Structural and Functional Analysis of a C3b-specific Antibody That Selectively Inhibits the Alternative Pathway of Complement

Kenneth J Katschke,Scott Stawicki,Jianping Yin,Micah Steffek,Hongkang Xi,Lizette Sturgeon,Philip E Hass,Kelly M Loyet,Laura Deforge,Yan Wu,Menno Van Lookeren Campagne,Christian Wiesmann

doi:10.1074/jbc.m809106200

Abstract

Amplification of the complement cascade through the alternative pathway can lead to excessive inflammation. Targeting C3b, a component central to the alternative pathway of complement, provides a powerful approach to inhibit complement-mediated immune responses and tissue injury. In the present study, phage display technology was employed to generate an antibody that selectively recognizes C3b but not the non-activated molecule C3. The crystal structure of C3b in complex with a Fab fragment of this antibody (S77) illustrates the structural basis for this selectivity. Cleavage of C3 to C3b results in a plethora of structural changes within C3, including the rearrangement of macroglobulin domain 6 enabling binding of S77 to the adjacent macroglobulin domain 7 domain. S77 blocks binding of factor B to C3b inhibiting the first step in the formation of the alternative pathway C3 convertase. In addition, S77 inhibits C5 binding to C3b. This results in significantly reduced formations of anaphylatoxins and membrane-attack complexes. This study for the first time demonstrates the structural basis for complement inhibition by a C3b-selective antibody and provides insights into the molecular mechanisms of alternative pathway complement activation.

Highlights

Up to 80% of total complement activation [2]
In addition to acting as the noncatalytic subunit of the protease factor B (fB), C3b serves as the binding partner for several complement regulators and receptors, including complement factor H and complement receptor 1 (CR1)
Because epitopes exposed after the first C3 cleavage step serve as binding sites for complement receptors and modulators, we further determined if S77 inhibits binding of complement regulators factor H (fH) and CR1 to C3b and whether S77 interferes with the formation of the complement convertase

Summary

Introduction

Up to 80% of total complement activation [2]. A first step in the AP of complement activation is the cleavage of C3 into C3a and C3b. Phage-display technology was employed to generate S77, an antibody that selectively binds C3 activation products but not native C3, targeting only a small portion (0.5%) of total C3 proteins present in serum.

Results

Conclusion