Epitope Mapping for Monoclonal Antibody Reveals the Activation Mechanism for αVβ3 Integrin

Epitopes for a panel of anti-alpha V beta 3 monoclonal antibodies (mAbs) were investigated to explore the activation mechanism of alpha V beta 3 integrin. Experiments utilizing alpha V/alpha IIb domain-swapping chimeras revealed that among the nine mAbs tested, five recognized the ligand-binding beta-propeller domain and four recognized the thigh domain, which is the upper leg of the alpha V chain. Interestingly, the four mAbs included function-blocking as well as non-functional mAbs, although they bound at a distance from the ligand-binding site. The epitopes for these four mAbs were further determined using human-to-mouse alpha V chimeras. Among the four, P3G8 recognized an amino acid residue, Ser-528, located on the side of the thigh domain, while AMF-7, M9, and P2W7 all recognized a common epitope, Ser-462, that was located close to the a-genu, where integrin makes a sharp bend in the crystal structure. Fibrinogen binding studies for cells expressing wild-type alpha V beta 3 confirmed that AMF-7, M9, and P2W7 were inhibitory, while P3G8 was non-functional. However, these mAbs were all unable to block binding when alpha V beta 3 was constrained in its extended conformation. These results suggest that AMF-7, M9, and P2W7 block ligand binding allosterically by stabilizing the angle of the bend in the bent conformation. Thus, a switchblade-like movement of the integrin leg is indispensable for the affinity regulation of alpha V beta 3 integrin.