BIVV001, a new class of factor VIII replacement for hemophilia A that is independent of von Willebrand factor in primates and mice

Abstract

Factor VIII (FVIII) replacement products enable comprehensive care in hemophilia A. Treatment goals in severe hemophilia A are expanding beyond low annualized bleed rates to include long-term outcomes associated with high sustained FVIII levels. Endogenous von Willebrand factor (VWF) stabilizes and protects FVIII from degradation and clearance, but it also subjects FVIII to a half-life ceiling of ∼15 to 19 hours. Increasing recombinant FVIII (rFVIII) half-life further is ultimately dependent upon uncoupling rFVIII from endogenous VWF. We have developed a new class of FVIII replacement, rFVIIIFc-VWF-XTEN (BIVV001), that is physically decoupled from endogenous VWF and has enhanced pharmacokinetic properties compared with all previous FVIII products. BIVV001 was bioengineered as a unique fusion protein consisting of a VWF-D'D3 domain fused to rFVIII via immunoglobulin-G1 Fc domains and 2 XTEN polypeptides (Amunix Pharmaceuticals, Inc, Mountain View, CA). Plasma FVIII half-life after BIVV001 administration in mice and monkeys was 25 to 31 hours and 33 to 34 hours, respectively, representing a three- to fourfold increase in FVIII half-life. Our results showed that multifaceted protein engineering, far beyond a few amino acid substitutions, could significantly improve rFVIII pharmacokinetic properties while maintaining hemostatic function. BIVV001 is the first rFVIII with the potential to significantly change the treatment paradigm for severe hemophilia A by providing optimal protection against all bleed types, with less frequent doses. The protein engineering methods described herein can also be applied to other complex proteins.

Graphical abstract

Figure 1.

Development of BIVV001. (A) Construct 1: rFVIIIFc-VWF(DʹD3). rFVIIIFc is stabilized by covalently attaching a D1D2DʹD3 (C1099A/C1142A) domain of VWF through immunoglobulin-G1 Fc molecules, which prevents interaction of rFVIIIFc with endogenous full-length VWF. A R1648A mutation in FVIII prevents FVIII processing into heavy and light chains. Construct 2: rFVIIIFc-VWF-XTEN. One 288-amino-acid XTEN polypeptide is inserted into the B-domain region of FVIII, a second 144-amino-acid XTEN polypeptide is inserted between DʹD3 and Fc. Construct 3: rFVIIIFc-VWF-XTEN. The LVPR thrombin site located between DʹD3 and Fc on constructs 1 and 2 was replaced with an FVIII acidic region 2 (a2) thrombin site. Construct 4: rFVIIIFc-VWF-XTEN (BIVV001). Three amino acid residues (GAP) from the FVIII/XTEN junction and 9 amino acids (PPVLKRHQA) from the FVIII B-domain linker were removed to avoid potential MHCII-binding sites. (B) BIVV001 is generated by coexpressing 2 polypeptide chains in HEK293 cells, a human cell line. Purified component parts of BIVV001 were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; BioRad stain-free gel; 4%-20%) under reduced (R) and nonreducing (NR) conditions. See supplemental Methods for more details. FVIII-XTEN-Fc portion is expressed in the first chain (band F8; NR and R lane) and DʹD3-XTEN-Fc portion (band VD; R lane) is expressed in the second chain. The 2 chains are held together by disulfide bonds in the Fc region (heterodimer band [HD]; NR lane). The D1D2 (propeptide) is removed during intracellular processing. Size-exclusion chromatography (SEC) was performed using high-performance liquid chromatography (1260 Bioinert) with a BEH450 column and run isocratically at 0.3 mL/min for 18 minutes. (See supplemental Methods for more details.) The SEC profile of BIVV001 under nonreducing conditions is shown; the predicted molecular mass of BIVV001 is ∼312 kDa, but, because of the presence of XTEN, it has a large hydrodynamic radius and elutes before 670 kDa. MW, molecular weight; PACE, paired basic amino acid cleaving enzyme; Tyr, tyrosine.

Figure 2.

Expression and PK properties of the VWF D′D3 domain. (A) Plasmids expressing VWF fragments were administered to VWF-knockout mice by hydrodynamic injection (HDI), to identify the VWF domains necessary for optimal endogenous FVIII stabilization. Plasma FVIII activity was measured in an FVIII chromogenic activity assay before and 48 hours after DNA injection. Individual data points are shown with the horizontal lines depicting the mean. (B) The rFVIIIFc half-life was compared in FVIII/VWF DKO mice expressing VWF-010 and VWF-013 to determine whether the dimeric (VWF-010) or monomeric (VWF-013: D1D2DʹD3C1099A/C1142A) DʹD3 is comparable in protecting FVIII. rFVIIIFc (200 IU/kg) was administered via the tail vein, and the plasma FVIII activity was measured with the chromogenic assay. FVIII activity in mU/mL was plotted against time. Data are means ± SD; n = 4 mice per time point. (C) The PKs of rFVIIIFc-VWF(DʹD3; 200 IU/kg IV) were evaluated in HemA and DKO mice, using a methodology similar to those described in panel B. Plasma FVIII activity was normalized to the dose of factor administered and was plotted against time. Factor half-life (t_1/2) was calculated by noncompartmental modeling using WinNonLin, version 5.2, (Pharsight Corp, Mountain View, CA). Data are means ± SD; n = 4 mice per time point.

Figure 3.

PK profiles of constructs 3 and 4. PK profiles were evaluated using methods described in Figure 2. (A) The PK of construct 3 was compared with rFVIIIFc and rFVIII in HemA mice. The PK of rFVIII (B), rFVIIIFc (C), and construct 3 (D) were compared in HemA, VWF heterozygous (VWF Het), and FVIII/VWF DKO mice. Doses from 125 to 200 IU/kg were used for IV injection, and plasma FVIII activity was normalized to the dose of factor administered and was plotted against time. Half-life (t_1/2) was calculated by noncompartmental modeling using WinNonLin, version 5.2 (Pharsight Corp, Mountain View, CA). Data are means ± SD; n = 3 to 4 animals per group for all mouse studies. (E) The PK of BIVV001 (construct 4; 100 and 300 IU/kg IV) was evaluated in cynomolgus monkeys. FVIII activity was measured with a capture chromogenic assay (Act) or enzyme-linked immunosorbent assay (ELISA) (Ag). For capture chromogenic assay, a biotinylated anti-XTEN monoclonal antibody was used to capture BIVV001 and prevent interference from endogenous FVIII. For ELISA, antihuman FVIII (GMA-8023) was used to capture BIVV001 and prevent interference from endogenous FVIII. Data are means ± SD; n = 4 per group with 4 animals per time point.

Figure 4.

HLA DR allele binding analysis of novel mutations and junctions in construct 3 to identify potential neoepitopes. (A) Construct 3, rFVIIIFc-VWF-XTEN, contains an R1648A mutation in the FVIII chain, introduced to avoid cleavage at R1648 and prevent intracellular processing into heavy and light chains. In the D3 domain, amino acids Cys1099 and Cys1142 were mutated to Ala to prevent DʹD3 dimerization. (B,C) Amino acid binding predictions to various HLA DR alleles were evaluated with the NetMHCIIpan, version 3.0, method, as described previously. Binding predictions are depicted before (B; red box; top) and after (B; red box, bottom) mutating WT-R1648 into R1648A in construct 3. After deleting the 9 amino acid residues from the FVIII B-domain linker that captures R1648A (C; green box, top), and before (C; red box; top; green box, bottom) deleting the GAP residues at the FVIII-XTEN junction. GAP residues were from XhoI restriction enzyme site, originally introduced to allow XTEN insertion into FVIII during cloning. IC₅₀, half-maximal inhibitory concentration.

Figure 4.

HLA DR allele binding analysis of novel mutations and junctions in construct 3 to identify potential neoepitopes. (A) Construct 3, rFVIIIFc-VWF-XTEN, contains an R1648A mutation in the FVIII chain, introduced to avoid cleavage at R1648 and prevent intracellular processing into heavy and light chains. In the D3 domain, amino acids Cys1099 and Cys1142 were mutated to Ala to prevent DʹD3 dimerization. (B,C) Amino acid binding predictions to various HLA DR alleles were evaluated with the NetMHCIIpan, version 3.0, method, as described previously. Binding predictions are depicted before (B; red box; top) and after (B; red box, bottom) mutating WT-R1648 into R1648A in construct 3. After deleting the 9 amino acid residues from the FVIII B-domain linker that captures R1648A (C; green box, top), and before (C; red box; top; green box, bottom) deleting the GAP residues at the FVIII-XTEN junction. GAP residues were from XhoI restriction enzyme site, originally introduced to allow XTEN insertion into FVIII during cloning. IC₅₀, half-maximal inhibitory concentration.

Figure 5.

In vitro hemostatic potential of BIVV001. (A) The effect of BIVV001 on whole-blood clotting time was assessed by using ROTEM and the extrinsically activated test with tissue factor assay. Whole blood from a blood donor with HemA was spiked with increasing concentrations of BIVV001 or rFVIII to obtain FVIII activities of 0% to 60% (based on a 1-stage activity assay). The procoagulant activity of BIVV001 and rFVIII was assessed by measuring peak thrombin concentration (B) and endogenous thrombin potential (C) using a thrombin generation assay. Congenitally FVIII-deficient plasma was spiked with BIVV001 and rFVIII (FVIII activities of 1.6%-100%). Representative curves are shown for ROTEM and thrombin generation assay. (D) The procoagulant properties of BIVV001 and BDD FVIII as precursors to the Xase complex (FVIIIa-FIXa) were assessed using an FXa generation assay, as previously described.^,, Xase complex K_m values were calculated by measuring the conversion of FX to FXa, by using an FXa-specific chromogenic assay. Xase activity was assessed in the presence of Ca²⁺ and resting (R) or SFLLRN-activated human platelets (in duplicate).

Figure 6.

Efficacy of BIVV001 in HemA mice. (A,B) Acute hemostatic efficacies of BIVV001 (37.5, 75, and 150 IU/kg; all IV and n = 14 for each dose), equivalent doses of rFVIII (n = 14 for each dose), and vehicle (n = 14) were evaluated in HemA mice in an acute-blood-loss, tail-clip model. C57BL/6 mice (n = 15) served as the positive control. (A) Individual median blood loss over 30 minutes was quantified gravimetrically and compared between groups with the Kolmogorov-Smirnov t test. Horizontal bars are medians. (B) The state of being “protected” was granted if blood loss was less than or equal to the mean volume of blood lost by C57BL/6 mice in the tail-clip model. The ED₅₀ was the dose of each treatment that achieved protection from acute bleeding in 50% of treated animals. ED₅₀ values were 81 IU/kg for BIVV001 and 105 IU/kg for rFVIII. (C) The effect of prophylactic single-dose BIVV001 (1.2-120 IU/kg; IV), equivalent doses of rFVIII, and vehicle on 24-hour survival rates of HemA mice (n = 15-20 per dose group) after tail vein transection were assessed. BIVV001 and rFVIII were administered at 96 and 24 hours, respectively, before tail vein transection. (D) Twenty-four-hour survival rates after BIVV001 and rFVIII administration were compared with those obtained with vehicle (log-rank Mantel-Cox test). FVIII led to significantly higher survival rates at 24 hours after TVT (P < .05) compared with vehicle. Direct survival comparison showed no significant difference between BIVV001 and rFVIII, indicating that BIVV001 is comparable to rFVIII; n = 15 to 20 mice per group. Similar survival protection ED₅₀ values for BIVV001 and rFVIII were calculated by the nonlinear fit equation.