We engineered a bi\paratopic sdAb able to efficiently neutralize the anticoagulant activity of antithrombin 10,000 and 1 25,000 males at birth, respectively (Bolton\Maggs & Pasi, 2003)

We engineered a bi\paratopic sdAb able to efficiently neutralize the anticoagulant activity of antithrombin 10,000 and 1 25,000 males at birth, respectively (Bolton\Maggs & Pasi, 2003). therapies for hemophilia, including non\factor replacement and gene therapy, are showing promising results in the clinic. However, challenges remain, including addressing patients with a history of factor VIII (FVIII) or IX (FIX) inhibitor development. Results Gemigliptin We have developed a novel therapeutic approach for hemophilia, based on llama\derived single\domain antibody fragments (sdAbs). We engineered a bi\paratopic sdAb able to efficiently neutralize the anticoagulant activity of antithrombin 10,000 and 1 25,000 males at birth, respectively (Bolton\Maggs & Pasi, 2003). Hemophilia A and hemophilia B Gemigliptin are clinically indistinguishable, and their treatment is dictated by the clinical severity. Bleedings are usually efficiently prevented or resolved via replacement therapy using plasma\derived or recombinant factor concentrates (Manco\Johnson < 0.0001 compared to FVIII\deficient plasma). Interestingly, the effect of KB\AT\23 was unaffected when anti\FVIII antibodies (titer 8?BU/ml) were present (ETP?=?0.9??0.1?M?min; = 0.998 versus KB\AT\23 alone). In contrast, the KB\AT\23\induced increased thrombin generation could be annulated by the?addition of antithrombin concentrate (2?M; ETP?=?0.2? 0.1?M?min: characteristics of KB\AT\23. To determine its circulatory survival (Fig?3A), we generated two distinct sdAb\fusion proteins. One consisting of KB\AT\23 fused to von Willebrand factor (VWF) residues 1261\1478 (designated KB\AT\23\fus), and a second consisting of the bivalent control sdAb KB\hFX\11 also fused to the same VWF polypeptide (KB\hFX\11\fus). KB\hFX\11 does not bind to murine proteins, while VWF polypeptide is used for detection of both sdAbs. Following intravenous tail vein infusion (10?mg/kg) in wild\type C57BL/6 mice, recovery at 5?min was 93??18% for KB\AT\23\fus and 47??7% for KB\hFX\11\fus (in the absence of FVIII. KB\AT\23 can be efficiently produced and secreted in hepatocyte cell lines Based on the promising results obtained with KB\AT\23 administered as a recombinant protein, we next explored the possibility of constitutively expressing the protein in liver using an AAV vector\mediated gene transfer. In order to develop hepatotropic AAV vectors expressing secretable nanobodies, we first cloned the KB\AT\23 coding sequence carrying a N\terminal His\tag and a C\terminal human influenza hemagglutinin (HA) tag in a hepatocyte\specific expression cassette (Fig?4A). We then generated 5 variants with different N\terminal signal peptides, either derived from heavy chain of human immunoglobulins (Haryadi and shown here. Based on kinetic progress curves in which KB\AT\23 neutralizes antithrombin\mediated inhibition of thrombin and FXa, it appears that KB\AT\23 behaves as a tight binding, competitive inhibitor. This suggests that KB\AT\23 interferes with complex formation between antithrombin and the enzymes. Future work will be aimed at elucidating the mechanism of action and the specific epitopes in more detail. It is worth noting that the duration of gene silencing by fitusiran is strongly dependent on the siRNA intracellular turnover (Bartlett & Davis, 2006), while KB\AT\23 activity depends entirely on its binding to circulating antithrombin. Free KB\AT\23 is rapidly eliminated, while antithrombin\bound KB\AT\23 is removed from the circulation in an antithrombin\dependent manner. This allows for a Gemigliptin rapid reversal of the treatment if needed, for instance in the event of thrombosis (Dargaud thrombin generation experiments, using a molar excess of sdAb over antithrombin, we noticed that the ETP was increased 1.5\ to 2\fold (Table?2), potentially raising Gemigliptin questions on whether this approach would pose an increased risk of thrombosis. However, it should be pointed out that in the plasma\based thrombin generation assay, the anticoagulant pathways are under\represented: Only 10% of the tissue Rabbit Polyclonal to JAK2 factor pathway inhibitor molecules is present, while cellular thrombomodulin (needed to activate the activated protein C pathway) and protease nexin\1 (a strong inhibitor of thrombin released from platelets) are both absent in these assays. It stands to reason therefore to assume that the thrombin generation assay in FIX\ or FVIII\deficient plasma in the presence of an antithrombin inhibitor can result in artificially exaggerated thrombin generation. Indeed, the presence of KB\AT\23 results in near\normalization of the bleeding tendency, while D\dimer levels (a marker for thrombosis) were not increased even after prolonged exposure to KB\AT\23. Based on these considerations, sdAbs could become a useful tool to improve the prophylactic treatment of hemophilia.. Gemigliptin