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.


Essentials
• Recombinant porcine factor VIII (rpFVIII) was evaluated in blood from patients with congenital hemophilia A and FVIII inhibitors.
• rpFVIII demonstrated a dose-dependent correction of thrombin generation and clot formation.
• Restoration of hemostatic markers depended on porcine-but not on human-FVIII inhibitor titers.
• In conclusion, responses to rpFVIII can occur in the presence of pre-existing FVIII inhibitors.

| INTRODUC TI ON
Congenital hemophilia A (CHA) is characterized by a deficiency of human factor VIII (FVIII), with most cases caused by inherited Xlinked mutations in the F8 clotting factor gene; approximately 30% of all hemophilia cases are caused by spontaneous mutations. 1,2 Development of inhibitory alloantibodies to FVIII is a major complication of replacement FVIII therapy for patients with hemophilia A because exogenous FVIII is no longer effective for control of bleeding in the presence of high titer inhibitors (>5 Bethesda units [BU]). [3][4][5] The lifetime risk (cumulative incidence) for development of anti-human FVIII inhibitory antibodies is approximately 30% in previously untreated patients with hemophilia A, 1 representing the most important challenge for the management of hemostasis in these patients. 6 Anti-human FVIII inhibitors are high-affinity polyclonal IgG neutralizing antibodies that primarily bind to the A2 and C2 domains and to the C-terminal region of the C1 domain of FVIII and inhibit FVIII hemostatic functions. [7][8][9] Bypassing agents such as recombinant activated factor VII or activated prothrombin complex concentrate are used for the management of acute bleeding in patients with hemophilia and human FVIII inhibitors. 10,11 There are reported variations in the clinical response to these agents 12 and, because routine assays are not able to accurately monitor treatment efficacy and inform treatment decisions, global coagulation assays may be required as an ancillary tool to support treatment decisions. 13,14 For patients with acquired hemophilia A (AHA), an additional disadvantage of therapy with bypassing agents is the risk of thrombosis that, although quite rare, is of importance when considering treatment of elderly patients with severe comorbidities. 13,15 Porcine FVIII (pFVIII) has been used in patients with hemophilia A and human FVIII (hFVIII) inhibitors on account of its 84% and 76% homology with human FVIII A2 and C2 domains, respectively. 16,17 This is similar enough to achieve hemostatic functions of the human protein but sufficiently different to avoid recognition by neutralizing human FVIII antibodies. [18][19][20] pFVIII derived from pig plasma, Hyate:C, carried a risk of allergic reactions and thrombocytopenia when used in human patients, and concerns over potential viral contamination of the product ultimately led to production being suspended. 13 Recombinant porcine factor VIII (rpFVIII, susoctocog alfa, Obizur, Baxalta US Inc., a Takeda company) is a B-domain deleted (BDD), 1448-amino acid heterodimer with a molecular mass of 170 kDa, composed of a 90-kDa heavy chain and an 80-kDa light chain. [21][22][23] In the clinical setting, rpFVIII has been evaluated in phase I and II studies in patients with CHA with inhibitors. 19,24,25 A phase II/III study reported the safety and efficacy of rpFVIII for the treatment of bleeding episodes in patients with AHA and pFVIII inhibitors (≤20 BU/ml). 26 These data supported regulatory approval of rpFVIII for the treatment of bleeding episodes in adults with AHA; rpFVIII is not approved for CHA.
Preclinical evidence suggests that the mode of action of rpFVIII is similar to that of human FVIII replacement products: the pairwise sequence homology is 86% compared with BDD human FVIII, thrombin activation of rpFVIII generates cleavage products comparable with human FVIII, and rpFVIII binds with high affinity to von Willebrand factor. 21,22,27 The functional characterization of rpFVIII demonstrated similar efficacy and half-life but higher recovery rate versus the plasma-derived pFVIII product Hyate:C. 19,22 RpFVIII displayed low cross-reactivity with human FVIII inhibitors and resulted in therapeutic levels of FVIII in patients with hemophilia A and in hemophilic plasmas in vitro, despite the presence of human FVIII inhibitors. 19,28 Global assays that measure thrombin generation provide information on the functional status of blood coagulation as inferred from the overall balance between pro-and anticoagulant factors. 29 Fibrin clot ultrastructure analysis can provide complementary information on whether a solid fibrin clot is formed. The objectives of this study were, first, to evaluate the hemostatic efficacy of rpFVIII in vitro, including effects on thrombin generation, clot formation, and fibrin clot structure, using blood samples from patients with hemophilia A with inhibitors to human FVIII. The second objective was to further assess the differences in hemostatic efficacy based on the level of human FVIII and porcine FVIII inhibitor titers and to determine the epitope-specificity of human FVIII inhibitors in patient samples.

| Patients and sample preparation
In this study, blood was obtained for in vitro analyses that included human and porcine FVIII inhibitors. The study was conducted across three clinical sites in three countries. The study protocol was approved by the institutional review board at each institution and written and signed informed consent was obtained from all study participants before enrollment.

K E Y W O R D S
factor VIII inhibitor, hemophilia A, porcine factor VIII, recombinant, thrombin Individuals with congenital hemophilia A with inhibitors to hFVIII were recruited during routine outpatient visits to hemophilia centers in Bonn, Germany; Lyon, France; and Tel Aviv, Israel, between January and November 2011. The study aimed to recruit consecutive patients who attended for routine visits at their center during the study period and consented to contribute to the study by providing blood samples in addition to samples taken for their routine laboratory parameters.
Whole blood samples (20 ml) were obtained during routine outpatient visits with written, informed patient consent. A 10-ml aliquot was collected in sodium citrate tubes (final concentration, 10.9 mM); 1.2 ml was used for thromboelastography studies using whole blood, with the remainder undergoing centrifugation for preparation of plasma for FVIII activity assays. An additional 10-ml aliquot was collected in sodium citrate tubes (final concentration, 10.9 mM) containing corn trypsin inhibitor 1.45 µM for separation of platelet-poor plasma (PPP) as required for the thrombin generation assay (TGA) and scanning electron microscopy. Samples were centrifuged at 2000g at room temperature for 10 min, then plasma was removed and centrifuged again at 10,000g for 3 min. The PPP was removed, aliquoted, and stored at ≤−60°C until assayed.

| FVIII activity (FVIII:C) measurement
FVIII activity was measured at both local and central laboratories.
A one-stage activated partial thromboplastin time-based assay was used. One-stage FVIII activity was measured at the central laboratory. In brief, citrated PPP samples were diluted in FVIII-deficient plasma and the activated partial thromboplastin time-reagent Actin FS (Siemens Healthcare Diagnostics, Marburg, Germany) was added.
After incubation for 3 min at 37°C, 25 mM CaCl 2 was added, and clotting times were measured. Analyses were performed using a Sysmex CA 1500 or BCS XP instrument (Siemens Healthcare Diagnostics).

| FVIII inhibitor assays
Inhibitory titers against human or porcine FVIII were determined using the Nijmegen-modified Bethesda assay (NBA) at both the local (site of sample collection) and central laboratories.
For the anti-human FVIII NBA, heat-inactivated citrated plasma samples were mixed 1:2 with human reference plasma and incubated for 2 h (37°C). Residual FVIII:C was measured using the Sysmex CA 1500 or BCS XP instrument (Siemens Healthcare Diagnostics). The anti-porcine FVIII NBA was conducted as for the anti-human FVIII NBA except that rpFVIII (diluted in hFVIII-deficient plasma to approximately 1 U/ml) was used in place of normal human plasma.

| Thrombin generation assay
Lag time, peak height, and endogenous thrombin potential (ETP) were assessed using citrated-CTI PPP samples. The time course of thrombin generation was determined at a low tissue factor concentration (1 pM) using calibrated automated thrombography (CAT) (Thrombinoscope bv) and a Fluoroskan Ascent fluorometer (Thermo LabSystems, Inc.), as described previously. 30 The responses were determined at rpFVIII equivalent concentrations of 0, 100, 200, and 400 U/kg. Using standard assumptions for body weight (70 kg), plasma volume (40 ml/kg) and FVIII molecular weight (200 ng is equivalent to 1 U), the appropriate rpFVIII concentrations were calculated to be 2.7, 5.4, and 10.8 U/ml. Reference values for a thrombin generation response with the TGA were determined at the central laboratory using data generated from 100 healthy volunteer men.
A full thrombin generation response referred to TGA results within the normal reference range (mean ± standard deviation) after in vitro addition of rpFVIII. A partial response referred to an increase or improvement of thrombin generation that could not reach the lower limit (mean ± standard deviation) of reference values.

| Thromboelasticity studies
Assessment of the viscoelastic properties of blood during clot formation and lysis were conducted on citrated whole blood at local study sites using either rotation thromboelastometry (ROTEM Pentapharm

| Fibrin clot structure assessment
Clots formed in the TGA assay were fixed with 1.5% glutaraldehyde (Sigma Aldrich) in phosphate buffered saline, pH 7.2, followed by a solution of OsO 4 in 1% sodium cacodylate 150 mM, pH 7.4. Fixed clots were dehydrated in serial ethanol solutions and hexamethyldisilazane and stored under vacuum. Samples were placed on carbon adhesive tabs, coated with gold-palladium ions, and viewed under a Hitachi S800 FEG scanning electron microscope (×10,000 magnification). Three to five representative images (mean of 250 fibrin fibers per sample) were analyzed using ImageJ software (National Institutes of Health). Clot quality was assessed according to the number, diameter, and density of fibrin fibers. For each patient sample, fibrin fiber diameters measured at baseline were compared with those measured after addition of rpFVIII 2.7, 5.4, and 10.8 U/ml. An increased number of thinner fibrin fibers with increased fiber density relative to baseline was considered an improvement in clot quality.

| Epitope mapping
Epitope mapping of inhibitors with respect to the A1, A2, A3, C1, and C2 domains of the human FVIII molecule was carried out at the central laboratory in Atlanta, Georgia, USA, using citrated PPP samples loaded onto direct enzyme-linked immunosorbent assay (ELISA) on half-area 96-well plates with purified single human domain hybrid FVIII proteins as test antigens and BDD human FVIII and BDD porcine FVIII as positive control antigens. 31 Plasma samples were serially diluted from 1:20 in blocking buffer (0.15 M NaCl/20 mM HEPES/5 mM CaCl 2 /0.05% sodium azide/0.05% Tween ® 80/0.25% BSA, pH 7.4) and dilutions added to the coated wells of the ELISA plate. Antibody binding was detected by goat anti-human IgG. Domain specificity was indicated by color reaction and ELISA titers.

| Statistical analyses
Samples were classified by human or porcine FVIII inhibitor titers as low titer (<5 BU/ml) or high titer (≥5 BU/ml) 32 and further stratified into the following subgroups: (1) low anti-human/low anti-porcine; (2) high anti-human/low anti-porcine; and (3) high anti-porcine inhibitor titers. Procoagulant response was classified as restored by a combination of increased peak height, increased ETP, and decreased time to peak and defined as positive, partial, or no response for each sample based on reference values.
Descriptive statistics were used for hemostatic assay results. In addition, Spearman rank correlation coefficients were used to analyze differences between rpFVIII inhibitor titers with respect to thrombogenic response. A Dunnett multiple comparison test was used to compare the differences between replicate clots used for analysis of clot formation within the same patient. A p value <0.05 was considered statistically significant.

| Patient characteristics, FVIII activity, and FVIII inhibitor levels
Although the study aimed to recruit consecutive patients who attended for routine visits at their center during the study period, some patients were excluded because they were noncompliant, did not visit the hemophilia center regularly, or declined to participate in the study. FVIII inhibitor titers were determined in samples collected from 20 participants with congenital hemophilia A. Patients were aged 11-70 years and FVIII activity was <0.01 IU/ml, indicative of severe hemophilia A. Results from one patient with AHA were also available but are not reported with the main results. The patient with AHA was aged 74 years; no FVIII activity data were available for this patient, anti-human FVIII inhibitor titer was 7 BU/ml (classified as high), and anti-porcine FVIII inhibitors were not detected (0 BU/ml). Thrombin generation normalization was not achieved for this patient and fiber diameter changes (mean difference) from baseline in response to rp- In total, measurements from 22 patient blood samples were recorded (two of 20 patients provided a second sample at a later visit).

| Effects of rpFVIII on thrombin generation
A total of 20 samples from 19 patients were analyzed using CAT to assess thrombin generation ( Table 2) to the addition of rpFVIII in vitro, 1/10 (10%) showed partial restoration, and 3/10 (30%) showed complete restoration (Table 2).
Data from 18 patients with complete data for both ETP and peak height generated at the central laboratory were used for the Spearman rank correlation analysis to examine the effect of porcine inhibitor titers on restoration of thrombin generation after addition of rpFVIII in vitro. ETP and peak height data also varied substantially.
A strong inverse relationship between porcine inhibitor titers and thrombin generation parameters was demonstrated using Spearman rank correlation coefficient (Table 3).

| Clot formation -thromboelasticity studies
Clot formation in response to increasing concentrations of rpFVIII (0, 2.7, 5.4, and 10.8 U/ml) was assessed by thromboelastography (Table 4). Overall, median quantifiable clotting times decreased with increasing concentrations of rpFVIII. Accordingly, observed values for maximum clot firmness increased with increasing concentrations of rpFVIII (Table 4).

| Fibrin clot structure assessment -scanning electron microscopy
Electron micrographs of clots formed in response to different concentrations of rpFVIII added to PPP samples ( Figure 2) were analyzed to assess clot quality (number, diameter, and density of fibrin fibers). Figure 2 shows examples of electron micrographs of clots from the same patient sample. An increase in the number of thinner fibrin fibers with increased fiber density was considered an improvement in clot quality.
The results of a Dunnett multiple comparison test on fibrin clot fiber diameters assessed by electron microscopy are shown in Table 5. Clot quality showed a dose-dependent improvement in response to rpFVIII added in vitro to samples with low porcine FVIII inhibitor titers. Clots formed in the presence of rpFVIII in samples with low porcine FVIII inhibitor titers were similar to those formed in control samples without inhibitors. In samples with high porcine FVIII inhibitor titers, little improvement in clot structure was observed, indicating that clots formed in response to rpFVIII were more vulnerable to fibrinolysis compared with noninhibitor control samples. Improvement in clot structure and thrombin generation were similarly dependent on rpFVIII dose and low anti-porcine inhibitor titer.   Abbreviations: ETP, endogenous thrombin potential; rpFVIII, recombinant porcine factor VIII. a Anti-porcine inhibitor values as measured by local laboratories and ETP and peak height as measured by central laboratory.

| Epitope mapping
titers. Ultrastructure analysis of fibrin fibers of the clots formed using scanning electron microscopy indicated that they were more vulnerable to fibrinolysis, as demonstrated in a previous report that showed a negative correlation between fibrin fiber diameters and clot resistance to fibrinolysis. 33 In general, rpFVIII restoration of hemostatic markers function in vitro appeared to be dependent on rpFVIII dose and porcine FVIII inhibitory antibody titer but not on the titer of human FVIII inhibitors.
These observations were supported by statistical testing (Spearman rank correlation analysis). An inverse correlation was found between the porcine FVIII inhibitor titer and thrombin generation response to rpFVIII as measured by ETP (2.7-5.4 U/ml rpFVIII) and peak height (2.7-10.8 U/ml rpFVIII) (p < 0.03).
Individual variability in responses to rpFVIII in vitro was noted. In some cases, hemostatic effects could be demonstrated in the presence of high human FVIII inhibitor titers ≥5 BU/ml (thrombin generation normalization was achieved in patients 1, 2, 9, 10, 13, and 14 with high human FVIII inhibitor titers; see Table 2), supportive of evidence that anti-human FVIII inhibitory antibodies might have a lower neutralizing capacity against porcine FVIII. 18-20 Furthermore, normalization of thrombin generation responses was also achieved in some patients from subgroup 3 with high titers for porcine FVIII inhibitors (patients 9, 10, 13, and 14; see Table 2).
In the present study, the observation that rpFVIII corrected thrombin generation responses in patient plasmas with low porcine F I G U R E 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

ACK N OWLED G M ENTS
The study and study report were funded by Baxalta US Inc., a Takeda