Targeting the Alternative Pathway of Complement in AMD With a Factor D Selective Antibody

Abstract

Purpose: : Beyond its role in innate host defense, accumulating genetic, preclinical, and clinical evidence suggests that complement activation through the alternative pathway (AP) contributes to the pathophysiology of age-related macular degeneration (AMD). AP activation is initiated by the binding of factor B to C3b, resulting in the formation of a C3bB pro-convertase. Factor D (fD), a rate-limiting serine protease of AP, converts the C3bB pro-convertase to the active C3bBb convertase. Here, we describe the structural and functional characteristics of FCFD4514S, a recombinant, humanized monoclonal antibody Fab fragment directed against fD (anti-fD) to inhibit AP activation.

Methods: : Human fD was expressed in Chinese hamster ovary cells and purified from cell supernatant. A complex of fD and anti-fD was purified and concentrated to 30mg/ml. Crystals were grown at 4oC using vapor diffusion method in sitting drops. A 2.4Å data set was collected at SSRL Synchrotron Source on beam line 9-2. Complement assays were performed using rabbit red blood cells and C1q-depleted human serum, or by measuring C3a des Arg formation in a C3 convertase assay.

Results: : To better understand how anti-fD inhibits AP activation at the molecular level, the structure of anti-fD in complex with fD was solved at 2.4Å resolution. Anti-fD binds to the C-terminal portion of fD. The binding interface is centered around Arg172 of fD which forms hydrogen bonds with heavy- and light-chain residues of anti-fD. Anti-fD does not alter the topology of the catalytic triad responsible for serine protease activity of fD. Rather than directly interfering with fD catalytic activity, binding studies indicate that anti-fD prevents docking of the fD molecule to the C3bB pro-convertase. As a result, proteolytic cleavage of factor B is prevented, and the formation of an active C3bBb AP convertase blocked.

Conclusions: : Structural and functional analysis explain the potent and selective blockade of AP activity by anti-fD. Based on its superior biological activity, anti-fD has been selected as our lead complement inhibitor for the potential treatment of patients with AMD.

Keywords: age-related macular degeneration • retina