Type: Oral
Session: 321. Coagulation and Fibrinolysis: Basic and Translational: Coagulation Protein: Molecular Insights and Molecular Therapies
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Translational Research
We first generated a single chain antibody fragment (scFv) of the FIXa binding arm of emicizumab by joining the variable heavy chain (VH) with the variable light chain (VL) to produce VH9VL. In line with previous observations that FIXa one arm antibodies of emicizumab that can only bind FIXa and impart a modest cofactor mimetic activity, VH9VL, facilitated FIXa-mediated FX activation at a much lower rate in comparison to bispecific emicizumab. The scFv, VH9VL, was used as a template to introduce membrane binding properties.
The Factor V C2-domain, which binds membrane, was fused at the C-terminus of VH9VL via a repeating Gly4Ser flexible linker of ~ 5.7 nm to allow the VH and VL domains of the scFv to potentially orient correctly at the membrane surface. The resulting construct VH9VLC2 showed a substantial amplification in cofactor mimetic activity as compared to VH9VL. In vitro kinetic analyses with purified proteins and synthetic phospholipid vesicles containing phosphatidylcholine (PC) and phosphatidylserine (PS) (PC:PS, 75:25 %) showed approximately 22-fold faster activation of FX by VH9VLC2 as compared to VH9VL. Remarkably, despite its inability to bind FX, cofactor mimetic activity of VH9VLC2 was about two-fold higher than that of emicizumab. Potent enhancement of FX activation by VH9VLC2 observed with purified proteins was also reflected in thrombin generation assays (TGA). VH9VLC2 added at 100 nM brought thrombin generation into the reference range in congenital FVIII-deficient HA plasma supplemented with 4 µM PC:PS and triggered with either low tissue factor (0.1 pM), or factor XIa (0.1 nM).
The role of C2 domain-mediated increase in cofactor activity of VH9VLC2 was further assessed by using an antibody fragment (E9scFv) that inhibits the binding of factor V to membranes. Increasing concentrations of E9scFv caused a dose-dependent reduction in the rate of FXa formation catalyzed by the complex of VH9VLC2: FIXa (20nM:25nM). Inhibition saturated at ~ 85% at 100 nM E9scFv, indicating a substantial contribution of membrane binding to the cofactor mimetic function of VH9VLC2.
To evaluate whether VH9VLC2 can restore clotting in HA plasma with FVIII inhibitors, normal pooled plasma was supplemented with a FVIII neutralizing antibody to mimic FVIII inhibitor plasma. Addition of VH9VLC2 to this plasma restored clotting time to the levels shown by normal pooled plasma, suggesting that VH9VLC2 can bypass FVIII inhibitor activity.
The engineered scFv with a factor V C2 domain fusion, VH9VLC2, demonstrates that mere addition of a membrane binding feature to the FIXa arm of the emicizumab surpasses the mimetic activity achieved by a bispecific antibody that exploits both bridging and allostery to be an effective cofactor. Such a construct also avoids the burden of productive assembly from a combination of three separate polypeptide chains, as in a functional bispecific antibody. Replicating the membrane binding feature into bispecific FVIIIa mimetic antibodies could further amplify function and more closely resemble FVIIIa in both activity and membrane dependent regulation. Our results provide surprising insights into how a single chain, monospecific, membrane-anchored FVIIIa mimetic enhances the catalytic activity of FIXa.
Disclosures: Kumar: Children's Hospital of Philadelphia: Patents & Royalties. Krishnaswamy: Bayer: Research Funding; VarmX: Consultancy.