Antihuman factor VIII C2 domain antibodies in hemophilia A mice recognize a functionally complex continuous spectrum of epitopes dominated by inhibitors of factor VIII activation

Abstract

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 beta-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 beta-sandwich. MAbs from groups A, AB, and B inhibit the binding of fVIIIa to phospholipid membranes. Group BC was the most common group and displayed the highest specific fVIII inhibitor activities. MAbs in this group are type II inhibitors that inhibit the activation of fVIII by either thrombin or factor Xa and poorly inhibit the binding of fVIII to phospholipid membranes or von Willebrand factor (VWF). Group BC MAbs are epitopically and mechanistically distinct from the extensively studied group C MAb, ESH8. These results reveal the structural and functional complexity of the anti-C2 domain antibody response and indicate that interference with fVIII activation is a major attribute of the inhibitor landscape.

Figure 1

Competition ELISA of C2 MAbs. (A) Venn diagram showing the overlap patterns of 30 anti-C2 MAbs in a competition ELISA. Groups A, B, and C are defined based on overlaps with the basis set MAbs, ESH4, 1B5, and ESH8, respectively. (B) Competition matrix showing the interaction of the 30 anti-C2 antibodies. The rows represent primary MAbs, and the columns represent biotinylated secondary MAbs. Noncompeting and competing MAbs are shown in gray and white, respectively. MAb numbering is defined in Table 1.

Figure 2

The spectrum of anti-C2 antibody epitopes. (A) Venn diagram representing overlapping anti-C2 MAbs. The overlap pattern of 18 ellipses is derived from the competition matrix of 30 MAbs in Figure 1B. Colors depict the 5 classes of MAbs: red, group A; orange, group AB; yellow, group B; green, group BC; and blue, group C. The thick lines represent the basis set MAbs: red, ESH4 (group A); yellow, 1B5 (group B); and blue, ESH8 (group C). The white ellipse represents the epitope of BO2C11. (B) Amino acid side chains recognized by anti-C2 MAbs. The strands representing the front and back faces of the β-sandwich are shown in green and red, respectively. Single-letter nomenclature is used to designate amino acids. Val2223 and Lys2227 are shown as van der Waals surfaces. The group AB MAbs, I109 and BO2C11, bind to the 2 phospholipid-binding β-hairpins containing Met2199 and Leu2251, respectively. The group B MAb, 3D12, binds to Phe2196. The group BC MAbs bind to Lys2227 and possibly Val2223.

Figure 3

Binding of anti-C2 MAbs to fVIII C2 mutants. The binding of anti-C2 antibodies to fVIII captured in microtiter wells using an anti-A2 MAb was detected using streptavidin–alkaline phosphatase conjugate. Gray boxes represent binding of the MAb detected by color development.

Figure 4

Functional assays of anti-C2 MAbs. (A) Representative Bethesda assays for a type I MAb (I89, ●) and a type II MAb (F178, ▵). (B) Inhibition of binding of fVIII to VWF by 3 representative anti-C2 MAbs. The MAbs shown are 3E6 (▿), F100 (○), and I89 (●). (C) Inhibition of binding of fVIII to phospholipid by the 3 anti-C2 MAbs shown in panel B. Error bars represent sample standard deviations of 3 to 12 replicates.

Figure 5

Functional properties of anti-C2 MAb groups. (A) Relationship between MAb group, fVIII inhibitor titer (●), and IC₅₀⁻¹ values for inhibition of binding of fVIII to phospholipid (○). (B) The inhibitor titers of MAbs with demonstrable inhibition of binding of fVIII to phospholipid (○) or VWF (●) (Table 1) are plotted versus IC₅₀⁻¹ for inhibition of binding.

Figure 6

Inhibition of intrinsic Xase activity by anti-C2 MAbs. FVIII (1 nM) was incubated for 2 hours at 37°C in the absence (closed symbols) or presence (open symbols) of 10 μg/mL VWF and in the absence (○) or presence of 20 nM anti-C2 MAbs: 3E6 (group A) (▿), 1B5 (group B) (◇), 2-77 (group BC) (▵), or 2-117 (group C) (□). Then fVIII was activated rapidly by thrombin (100 nM) for 30 seconds, followed by addition of desulfatohirudin (150 nM) to inhibit thrombin. At this concentration of thrombin, fVIII is activated completely within 10 seconds. The fVIIIa sample was added to 2 nM factor IXa/20 μM PCPS, followed immediately by addition of 300 nM factor X and measurement of the initial velocity of factor X activation as described in “Intrinsic fXase assays for measurement of fVIIIa activity.”

Figure 7

Inhibition by anti-C2 MAbs of the activation of fVIII by thrombin or factor Xa. (A) FVIII (1 nM) was incubated for 2 hours at 37°C in the absence (closed symbols) or presence (open symbols) of 10 μg/mL VWF and in the absence (○) or presence of 130 nM 2–77 (group BC) (▵) or ESH8 (group C) (□). The fVIII sample was diluted 0.5 nM in 1.5 nM factor IXa/20 μM PCPS and thrombin (0.55 nM) was added for the indicated times, followed by measurement of the initial velocity of factor X activation as described in “Intrinsic fXase assays for measurement of fVIIIa activity.” (B) FVIII (4 nM) was incubated for 2 hours at 37°C in the absence (closed symbols) or presence (open symbols) of 40 μg/mL VWF and in the absence (○) or presence of 520 nM 2–77 (group BC) (▵) or ESH8 (group C) (□). The fVIII sample was diluted to 2 nM and activated with 2.5 nM factor Xa in the presence of 1.5 nM factor IXa/20 μM PCPS for the indicated times, followed by measurement of the initial velocity of factor X activation as described in “Intrinsic fXase assays for measurement of fVIIIa activity.”