The diversity of factor VIII (fVIII) C2 domain antibody epitopes was investigated by competition enzyme-linked immunosorbent assay (ELISA) using a panel of 56 antibodies. The overlap patterns produced 5 groups of monoclonal antibodies (MAbs), designated A, AB, B, BC, and C, and yielded a set of 18 distinct epitopes. Group-specific loss of antigenicity was associated with mutations at the Met2199/ Phe2200 phospholipid binding ␤-hairpin (group AB MAbs) and at Lys2227 (group BC MAbs), which allowed orientation of the epitope structure as a continuum that covers one face of the C2 ␤-sandwich.

Treatment of patients with hemophilia A and B has undergone significant advances during the past 2 decades. However, despite these advances, the development of antibodies that inhibit the function of infused clotting factor remains a major challenge and is considered the most significant complication of hemophilia treatment. This chapter reviews current tools available for the care of patients with inhibitors and highlights areas where progress is imminent or strongly needed. For management of bleeding, bypassing agents remain the mainstay of therapy. Recombinant factor VIIa and activated prothrombin complex concentrates are similarly effective in populations of patients with hemophilia and inhibitors; however, individuals may show a better response to one agent over another. Recent studies have shown that prophylaxis with bypassing agents can reduce bleeding episodes by ∼50%-80%. The prophylactic use of bypassing agents is an important tool to reduce morbidity in patients before they undergo immune tolerance induction (ITI) and in those with persistent high titer inhibitors, but cost and lack of convenience remain barriers. Because of the significant burden that inhibitors add to the individual patient and the health care system, inhibitor eradication should be pursued in as many patients as possible. ITI is an effective tool, particularly in patients with severe hemophilia A and good risk profiles, and leads to a return to a normal factor VIII response in ∼60% of patients. However, for the group of patients who fail to respond to ITI or have hemophilia B, new and improved tools are needed.

Evidence-based recommendations to guide treatment decisions may increase the likelihood of successful inhibitor eradication and the induction of FVIII tolerance in patients with hemophilia A who develop inhibitory antibodies.

Optimization of a protein’s pharmaceutical properties is usually carried out by rational design and/or directed evolution. Here we test an alternative approach based on ancestral sequence reconstruction. Using available genomic sequence data on coagulation factor VIII and predictive models of molecular evolution, we engineer protein variants with improved activity, stability. biosynthesis potential, and reduced inhibition by clinical anti-drug antibodies. In principle, this approach can be applied to any protein drug based on a conserved gene sequence.

Treatment of patients with hemophilia A and B has undergone significant advances during the past 2 decades. However, despite these advances, the development of antibodies that inhibit the function of infused clotting factor remains a major challenge and is considered the most significant complication of hemophilia treatment. This chapter reviews current tools available for the care of patients with inhibitors and highlights areas where progress is imminent or strongly needed. For management of bleeding, bypassing agents remain the mainstay of therapy. Recombinant factor VIIa and activated prothrombin complex concentrates are similarly effective in populations of patients with hemophilia and inhibitors; however, individuals may show a better response to one agent over another. Recent studies have shown that prophylaxis with bypassing agents can reduce bleeding episodes by ∼50%-80%. The prophylactic use of bypassing agents is an important tool to reduce morbidity in patients before they undergo immune tolerance induction (ITI) and in those with persistent high titer inhibitors, but cost and lack of convenience remain barriers. Because of the significant burden that inhibitors add to the individual patient and the health care system, inhibitor eradication should be pursued in as many patients as possible. ITI is an effective tool, particularly in patients with severe hemophilia A and good risk profiles, and leads to a return to a normal factor VIII response in ∼60% of patients. However, for the group of patients who fail to respond to ITI or have hemophilia B, new and improved tools are needed.

Key Points The multivariate mechanism of FeCl3-induced thrombosis is rooted in colloidal chemistry, mass transfer, and biological clotting. FeCl3-induced thrombosis is mediated by charge-based binding of proteins (cell surface bound and soluble) to the Fe3+ ion.

The immune response to infused factor concentrates remains a major source of morbidity and mortality in the treatment of patients with hemophilia A and B. This review focuses on current treatment options and novel therapies currently in clinical trials. After a brief review of immune tolerance regimens, the focus of the discussion is on preventing bleeding in patients with hemophilia and inhibitors. Recombinant factor VIIa and activated prothrombin complex concentrates are the mainstays in treating bleeds in patients with inhibitors. Both agents have been shown to reduce bleeding episodes to a similar degree when infused prophylactically; however, individual patients may respond better to one agent over the other at any given time. The international immune tolerance trial revealed that a high-dose factor VIII regimen provided significantly better bleeding protection than the low-dose regimen. Given the high cost of treatment and the potential for a high-dose immune tolerance regimen to prevent bleeding in some patients, we discuss how we treat patients to maximize the prevention of bleeds while minimizing cost. Novel approaches to treatment of these patients are in development. These include agents that mimic factor VIII or augment thrombin generation by bypassing the inhibitor, as well as agents that inhibit the natural anticoagulants.

IntroductionApproximately 30% of patients with hemophilia A develop detectable antifactor VIII (fVIII) antibodies in response to infusions of fVIII. [1][2][3][4] The immune response to fVIII currently is the most significant complication in the management of patients with hemophilia A. In addition, autoimmune antibodies to fVIII can develop in nonhemophiliacs, producing acquired hemophilia A, which frequently produces life-or limb-threatening bleeding. Most inhibitory antibodies are directed at either the 40-kDa A2 or the 15-kDa C2 domains of the A1-A2-B-ap-A3-C1-C2 fVIII sequence. 5 fVIII inhibitors can either inhibit fVIII completely or incompletely at saturating concentrations, corresponding to type I and type II behavior, respectively. 6 Classical anti-C2 antibodies inhibit binding of fVIIIa to negatively charged phospholipid membranes. [7][8][9] The binding of fVIII to phospholipid membranes and to von Willebrand factor (VWF) is mutually exclusive, and antibodies have been shown to block binding to both phospholipid and/or VWF. [10][11][12][13][14] In addition, murine anti-C2 monoclonal antibodies (mAbs) 15,16 and anti-C2 antibodies in 2 polyclonal patient plasmas 16,17 have been identified that interfere with the activation of fVIII by thrombin or factor Xa.We recently characterized the diversity of a large panel of murine anti-C2 mAbs. 18 Five groups of structural epitopes were defined based on patterns of overlapping epitopes. Group A, AB, and B antibodies correspond to classical inhibitors that inhibit the binding of fVIII to phospholipid and VWF. Group BC antibodies are the most frequent and are type II inhibitors with inhibitory titers usually greater than 10 000 Bethesda units per mg immunoglobulin G. These antibodies inhibit the activation of fVIII by thrombin and factor Xa in the presence and absence of VWF. ESH8, a wellcharacterized murine anti-C2 mAb, which blocks the release of VWF from fVIII after thrombin activation, is a group C mAb. 16 In this study, we used murine group-specific antihuman C2 mAbs in a competition enzyme-linked immunosorbent assay (ELISA) to determine whether nonclassical group BC and C antibodies are present in human fVIII inhibitor patients. MethodsfVIII inhibitor plasmas from 26 patients with congenital hemophilia A or acquired hemophilia A were obtained either as described previously 19,20 from the Emory Comprehensive Hemophilia Center or from George King Bio-Medical (Overland Park, KS). Recombinant full-length human fVIII was a gift from Baxter Biosciences (Duarte, CA). mAbs ESH-4 (group A) and ESH-8 (group C) were purchased from American Diagnostica (Greenwich, CT). mAbs 3E6 (group A), I109 (group AB), 1B5 (group B), 2-77 (group BC), and 2-117 (group C) were isolated as described previously. 18 mAbs were biotinylated as previously described. 18 Anti-fVIII ELISAs were performed as a modification of previously described procedures. 18 Briefly, ELISA plates were coated with fVIII, preincubated with 3 g/mL of a nonbiotinylated murine antihuman C2 "blocking" mAb, followed b...