Developing A Novel Antibody Specifically Targeting The Active VWF A1 Domain

BackgroundVWF is a critical adhesive glycoprotein that plays a crucial role in the process of coagulation and thrombosis.Under normal physiological conditions,VWF stays as a globular inactive form that is incapable of binding to platelet receptor GPIba,as the platelet-binding site on A1 domain is masked and thus does not form clot.Facing with high shear stress in physiological or pathological hemostasis process,VWF is anchored to exposed collagen and its elongation and relaxation is enough to expose VWF A1 domain sequences,which is considered as active form,thus tethering platelets adhesion to the damaged vessels to form clot.Therefore,platelet clot formed on damaged vessels is mediated only by active VWF A1 domain.The deficiency or abnormal of VWF is considered to be a common cause of blood diseases like thrombosis and thrombotic thrombocytopenic purpura(TTP).The advent of FDA-approved humanized Nanobody Caplacizumab(Cablivi)in 2009 has brought breakthrough for TTP treatment as it was designed to prevent the interaction between VWF A1 domain and platelet.However,the non-selective binding of Cablivi to active and inactive VWF A1 domain,resulting in the significant decrease in plasma VWF and FⅧlevels,which is associated with mild or severe epistaxis,gingival bleeding symptoms in patients received Cablivi therapy.This non-specific combination also leads to frequent and high-dose injection of Cablivi,which increases the treatment cost.Imagine that if a Cablivi-like antibody that specifically targets active VWF A1 domain,the additional bleeding tendency during therapy would be significantly reduced.The introduction of such antibodies that specifically targeted active VWF A1 domain into clinical therapy may lead to dramatic improvement of drug development for VWF-related diseases such as thrombosis and TTP.ObjectivesThis study aims to develop a novel antibody specifically targeting the active VWF A1 domain for thrombosis and TTP treatment.Methods1.Design of 11A5 antibodyWe identified a conformation-sensitive loop region(residues 1330-1340),which is only exposed in the active VWF A1 domain on the basis of hydrogen-deuterium exchange mass spectrometry analysis.We used this sequence as an antigen and injected it into mice to screen a monoclonal antibody named 11A5 by hybridoma technique.2.The binding specificity of 11A5 to active and inactive VWFIt has been reported that residues 1261-1472 on A1 domain represent active VWF form that can spontaneously bind to platelets.The binding specificity of 11A5 to active and inactive VWF forms was evaluated by ELISA and an agarose gel.3.Assessment of the bleeding risk associated with 11A5We used a polyclonal antibody(pAb)with similar effects to Cablivi and compared with 11A5 in terms of bleeding risk.PBS,pAb and 11A5 were injected through the tail vein,respectively.The fluoresecent emission spectrum of GFP-VWF mouse plasma was recorded by a fluorescence spectrophotometer.The activity of FⅧ in WT mouse plasma was detected by the FⅧ:C kit.The tail bleeding time was calculated.4.Therapeutic effects of 11A5 on TTP mouse modelTwo groups of Adamts13-/-mice were treated with PBS and 11A5,respectively.TTP mouse model was induced by a mixture of DDAVP and Collagen through intraperitoneal injection.Platelet counts were analyzed using an automated hematology analyzer and hemolysis was analyzed via preparing blood smear.5.Effects of 11A5 on FeCl3-induced mouse carotid arterial thrombosis formationTwo groups of GFP-VWF mice were treated with PBS and 11A5,respectively.To induce the formation of carotid artery thrombus in mice,a filter paper strip that soaked in 5%FeCl3 was placed on the left carotid artery of mice after isolated.The effects of 11A5 on arterial thrombosis were analyzed by thrombus fluorescence intensity and area.6.Effects of 11A5 on mouse inferior vena cava thrombosis formationWild-type mice were injected with PBS and 11A5 through the tail vein,respectively.Mouse deep venous thrombosis formation model was induced by inferior vena cava ligation.The weight and size of thrombus were weighed and measured.Results1.11A5 recognizes only active VWF within a certain concentration rangeThe results of agarose gel showed that the different distribution of active rVWF multimers can be stained by 11A5.ELISA analysis showed that 11A5 can recognize only active VWF forms in the concentration range of 10-9～10-6 g/L.2.11A5 is associated with a lower risk of bleedingOn one hand,plasma levels of VWF(1 h,p=0.8338;4 h,p=0.7534;8 h,p=0.9132)and FⅧ(p=0.7390)had no significant changes in 11A5-treated group compared with blank control,while a significant decrease of plasma VWF(1 h,p=0.1200;4 h,p=0.0442;8 h,p=0.0037)and FⅧ(p=0.0033)levels was found in pAb-treated group due to non-specific binding between pAb and inactive VWF;On the other hand,the tail bleeding time counted in 11A5-treated group was dramatically reduced than that in pAb-treated group(p=0.0027).Therefore,11A5 is more biosafety that causes less bleeding risk than non-specific antibodies.3.11A5 alleviates thrombocytopenia and inhibits hemolysis in TTP mouse modelIn TTP mouse model,we found that the administration of 11A5 alleviated thrombocytopenia and inhibited hemolysis symptoms(p<0.0001).The results showed that 11A5 has preventive and therapeutic effects on TTP mouse model.4.11A5 inhibits FeCl3-induced mouse carotid thrombosis formationIn FeCl3-induced mouse carotid thrombosis model,we found that the onset of thrombus formation was delayed in the 11 A5-treated group and the maximum fluorescence intensity(p=0.0118)and area(p=0.0074)of thrombosis was significantly reduced than those in blank control.It is suggested that 11A5 shows a prominent positive effect on preventing arterial thrombosis formation.5.11A5 inhibits mouse inferior vena cava thrombosis formationIn the mouse deep vein thrombosis model,the results showed that the frequency of thrombus formation,the weight(p=0.0391)and size(p=0.0020)of thrombosis dramatically decreased in the 11A5-treated group than those in the blank control.These date suggest that 11A5 can effectively inhibit venous thrombosis formation.ConclusionsThe main conclusions are:1)11A5 specifically targets the active VWF A1 domain;2)11A5 has little effect on plasma VWF and FⅧ levels,significantly reducing bleeding risk;3)The application of 11A5 can alleviate thrombocytopenia and hemolysis in TTP mouse model;4)The application of 11A5 can significantly reduce mouse arterial thrombosis formation;5)The application of 11A5 can effectively reduce mouse deep venous thrombosis formation.