Recombinant porcine factor VIII corrects thrombin generation in vitro in plasma from patients with congenital hemophilia A and inhibitors

Abstract

Background: Neutralizing factor VIII (FVIII) antibodies are a major complication in hemophilia A. Antihemophilic factor VIII (recombinant), porcine sequence (rpFVIII; susoctocog alfa; Baxalta US Inc., a Takeda company) has low cross-reactivity to anti-human FVIII antibodies and can provide functional FVIII activity in the presence of FVIII inhibitors.

Objectives: Evaluate in vitro thrombin generation and clot formation responses to rpFVIII in blood from patients with congenital hemophilia A.

Methods: In this multicenter study, blood was obtained for in vitro analyses that included human and porcine FVIII inhibitors, low <5 Bethesda units (BU)/ml or high ≥5 BU/ml titer (Nijmegen-modified Bethesda assay); thrombin generation assay (TGA), clot viscoelasticity (thromboelastography), fibrin clot structure analysis (scanning electron microscopy), and epitope mapping.

Results: Blood samples were from 20 patients with congenital hemophilia A (FVIII activity <1%, mean [range] inhibitor titers: anti-human FVIII, 14 [1-427] BU/ml [n = 13 high, n = 6 low, n = 1 data unavailable]); anti-porcine FVIII, 12 (0-886) BU/ml (n = 11 high, n = 8 low, n = 1 data unavailable). Porcine inhibitor titer and TGA response measured by endogenous thrombin potential showed an inverse correlation (2.7-10.8 U/ml rpFVIII Spearman correlation coefficient: -0.594 to -0.773; p < 0.01). Clot structures in low anti-porcine inhibitor titer plasmas were similar to those in noninhibitor plasma.

Conclusions: Recombinant porcine factor VIII demonstrated a dose-dependent correction of thrombin generation and clot formation in vitro, dependent on the anti-porcine FVIII inhibitor titer. Procoagulant responses to rpFVIII occurred in plasma containing FVIII inhibitors.

Keywords: factor VIII inhibitor; hemophilia A; porcine factor VIII; recombinant; thrombin.

FIGURE 1

Correction of thrombin generation parameters in patient plasma. Thrombin generation profiles for two patient sample plasmas from local laboratories in Lyon after addition of varying recombinant porcine factor VIII (rpFVIII) concentrations (0–10.8 U/ml) in vitro. (A) Restoration of thrombin generation was dose dependent in a sample plasma with high human FVIII inhibitor titer and low porcine inhibitor titer. (B) In a sample plasma with high human FVIII inhibitor titer and high porcine inhibitor titer, there was a slight increase in thrombin generation observed with the highest rpFVIII concentration (10.8 U/ml) compared with vehicle control

FIGURE 2

Scanning electron micrographs (×10,000; representative images) of fibrin clots formed in platelet‐poor plasma from patient 2 with hemophilia A (high hFVIII inhibitor titer and low pFVIII inhibitor titer) in response to increasing concentrations of recombinant porcine factor VIII (rpFVIII), 0–10.8 U/ml. Fibrin clot structure assessment showed a dose‐dependent improvement in clot quality with significant fiber and hole diameter reductions achieved with 5.4 and 10.8 U/ml of rpFVIII added in vitro. Thrombin generation normalization occurred at the lowest rpFVIII concentration (2.7 U/ml) for this patient sample (see Table 2)

FIGURE 3

FVIII domain dominance and thrombin generation response map. Epitope mapping of inhibitor plasmas to domains of the human FVIII molecule analyzed by direct enzyme‐linked immunosorbent assay. Dominant hFVIII domain(s) indicated in the top row of text in each circle (second text row is patient number). In some patients, >1 domain was dominant. Domains in parentheses indicate low signal. Thrombogenic response was defined as a positive response or no response for each sample. Results are shown only for samples with inhibitor titer information available for both hFVIII and pFVIII inhibitors (20 samples were tested from 18 patients). Excludes secondary sample results from (1) Patient 17 (high hFVIII inhibitor titer, pFVIII inhibitor titer not determined, thrombin generation response not restored): A2 + C2 dominant epitopes with high porcine antibody cross‐reactivity, and (2) Patient 16 (low hFVIII inhibitor titer, porcine inhibitor titer not determined, no thrombin generation data): polyclonal. Abbreviations: BU, Bethesda units; FVIII, factor VIII; hFVIII, human factor VIII inhibitor; none, no predominant epitope; pFVIII, porcine factor VIII; poly, polyclonal antibodies; P xR, porcine crossreactive antibodies