Nonclassical anti-C2 domain antibodies are present in patients with factor VIII inhibitors

Abstract

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...

Structure of the factor VIII C2 domain in a ternary complex with 2 inhibitor antibodies reveals classical and nonclassical epitopes

“…Notably, classical C2 antibodies typically are low-titer type I inhibitors, and nonclassical antibodies typically are high-titer type II inhibitors, yet classical antibodies are more pathogenic in bleeding models. [38][39][40]60 One dramatic exception to this observation is the BO2C11 antibody inhibitor. The BO2C11 is a classical anti-C2 inhibitor that buries significantly more surface area (;1100Å…”

“…25 However, recent evidence suggests that a separate class of "nonclassical" anti-C2 antibodies allows binding of the C2 domain to PL/VWF, but inhibits the activation of fVIII by thrombin or factor Xa. [38][39][40] Interestingly, a large panel study showed that nonclassical inhibitors represented the highest frequency of occurrence among anti-C2 antibodies in inhibitor plasma from hemophilia A mice inoculated with human fVIII, and they are pathogenic in a murine tail snip bleeding model. 38,40 Moreover, it was also found that nonclassical anti-C2 antibodies are present in the majority of human polyclonal anti-C2 inhibitor plasmas.…”

“…38,40 Moreover, it was also found that nonclassical anti-C2 antibodies are present in the majority of human polyclonal anti-C2 inhibitor plasmas. 39 Therefore, nonclassical inhibitors represent a significant component of the human pathogenic anti-fVIII immune response.…”

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors

“…35 Hemophilia A mouse tail snip bleeding model An in vivo tail snip assay was used to determine bleeding phenotype of hemophilia A mice in the presence and absence of anti-C1 mAbs as previously described. 36,37 fVIII clearance in vivo To determine fVIII clearance, mice were killed 2 hours following BDD fVIII injections and plasma samples were collected by cardiac puncture.…”

A subset of high-titer anti–factor VIII A2 domain antibodies is responsive to treatment with factor VIII

“…9 Nonclassical group BC/C antibodies are present in the plasmas of most human fVIII inhibitor patients. 10 Group BC antibodies have inhibitor titers on an equimolar basis that are usually at least 10-fold higher than classical anti-C2 antibodies. However, at saturating concentrations, they produce residual fVIII levels of 20% to 40%.…”