A Novel Antibody against Human Factor B that Blocks Formation of the C3bB Proconvertase and Inhibits Complement Activation in Disease Models

Abstract

The alternative pathway (AP) is critical for the efficient activation of complement regardless of the trigger. It is also a major player in pathogenesis, as illustrated by the long list of diseases in which AP activation contributes to pathology. Its relevance to human disease is further emphasized by the high prevalence of pathogenic inherited defects and acquired autoantibodies disrupting components and regulators of the AP C3-convertase. Because pharmacological downmodulation of the AP emerges as a broad-spectrum treatment alternative, there is a powerful interest in developing new molecules to block formation and/or activity of the AP C3-convertase. In this paper, we describe the generation of a novel mAb targeting human factor B (FB). mAb FB48.4.2, recognizing with high affinity an evolutionary-conserved epitope in the Ba fragment of FB, very efficiently inhibited formation of the AP C3-proconvertase by blocking the interaction between FB and C3b. In vitro assays using rabbit and sheep erythrocytes demonstrated that FB28.4.2 was a potent AP inhibitor that blocked complement-mediated hemolysis in several species. Using ex vivo models of disease we demonstrated that FB28.4.2 protected paroxysmal nocturnal hemoglobinuria erythrocytes from complement-mediated hemolysis and inhibited both C3 fragment and C5b-9 deposition on ADP-activated HMEC-1 cells, an experimental model for atypical hemolytic uremic syndrome. Moreover, i.v. injection of FB28.4.2 in rats blocked complement activation in rat serum and prevented the passive induction of experimental autoimmune Myasthenia gravis. As a whole, these data demonstrate the potential value of FB28.4.2 for the treatment of disorders associated with AP complement dysregulation in man and animal models.

Identification of C3b-Binding Small-Molecule Complement Inhibitors Using Cheminformatics

“…Despite this large contact area, the initial C3b/fB interaction occurs via a fast metal-independent binding event involving the Ba fragment of fB (49, 50). Importantly, a monoclonal antibody which targets an epitope on the Ba fragment is a potent inhibitor of the AP (51) and mutagenesis data suggests that C3b/Ba complex formation is driven by three relatively small interaction sites comprised of roughly 523, 111, and 181 Å 2 , respectively (35, 52). To improve the chance of identifying small molecules capable of impeding the C3b/fB interaction, we targeted a region on C3b which corresponds to a C3b/Ba interaction site formed by the CCP3 domain and centered on the C3b residues His-870, Asn-871, Pro-872, Ala-873, Lys-906, Gly-908, Leu-909, Gln-910, Glu-911, Glu-913, Arg-928, Ser-930 (UNIPROT#: P01024) (Fig.…”

Compstatin analog Cp40 inhibits complement dysregulation in vitro in C3 glomerulopathy

“…These drugs can be classified broadly as antibody-based therapeutics, which aim to control complement by blocking specific components of C3 convertase to prevent its formation and/or function ( e.g. , anti-C3b monoclonal antibody (Paixao-Cavalcante, et al, 2014); anti-FB antibody (Subias, et al, 2014); anti-properdin antibody (Pauly, et al, 2014)), and regulation-based strategies, which aim to control complement by augmenting control of the C3 convertase ( e.g. , soluble CR1 (Zhang, et al, 2013); mini-FH (directly links the regulatory and surface targeting domains of FH) (Hebecker, et al, 2013); TT30 (combines the regulatory domain of FH with the iC3b/C3d-binding domain of CR2) (Risitano, et al, 2012)).…”

Functional and structural characterization of four mouse monoclonal antibodies to complement C3 with potential therapeutic and diagnostic applications

“…The capacity of the mAbs to prevent the activation of AP on cellular surfaces was assessed with the classical AP hemolytic assay using rabbit erythrocytes as previously described , and the hemolytic assay using sheep erythrocytes to test sera from atypical uremic syndrome(aHUS) patients . For this assay, we used an aHUS‐like serum consisting of a normal human serum that has been depleted of 75% of the FH.…”

“…For the determination of binding constants (KD) of the mAbs we used a single‐cycle‐kinetics method. This approach consisted of five consecutive injections of C3b or mouse C3 (analyte) at increasing concentrations, over a surface in which the mAbs were captured using an anti‐mouse antibody as described previously . Each injection lasted for 150 s separated by a dissociation period of 180 s, during which C3‐free buffer was injected.…”