Background: Unregulated plasma kallikrein proteolytic activity can result from C1-inhibitor deficiency, causing excessive and potentially fatal edema.Results: The antibody DX-2930 potently and specifically inhibits plasma kallikrein and exhibits a long plasma half-life.Conclusion: An antibody protease inhibitor can lead to potent and specific bioactivity.Significance: DX-2930 could be an effective therapeutic for the prophylactic inhibition of plasma kallikrein-mediated diseases.

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The neonatal Fc receptor FcRn plays a critical role in the trafficking of IgGs across tissue barriers and in retaining high circulating concentrations of both IgG and albumin. Although generally beneficial from an immunological perspective in maintaining IgG populations, FcRn can contribute to the pathogenesis of autoimmune disorders when an abnormal immune response targets normal biological components. We previously described a monoclonal antibody (DX-2507) that binds to FcRn with high affinity at both neutral and acidic pH, prevents the simultaneous binding of IgG, and reduces circulating IgG levels in preclinical animal models. Here, we report a 2.5 Å resolution X-ray crystal structure of an FcRn-DX-2507 Fab complex, revealing a nearly complete overlap of the IgG-Fc binding site in FcRn by complementarity-determining regions in DX-2507. This overlap explains how DX-2507 blocks IgG binding to FcRn and thereby shortens IgG half-life by preventing IgGs from recycling back into circulation. Moreover, the complex structure explains how the DX-2507 interaction is pH-insensitive unlike normal Fc interactions and how serum albumin levels are unaffected by DX-2507 binding. These structural studies could inform antibody-based therapeutic approaches for limiting the effects of IgG-mediated autoimmune disease.

Factor XII (FXII), also called Hageman Factor, is a key component of the plasma contact system. When blood is exposed to artificial surfaces or polyanionic substances, the zymogens FXII and prekallikrein (PK) undergo reciprocal activation to the proteases FXIIa and plasma kallikrein, respectively. FXIIa initiates coagulation through the intrinsic pathway by activating factor XI, while plasma kallikrein mediates generation of the potent vasodilator bradykinin. Patients lacking FXII or PK do not experience abnormal bleeding, indicating these proteins are not required for hemostasis. However, FXIIa and plasma kallikrein are required for formation of occlusive clots in animal thrombosis models, and FXIIa likely contributes to thrombus formation in humans when blood is passed through extracorporeal circuits (e.g. cardiopulmonary bypass). These observations suggest that FXIIa inhibition could be an effective antithrombotic strategy that would not have bleeding side effects associated with current approved anticoagulants. We used our human antibody phage display library to identify a highly selective and potent monoclonal antibody inhibitor of FXIIa, DX-4012. DX-4012 inhibits the proteolytic activity of FXIIa with an apparent Ki of ~15 pM, and does not inhibit closely related sequence homologs or other coagulation factors at concentrations up to 1 µM. When tested at 1 µM in human plasma, DX-4012 prolonged the activated partial thromboplastin time (aPTT) 3.4-fold, with no effect on the prothrombin time (PT). In a non-human primate pharmacokinetic study, an intravenous infusion of 10 mg/kg DX-4012 prolonged the aPTT 2.4-fold but had no effect on the PT. Given the importance of plasma kallikrein to FXII activation, we reasoned that the antithrombotic effect of DX-4012 could be augmented by combination with a kallikrein inhibitor. To test this, a variant of DX-4012 was converted into a single chain variable fragment (scFv) and combined with DX-2930, a potent and specific monoclonal antibody inhibitor of plasma kallikrein, to generate a "Morrison format" bispecific antibody. Enzyme inhibition assays determined that the apparent Ki values of the individual anti-kallikrein and anti-FXIIa components of the bispecific antibody were similar to the parent molecules (apparent Ki 389 pM and 73 pM, respectively). In contact-activated dilute plasma, the bispecific antibody was > 5 times more effective at preventing kallikrein generation than a 1:1 combination of DX-4012 and DX-2930, and more than 20-fold more effective than either DX-4012 or DX-2930 alone. Our data indicate that DX-4012, either as a monoclonal antibody or as a component of a bispecific antibody, shows potential as a novel antithrombotic therapy. Simultaneous inhibition of FXIIa and plasma kallikrein may be a uniquely potent method of blocking FXIIa activity through inhibition of the positive feedback loop during contact activation. Disclosures Mason: Dyax Corp: Employment. Kenniston:Dyax Corp: Employment. Comeau:Dyax Corp: Employment. Conley:Dyax Corp: Employment. Kastrapeli:Dyax Corp: Employment. Kopacz:Dyax Corp: Employment. Lindberg:Dyax Corp: Employment. Cosic:Dyax Corp: Employment. Kivaa:Dyax Corp: Employment. Qiu:Dyax Corp: Employment. Faucette:Dyax Corp: Employment. Sexton:Dyax Corp: Employment. Tenhoor:Dyax Corp: Employment. Wallisch:Aronora: Employment. Gruber:Aronora, Inc.: Employment, Equity Ownership, Patents & Royalties, Research Funding. Adelman:Dyax Corp: Employment. Nixon:Dyax Corp: Employment.

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