Non-classical Antibodies Research Articles

Structural Studies Of The Factor VIII C2 Domain In a Ternary Complex With Two Inhibitory Antibodies Reveals Classical and Non-Classical Epitopes

The factor VIII C2 domain is a highly immunogenic domain, whereby inhibitory antibodies develop following factor VIII replacement therapy for congenital hemophilia A patients. Inhibitory antibodies also arise spontaneously in cases of acquired hemophilia A. The structural basis for molecular recognition by two classes of anti-C2 inhibitory antibodies that bind to factor VIII simultaneously has been investigated by small angle X-ray scattering and X-ray crystallography. The C2 domain/3E6 FAB/G99 FAB stable ternary complex, both in solution and in its crystalline state, illustrates that each antibody epitope resides on opposing faces of the factor VIII C2 domain. The 3E6 epitope is a classical antibody that forms direct contacts to the C2 domain at two loops consisting of Glu2181-Ala2188 and Thr2202-Arg2215, which inhibits the binding of the C2 domain to von Willebrand Factor and phospholipid surfaces. The G99 is a non-classical antibody that prevents proteolytic activation of factor VIII, and its epitope centers on Lys2227 and also makes direct contacts with loops Gln2222-Trp2229, Leu2261-Ser2263, His2269-Val2282 and Arg2307-Gln2311. Each binding interface is highly electrostatic, with positive charges present on both C2 epitopes and complementary negative charges on each antibody. A new model of phospholipid membrane association is also presented, where the 3E6 epitope faces the negatively charged membrane surface and Arg2320 is poised at the center of the binding interface. Furthermore, a 1.7 Å X-ray crystal structure of the porcine factor VIII C2 domain has also been determined, which supports the presented model for phospholipid binding. These results illustrate the complex nature of the polyclonal immune response against the factor VIII C2 domain, and further define the epitopes for both classical and non-classical inhibitory antibodies. Disclosures:No relevant conflicts of interest to declare.

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

AbstractThe antihuman factor VIII (fVIII) C2 domain immune response in hemophilia A mice consists of antibodies that can be divided into 5 groups of structural epitopes and 2 groups of functional epitopes. Groups A, AB, and B consist of classical C2 antibodies that inhibit the binding of fVIII to phospholipid and von Willebrand factor. Groups BC and C contain nonclassical C2 antibodies that block the activation of fVIII by thrombin or factor Xa. Group BC antibodies are the most common and display high specific inhibitory activity and type II kinetics. The C2 epitope groups recognized by 26 polyclonal human anti-fVIII inhibitor plasmas were identified by a novel competition enzyme-linked immunosorbent assay using group-specific murine monoclonal antibodies. Most of the anti-C2 inhibitor plasmas inhibited the binding of both classical and nonclassical antibodies. These results suggest that nonclassical anti-C2 antibodies contribute significantly to the pathogenicity of fVIII inhibitors.

Non-Classical Anti-Factor VIII C2 Domain Inhibitors Are Present in the Plasmas of Most Factor VIII Inhibitor Patients.

Approximately 30% of patients with severe hemophilia A will develop inhibitory antibodies to factor VIII (fVIII inhibitors). 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 non-hemophiliacs, producing acquired hemophilia A, which frequently produces life- or limb-threatening bleeding. These inhibitors primarily are directed against the A2 or C2 domains of fVIII. The human response to the C2 domain of fVIII classically has been thought to inhibit fVIII activity by blocking its binding to phospholipid. We recently characterized the antibody response to the C2 domain of human fVIII in a murine hemophilia model and described 5 structural groups of antibodies. Groups A, AB, and B are classical anti-C2 antibodies. Groups BC and C consist of non-classical anti-C2 antibodies that inhibit the proteolytic activation of fVIII but do not block the binding of fVIII to phospholipid. Most non-classical antibodies have inhibitor titers greater than 10,000 Bethesda units/mg IgG. To determine if non-classical antibodies are present in fVIII inhibitor patients, patient plasmas were tested in an ELISA for their ability to block the binding of representative antibodies from the different anti-human fVIII C2 antibody groups. Classical and non-classical monoclonal antibodies (MAbs) were biotinylated and serially diluted into either fVIII deficient plasma or patient inhibitor plasma and then added to microtiter wells coated with fVIII. The ability of patient plasma to block the binding of the murine MAbs to fVIII was determined. A total of 16 patient plasmas were assessed: 4 from patients with a C2 predominant response, 2 with a non-C2 predominant response, and 10 with unknown specificities. Three of the 4 patients with C2 predominant responses had non-classical anti-C2 antibodies, while the 2 with non-C2 predominant responses did not. In the unknown plasmas, 6 of 10 had evidence of non-classical antibodies. Figure 1 shows representative results of the effect of 3 patient plasmas on the binding of a biotinylated non-classical MAb to fVIII. Patient plasmas 1 and 2 blocked MAb binding while patient plasma 3 did not. This study indicates that the majority of patients with fVIII inhibitors have non-classical anti-C2 antibodies in their response to fVIII. [Display omitted]