Antiaggregatory and proangiogenic effects of a novel recombinant human dual specificity anti‐integrin antibody

Abstract

To cite this article: Escher R, Cung T, Stutz M, Haeberli A, Djonov V, Berchtold P, Hlushchuk R. Antiaggregatory and proangiogenic effects of a novel recombinant human dual specificity anti-integrin antibody. J Thromb Haemost 2009; 7: 460-9.Summary. Background: b 3 -Integrins are involved in platelet aggregation via a IIb b 3 [glycoprotein (GP)IIb-GPIIIa], and in angiogenesis via endothelial a V b 3 . Cross-reactive ligands with antiaggregatory and proangiogenic effects, both desirable in peripheral vasculopathies, have not yet been described. Objectives: In vitro and in vivo characterization of antiaggregatory and proangiogenic effects of two recombinant human Fab fragments, with emphasis on b 3 -integrins. Methods: Recombinant Fab fragments were obtained by phage display technology. Specificity, affinity and IC 50 were determined by immunodot assays, enzyme-linked immunosorbent assay (ELISA), and Scatchard plot analysis, and by means of human umbilical vein endothelial cells (HUVECs). Functional analyses included ELISA for interaction with fibrinogen binding to GPIIbGPIIIa, flow cytometry for measurement of activation parameters and competitive inhibition experiments, human platelet aggregometry, and proliferation, tube formation and the chorioallantoic membrane (CAM) assay for measurement of angiogenic effects. Results: We observed specific and highaffinity binding to an intact GPIIb-GPIIIa receptor complex of two human Fab autoantibody fragments, with no platelet activation. Dose-dependent fibrinogen binding to GPIIbGPIIIa and platelet aggregation were completely inhibited. One Fab fragment was competitively inhibited by abciximab and its murine analog monoclonal antibody (mAb) 7E3, whereas the other Fab fragment bound to cultured HUVECs, suggesting cross-reactivity with a V b 3 , and also demonstrated proangiogenic effects in tube formation and CAM assays. Conclusions: These Fab fragments are the first entirely human anti-GPIIb-GPIIIa Fab fragments with full antiaggregatory properties; furthermore, they do not activate platelets. The unique dual-specificity anti-b 3 -integrin Fab fragment may represent a new tool for the study and management of peripheral arterial vasculopathies.