The diversity of the immune response to the A2 domain of human factor VIII

Abstract

Approximately 30% of patients with severe hemophilia A develop inhibitory anti-factor VIII (fVIII) antibodies (Abs). We characterized 29 anti-human A2 monoclonal Abs (mAbs) produced in a murine hemophilia A model. A basis set of nonoverlapping mAbs was defined by competition enzyme-linked immunosorbent assay, producing 5 major groups. The overlapping epitopes covered nearly the entire A2 surface when mapped by homolog-scanning mutagenesis. Most group A mAbs recognized a previously described epitope bounded by Arg484-Ile508 in the N-terminal A2 subdomain, resulting in binding to activated fVIII and noncompetitive inhibition of the intrinsic fXase complex. Group B and C mAbs displayed little or no inhibitory activity. Group D and E mAbs recognized epitopes in the C-terminal A2 subdomain. A subset of group D mAbs inhibited the activation of fVIII by interfering with thrombin-catalyzed cleavage at Arg372 at the A1-A2 domain junction. Other group D mAbs displayed indeterminate or no inhibitory activity despite inhibiting cleavage at Arg740 at the A2-B domain junction. Group E mAbs inhibited fVIII light-chain cleavage at Arg1689. Inhibition of cleavages at Arg372 and Arg1689 represent novel mechanisms of inhibitor function and, along with the extensive epitope spectrum identified in this study, reveal hitherto unrecognized complexity in the immune response to fVIII.

Figure 1

ELISA competition matrix and overlap pattern of anti-A2 mAbs. (A) Competition matrix compiling the results of competition ELISA among 29 anti-A2 mAbs. Rows represent primary mAbs and columns represent biotinylated secondary mAbs. Yellow and white squares represent binding and no binding (no overlap and overlap), respectively, of secondary mAbs. (B) Venn diagram representing overlaps produced by the competition matrix. Each mAb was assigned a group based on its overlap with a basis set of 5 mAbs 4A4—2-93, G48, 2-54, and 1D4—that are underlined and italicized. Colors depict the 8 groups of mAbs defined by their overlap with basis set mAbs: dark red, group A; red, group AB; orange, group B; yellow, group C; green, group BCD; light blue, group D; blue, group DE; and purple, group E.

Figure 2

Epitope mapping of anti-A2 mAbs by homolog-scanning mutagenesis. (A) BDD hybrid human/porcine fVIII constructs. Shaded areas represent porcine fVIII substitutions. (B) Binding of anti-A2 mAbs to human/porcine fVIII. fVIII was captured on a microtiter plate using immobilized anti-C2 mAb, followed by addition of biotinylated anti-A2 mAb and detection using streptavidin–alkaline phosphatase. Bars represent the range of duplicate determinations.

Figure 2

Epitope mapping of anti-A2 mAbs by homolog-scanning mutagenesis. (A) BDD hybrid human/porcine fVIII constructs. Shaded areas represent porcine fVIII substitutions. (B) Binding of anti-A2 mAbs to human/porcine fVIII. fVIII was captured on a microtiter plate using immobilized anti-C2 mAb, followed by addition of biotinylated anti-A2 mAb and detection using streptavidin–alkaline phosphatase. Bars represent the range of duplicate determinations.

Figure 3

Map of overlapping mAb epitopes onto the A2 surface. (A) X-ray structure of BDD fVIII (PDB 2R7E) showing regions recognized by group A mAbs (Asp403-His444 and Arg484-Ile508) and group E mAbs (Glu604-Cys711). A2 residues Asp712-Arg740 were not identified in the structure. (B) Regions with the A2 polypeptide chain identified by homolog-scanning mutagenesis were used to anchor the Venn diagram onto the A2 surface.

Figure 4

Anticoagulant properties of anti-A2 mAbs. (A) Residual fVIII activity was measured by 1-stage coagulation assay following incubation of normal human plasma with varying concentrations of mAbs for 2 hours at 37°C. Data represent sample means and sample standard deviations. The curves represent least-squares fits to the data. The inhibitor titer in Bethesda units per milliter was obtained by determining the dilution of mAb producing 50% inhibition and converted to Bethesda units per milligram using the mAb concentration. (B) BDD human fVIII (50 nM) was incubated with the indicated concentrations of anti-A2 mAbs for 30 minutes and then thrombin for 60 seconds, followed by sample dilution into fIXa/PCPS phospholipid vesicles, addition of fX, and measurement of fXa as described in “Intrinsic fXase assay.” Results are presented as the percentage of fXa formed in the absence of mAb.

Figure 5

Inhibition of thrombin-catalyzed activation of fVIII by group D and E mAbs. The activation of full-length fVIII (A1-A2-B/ap-A3-C1-C2) is associated with proteolytic cleavages catalyzed by thrombin. The fastest cleavage occurs at Arg740 in the A1-A2-B heavy chain, producing the A1-A2 fragment and the ap-A2-C1-C2 light chain. Cleavage then occurs at Arg372 or Arg1689. Cleavage at Arg372 is necessary for the development of fIXa cofactor activity of fVIIIa, whereas cleavage at Arg1689 is necessary for the dissociation of fVIII from VWF. Cleavage at Arg740 is not necessary for either fVIIIa formation or the dissociation of fVIII from VWF.

Figure 6

Effect of group D and E anti-A2 mAbs on thrombin-catalyzed proteolytic cleavage of full-length fVIII. fVIII (100 nM) was incubated with 0.5 nM thrombin for 10 minutes at 37°C in the presence of the indicated mAb concentrations. Proteolytic cleavages were analyzed by SDS-PAGE as described in “Effect of anti-A2 mAbs on thrombin-catalyzed proteolytic cleavage of fVIII.” Ctrl, fVIII control not exposed to thrombin or anti-A2 mAbs; Stds, Molecular weight standards are 250, 150, 100, 75, 50, 37, and 25 kDa.

Figure 7

Effect of group D and E anti-A2 mAbs on thrombin-catalyzed proteolytic cleavage of the full-length fVIII/VWF complex. fVIII (100 nM) was incubated for 10 minutes at 37°C with VWF (1000 nM monomer equivalents) and then incubated for an additional 10 minutes in the presence of increasing mAb concentrations. Thrombin (0.5 nM) was added for 10 minutes and the reaction products were analyzed by SDS-PAGE as described in “Effect of anti-A2 mAbs on thrombin-catalyzed proteolytic cleavage of fVIII.” Ctrl, fVIII control not exposed to thrombin or anti-A2 mAbs; Stds, Molecular weight standards are 250, 150, 100, 75, 50, 37, and 25 kDa.