Molecular Design and Characterization of a Novel, Vwf Independent Coagulation Factor VIII (LAFATE)

We have known for long that most FVIII is carried by vWF in the blood stream, and began to aware that vWF determines the fate of FVIII whatever modification we make on it for its prolonged circulation. Thus, the consideration that vWF binding property of FVIII is to be conserved is now facing change.

The aim of this study is to find out a way to unbind FVIII from endogenous vWF but to stabilize it to generate a novel longer-acting coagulation FVIII. We took a way to shield three major LRP1 binding sites by conjugating PEG and at the same time vWF binding sites by connecting D'D3 domains, the FVIII binding domains of vWF, or its variants using a flexible amino acid linker. We constructed a stable single chain form of FVIII (scFVIII) and employed it as a basic FVIII backbone structure for doing so.

We generated FVIII variants according to this strategy, and evaluated their pharmacokinetic property in hemophilia A mice. Unmodified rFVIII (Advate^®) was used as the reference. The mice were intravenously injected with the reference and test articles at a dose of 125 IU/kg rFVIII, respectively, and blood was sampled 5 min to 72 h or further after administration. Citrated plasma was prepared and analyzed for FVIII activity (Coamatic^®, Chromogenix). PK parameters were determined using non-compartmental modeling with Phoenix WinNonlin 6.4 (Pharsight).

We first generated three different types of FVIII variants containing vWF D'D3 domains (D'D3 monomer, D'D3 dimer, D'D3-Fc dimer linked to C-terminus of C2 domain or N-terminus of A1 domain of FVIII), and then conjugated a 40kDa PEG to the B-domain of each variant site-specifically. In the subsequent pharmacokinetic study in hemophilia A mice, we observed significant prolongation of half-lives from 1.8- to 3.6-fold comparing with Advate^®. The D'D3 monomer linked FVIII exhibited most prolonged half-life, suggesting that a D'D3 monomer is active enough to substantially attenuate binding of endogenous vWF to the chimera.

Recently published electron microscopy and hydrogen-deuterium exchange analyses established the C1 domain as the major binding site for the vWF D'D3 domains on FVIII, and noted additional sites located within the a3 acidic peptide and the A3 and C2 domains of FVIII as being implicated in the FVIII-vWF interaction. In the following experiment, we aimed to find out optimal length of the amino acids linker that would secure easy access of D'D3 domains to A3 and C2 domains of FVIII. Considering the distance between these two domains measurable from the structural information of FVIII, we generated D'D3 monomer connected with 60, 120 and 300 amino acids linkers to scFVIII, respectively, PEGylated them and evaluated their pharmacokinetic properties in hemophilia A mice again. These molecules showed 2.9-, 3.5-, and 2.5-fold more prolonged half-lives than Advate^®, respectively, and the 120 amino acids linker seemed optimal.

Finally we conjugated several PEGs of different type and size, respectively, to the FVIII-D'D3 connected with this optimal linker, and evaluated their PK properties in hemophilia A mice in the next experiment. Consequently, we identified four different PEGs that support best performance, and named these compounds as LAFATE. LAFATE corrected impaired thrombin generation of the FVIII deficient plasma in a concentration-dependent manner, and stopped acute bleeding as effectively as Advate^®, evaluated by the amount of hemoglobin loss for a given time period in a tail-clip bleeding model of hemophilia A mice. LAFATE has a good potential to bring in possible administration of a hemophilia A drug on weekly bases and therefore, warrants further investigation.

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