Rational Engineering of a Minimized Immune Inhibitor with Unique Triple-Targeting Properties

Abstract

Inadequate control of the complement system is the underlying or aggravating factor in many human diseases. While treatment options that specifically target the alternative pathway (AP) of complement activation are considered highly desirable, no such option is available in the clinic. Here we present a successful example of protein engineering, guided by structural insight on the complement regulator factor H (FH), yielding a novel complement-targeted therapeutic (mini-FH) with clinical potential. Despite a 70% reduction in size, mini-FH retained and in some respects exceeded the regulatory activity and cell surface-recognition properties of its parent protein FH, including the recently described recognition of sites of oxidative stress. Importantly, the chosen design extended the functional spectrum of the inhibitor, as mini-FH showed increased binding to the surface-bound opsonins iC3b and C3dg when compared to FH. Thus, mini-FH is equipped with a unique and clinically valuable triple-targeting profile towards diseased host cells, through its binding to sites of ongoing complement activation, markers of oxidative damage, and host surface-specific polyanions. When assessed in a clinically relevant AP-mediated disease model of paroxysmal nocturnal hemoglobinuria, mini-FH largely outperformed factor H and indicated advantages over clinically evaluated AP inhibitors. Thus, the rational engineering of a streamlined FH construct not only provided insight into the function of a key complement regulator but also yielded a novel inhibitor that combines a triple targeting approach with high AP-specific inhibitory activity (IC50 ~ 40 nM), which may pave the way towards new options for the treatment of complement-mediated diseases.

Incomplete inhibition by eculizumab: mechanistic evidence for residual C5 activity during strong complement activation

“…This is consistent with previous data showing that the C3-opsonintargeting inhibitors mini-FH and TT30 efficiently control AP activation in several assays. 38,44,45,49,50 As observed for the double inhibition of C5 in NHS (above), addition of coversin or PAS-coversin to the patientderived, eculizumab-containing serum completely stopped the residual hemolysis if compared with eculizumab alone. We verified that the patient sera indeed contained an excess amount of eculizumab over C5 ( Figure 1F).…”

“…Immobilization of 800 response units (RUs) of C3b onto the carboxymethyldextran surface of a sensor chip via standard amine coupling enabled binding of mini-FH ( Figure 3A) as expected. 38,45 In contrast, no binding of C5 to amine coupled C3b was observed. However, when additional 900 RUs of C3b molecules were immobilized in a more physiological manner employing onchip assembled C3 convertases, C5 did efficiently adhere to such "physiologically" immobilized C3b in a concentration-dependent way ( Figure 3B).…”

“…As a control, the minimized, engineered version of the natural AP-specific regulator FH, mini-FH, was included; mini-FH regulates the complement cascade upstream of C5 activation and inhibits at the level of activated C3. 38 All inhibitors showed a clear concentration-dependent inhibition of hemolysis, but only mini-FH inhibited lysis completely, while all C5 inhibitors in the assay reached a plateau of background lysis and failed to completely inhibit hemolysis, even at higher inhibitor concentrations ( Figure 1B). Of note, the level of background hemolysis was different for the C5 inhibitors.…”

“…The minimized engineered version of the natural AP regulator FH, mini-FH, and the C-terminal FH fragment, FH18-20, which harbors the FH host surface recognition patch, were produced as described previously. 38,39 Hemolysis and bacterial lysis assays CP activity was measured with a standard heterologous hemolysis assay using antibody-sensitized sheep erythrocytes (shRBCs). 40 Alternatively, a very sensitive homologous assay with antibody-sensitized PNH-RBCs was adapted from the procedure of Rosse.…”

Complement inhibitors to treat IgM-mediated autoimmune hemolysis

“…Mini-FH has high affinity for C3b and C3d and showed better inhibitory function in vitro compared to native FH. 56,57 In addition, as with eculizumab, safety issues still have to be defined with regard to infectious complications. A more specific approach to inhibit complement activation via the classical pathway on the level of C3 has been described by Yazdanbaksh and co-workers.…”