150, which normally coordinates Arg of the ligand Arg-Gly-Asp motif, formed contacts with Arg-54 of the Fab that were expected to reduce soluble FN10 binding to cellular αVβ3 complexed with 17E6. This was confirmed in direct binding studies, suggesting that 17E6 is an allosteric inhibitor of αV integrins.

Highlights

17E6, a primate-specific mouse mAb that inhibits ␣V integrins, is in phase II trials for treating cancer
We describe the first crystal structure of an integrin ectodomain complexed to a function-blocking mAb, the Fab fragment of 17E6, in presence of Mn2ϩ, an integrinactivating cation
The integrin in the complex assumes a bent conformation and in solution, suggesting that the well established integrin activator Mn2ϩ does not necessarily switch the ␣V␤3 ectodomain to a linear conformation as has been suggested [36]. This finding is consistent with our earlier fluorescent lifetime imaging study revealing a bent 17E6-bound cellular ␣V␤3 in Mn2ϩ [15] and a cryoelectron tomography study showing no dramatic genu extension upon Mn2ϩ-induced activation of membrane-bound unliganded ␣IIb␤3 integrin [37]

Summary

Background

17E6, a primate-specific mouse mAb that inhibits ␣V integrins, is in phase II trials for treating cancer. The Propeller residue Asp-150, which normally coordinates Arg of the ligand Arg-Gly-Asp motif, formed contacts with Arg-54 of the Fab that were expected to reduce soluble FN10 binding to cellular ␣V␤3 complexed with 17E6. This was confirmed in direct binding studies, suggesting that 17E6 is an allosteric inhibitor of ␣V integrins. The new structure assumed a bent conformation, exhibited some flexibility at the ␣-genu, revealed the structural basis for 17E6 selectivity to the ␣V subunit of primates, and defined the antibody-integrin binding interface The significance of these findings is discussed

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION