Blocking Monoclonal Antibodies to αVβ3 Integrin: A Unique Epitope of αVβ3 Integrin Is Present on Human Osteoclasts

Abstract

Integrins are a family of cell surface glycoproteins that promote cell adhesion. The integrin αVβ3, vitronectin receptor, is a major integrin expressed by osteoclasts. To further investigate the role of αVβ3 in cell adhesion, we generated and characterized monoclonal antibodies to αVβ3 by immunizing BALB/c mice with purified αVβ3 protein. Three monoclonal antibodies (mAbs), 9D4.9.1, 9G2.1.3, and 10C4.1.3, from a total of more than 1100 positive cultures which bound αVβ3, were characterized extensively: mAbs 9G2.1.3 and 10C4.1.3 recognize the αVβ3 complex whereas mAb 9D4.9.1 reacts with the β3-chain shared between the αVβ3 complex and gpIIbIIIa. Further epitope mapping using flow microfluorometry analysis and histochemical staining of various tissues showed that 9D4.9.1 and 10C4.1.3 recognized distinct epitopes. Ligand-binding studies using cell-bound and purified αVβ3 demonstrated that all three mAbs blocked fibrinogen binding. Vitronectin binding was blocked by mAb 9D4.9.1 and, less effectively, by mAb 10C4.1.3; mAb 9G2.1.3 was without effect. All three mAbs recognized osteoclasts from human tissues; mAb 9G2.1.3 also stained osteoclasts from a wide range of nonhuman species. Monoclonal antibodies 9D4.9.1 and 9G2.1.3 bound to a panel of cultured cell lines and various tissues. In contrast, mAb 10C4.1.3 bound only weakly or not at all to tissues expressing αVβ3 with the exception of osteoclasts. Thus, mAb 10C4.1.3 showed a very narrow tissue specificity being restricted to high-level expression on human osteoclasts.