Abstract
Hemophilia is an X-linked bleeding disorder resulting in deficiency of FVIII (hemophilia A; HA) or FIX (HB). Despite treatment with infused FVIII or FIX, people with hemophilia still experience joint bleeding and impaired quality of life. Furthermore, the development of neutralizing antibodies to FVIII or FIX remains a major complication of therapy. To this end, several innovative therapeutic advances which rebalance the coagulation system by targeting anticoagulant pathways (antithrombin, tissue factor pathway inhibitor, and activated protein C (APC)) are particularly interesting as they should be effective for both HA and HB, with and without inhibitors, and may also be useful to treat certain rare bleeding disorders. Many of these approaches are in clinical trials and show promise. However, each of these anticoagulant pathways target multiple coagulation factors, and APC plays an important cellular signaling role. In the current study, we developed a monoclonal antibody (GB5) that targets human FV/FVa which could serve as an alternative therapeutic approach to enhance thrombin generation and prevent bleeding in different clinical situations.
Monoclonal antibody GB5 was identified following screening of hybridoma clones derived from mice immunized with human FV. The initial screen identified GB5 as an antibody that could promote thrombin generation (TG) in the presence of APC. To characterize the antibody, purified GB5 was attached to a biosensor tip and evaluated using biolayer interferometry. Analysis of biosensor tracings revealed GB5 bound specifically and with high affinity to both FV (K d = 0.6 nM; 2 experiments) and FVa (K d = 0.7 nM; 2 experiments). A TG assay with pooled normal human plasma (NHP) or HA plasma in the absence or presence of APC was used to assess the functional effects of GB5. In the absence of APC, GB5 had a minimal effect on the TG profile initiated with low tissue factor (0.2 pM). As expected, the addition of APC (2 nM, final) to NHP or HA plasma substantially reduced peak thrombin. However, GB5 dose-dependently increased peak thrombin in the presence of APC in NHP or HA plasma and restored TG to normal levels at ~20 nM. GB5 also protected FV from APC when assessed using a purified prothrombin activation assay. Western blotting experiments using FV proteolyzed with thrombin, APC, or plasmin revealed that GB5 binds the light chain A3-C1-C2.
Preliminary experiments revealed that GB5 does not cross react with mouse FV. To evaluate the effect of GB5 in vivo, wild-type (WT) mice were treated with a liver targeted antisense oligonucleotide (ASO) to knockdown mouse plasma FV (ASO-FV; 40 mg/kg; FV-ASO mice). Mouse platelet FV was not impacted. To supplement the plasma compartment with FV, the FV-ASO mice were administered human FV (hFV-mice). This experimental set-up allowed for clot formation to largely be dependent on hFV using the mouse laser injury model. In these studies, we compared the kinetics of thrombus formation in WT mice, FV-ASO mice, or hFV-mice (n=2-3/group; 7-10 thrombi per group) for 10-15 minutes following laser injury. We found that platelet and fibrin accumulation was robust in WT mice and almost undetectable in FV-ASO mice (consistent with the lack of plasma FV), while clot formation was low but detectable in hFV-mice. There was no obvious difference in thrombus size when GB5 was given to WT or FV-ASO mice. However, in hFV-mice (200 μg/kg hFV), GB5 increased platelet and fibrin accumulation to levels seen with WT mice. Quantitative analysis revealed that compared to hFV mice alone, GB5 increased platelet accumulation 3-fold and fibrin (3-6-fold accumulation. Significantly greater accumulation of platelets and fibrin (~30-fold) was observed when higher amounts of hFV (400 μg/kg) were co-infused with GB5 compared to hFV-mice without antibody.
Together, these results demonstrate that monoclonal antibody GB5 binds to hFV/FVa with high affinity and confers APC resistance. Using a unique mouse model to assess human FV, we found that GB5 enhanced clot formation in vivo using the laser injury model. From a mechanistic standpoint, these data show that protecting FV from APC translates to greater thrombin generation in vivo and suggest this approach may be useful to treat bleeding disorders such as HA and HB. Additional injury models using the hFV-mice and hemophilic mice to assess the effectiveness of GB5 are ongoing.
Disclosures
Crosby: Ionis Pharmaceuticals: Other: Jeffrey is a current employee of Ionis Pharmaceuticals. Revenko: Ionis Pharmaceuticals: Other: Alexey is a current employee of the company.. MacLeod: Ionis Pharmaceuticals: Other: Robert is a current employee of the company. Monia: Ionis Pharmaceuticals: Other: Brett is a current employee of the company. .
Anticoagulant and fibrinolytic activities are promoted, not retarded, in vivo after thrombin generation in the presence of a monoclonal antibody that inhibits activation of protein C
