The development and characterization of a long acting anti‐thrombotic von Willebrand factor (VWF) aptamer

Abstract Background Thrombus formation involves coagulation proteins and platelets. The latter, referred to as platelet‐mediated thrombogenesis, is predominant in arterial circulation. Platelet thrombogenesis follows vascular injury when extracellular von Willebrand factor (VWF) binds via its A3 domain to exposed collagen, and the free VWF A1 domain binds to platelet glycoprotein Ib (GPIb). Objectives To characterize the antiplatelet/antithrombotic activity of the pegylated VWF antagonist aptamer BT200 and identify the aptamer VWF binding site. Methods BT100 is an optimized aptamer synthesized by solid‐phase chemistry and pegylated (BT200) by standard conjugation chemistry. The affinity of BT200 for purified human VWF was evaluated as was VWF inhibition in monkey and human plasma. Efficacy of BT200 was assessed in the monkey FeCl3 femoral artery thrombosis model. Results BT200 bound human VWF at an EC50 of 5.0 nmol/L and inhibited VWF A1 domain activity in monkey and human plasma with mean IC50 values of 183 and 70 nmol/L. BT200 administration to cynomolgus monkeys caused a time‐dependent and dose‐dependent effect on VWF A1 domain activity and inhibited platelet function as measured by collagen adenosine diphosphate closure time in the platelet function analyzer. BT200 demonstrated a bioavailability of ≥77% and exhibited a half‐life of >100 hours after subcutaneous injection. The treatment effectively prevented arterial occlusion in an FeCl3‐induced thrombosis model in monkeys. Conclusions BT200 has shown promising inhibition of human VWF in vitro and prevented arterial occlusion in non‐human primates. These data including a long half‐life after subcutaneous injections provide a strong rationale for ongoing clinical development of BT200.


| INTRODUC TI ON
von Willebrand factor (VWF) is essential for the first step in maintaining hemostasis, ie, arresting hemorrhage at sites where there has been a break in the integrity of the vasculature. 1 VWF makes a molecular bridge between the collagen structural matrix that underlies the vascular endothelium and circulating blood platelets, 2 enabling platelets to adhere to sites of vessel damage and initiate formation of thrombus in order to arrest hemorrhage. 3 In addition to its role in maintaining hemostasis, VWF plays a role in shear-dependent thrombogenesis, 4 which can occur in stenotic coronary arteries or ruptured atherosclerotic plaque lesions. 3,5 An exaggerated platelet aggregation response at sites of vascular damage or artherosclerotic plaque rupture can lead to the development of vascular occlusive thrombi. 6 Animal models in mice, pigs, and dogs have shown that VWF deficiency protects against occlusive arterial thrombi. 6,7 Thrombus development in the coronary and cerebral arteries is the most common cause of mortality and morbidity, worldwide, 8 precipitating myocardial infarction or ischemic stroke, respectively. In fact, atherosclerotic lesions causing myocardial infarction or stroke may be ideal targets for VWF inhibitors. 6,[9][10][11][12] Recently, a VWF inhibitor has been authorized for acquired thrombotic thrombocytopenic purpura, 13,14 a rare disease which is complicated by microthrombotic 15,16 or more rarely by even fatal macrothrombotic events such as myocardial 17,18 or cerebral infarction. 14 This could encourage broader development of VWF inhibitors against arterial thrombosis. In this study, we characterized the anti-VWF aptamer BT200 using purified human VWF and plasma from both cynomolgus monkeys and humans. The influence of BT200 on VWF-platelet interaction was evaluated in vitro using human and cynomolgus monkey whole blood and in vivo following intravenous and subcutaneous administration to monkeys. In addition, the effect of BT200 on thrombus formation was evaluated in a femoral artery thrombosis model.

| Discovery of BT100
ARC15105 is a second-generation VWF inhibition aptamer with fully 2′OME modified chemistry that demonstrated inhibition of shear-induced platelet plug formation with a prolonged a half-life. 19 With a very short five base-pair in a typical stem-loop structure, ARC15105 is not stable especially at elevated temperature at 37°C.
In order to increase the structural stability, four more extra basepairs were added to generate BT100 with improved potency (patent #WO/2018/213697).

| Cocrystallization of BT100 with VWF A1 domain
The VWF A1 domain has been previously purified and cocrystallized with a DNA aptamer ARC1172. 20 Similar protein purification, crystallization, and data collection method was used. The diffraction data on the crystal of VWF A1/BT-100 RNA was collected at SSRF beam line 17U1 and processed to 2.09 Angstrom resolution in space group P21212 using XDS. A heterodimer was located in the asymmetric unit when using Phaser as a molecular replacement program with search model PDB ID 1SQ0. After the initial refinement using program Refmac5, the density of RNA was observed. Then, cycles of model building using Coot and refinement was iterated to complete the final structure of VWF A1/BT-100 aptamer RNA.

| In vitro binding of BT200 to purified human VWF
The affinity of BT200 for purified human VWF was evaluated using an enzyme-linked immunosorbent assay (ELISA) method. In To detect HRP, 100 µL of Slow TMB solution was added to each F I G U R E 1 A, Secondary structure of the anti von Willebrand factor aptamer BT200. The letter "m" stands for methylated and the letters ACGU for the nucleobases adenine, cytosine, guanine, and uracil. B, Cocrystal structure of the unpegylated aptamer BT100 with the von Willebrand factor A1 domain

| In vitro inhibition of antibody binding to the VWF A1 domain in cynomolgus monkey and human plasma
Whole blood was drawn from three cynomolgus monkeys into blood collection tubes containing 3.2% sodium citrate (9:1) and plasma was separated by centrifugation at 1800 g. A BT200 stock solution of 1 mg/mL in PBS was diluted into plasma to final concentrations of 1, 3, 10, 30, 100, 300 nmol/L, and 1 and 3 µmol/L.

| Effect of BT200 on VWF-dependent platelet plug formation in cynomolgus monkey and human whole blood
Sixteen cynomolgus monkeys (2.6-3.9 kg, 3-5 years of age) were ob-

| Pharmacokinetics of BT200 in cynomolgus monkeys
In a single-dose pharmacokinetics (PK) study in cynomolgus monkey, BT200 was injected SC at single doses of 0.5, 2, 10 mg/kg (three monkeys per sex and dose group). In addition, BT200 was infused IV at a single dose of 2 mg/kg (three monkeys per sex and dose group) to investigate the bioavailability of BT200.

| Effect of BT200 on FeCl 3 -induced femoral artery thrombosis
The effects of three different single doses of BT200 and a reference to 3% isoflurane with an oxygen exchange rate of 2 L/min. Once animals were fully anesthetized, 1.5% isoflurane was used for maintenance. The experimental animals were placed on a constant-temperature warming plate to maintain a constant body temperature.
Blood pressure, heart rate, and body temperature were monitored throughout the procedure. A blunt instrument was used to expose approximately 2 to 3 cm of the left femoral artery, and the exposed blood vessel was covered with gauze soaked in (37°C) normal saline, to maintain moisture and protect the tissue at the site of vascular separation. A Doppler probe was placed on the exposed left femoral artery. Before placement, the probe was filled with acoustic coupling gel to prevent air bubbles in the gel. The probe was placed perpendicular to the blood vessel. Blood flow was continuously monitored and recorded.
Prior to FeCl 3 treatment, blood flow was monitored for a minimum of 5 minutes; this record served as the baseline blood flow. Two

| BT200 binds to purified human VWF with high affinity
BT200 binding to purified human VWF protein was evaluated at concentrations ranging from 0.03 to 1000 nmol/L. BT200 bound to purified human VWF in a concentration-dependent manner (Figure 2).
The mean concentration resulting in 50% maximal absorbance (EC 50 ) in three independent experiments was 5.0 nmol/L (51 ng/mL) with a coefficient of variation (CV) value of 11%. The results of this in vitro binding study demonstrate that BT200 binds to VWF with high affinity. The results suggest that inhibition of VWF by BT200 in both monkey and human plasma was mediated by blocking the A1 domain.  concentration-dependent increase in CADP-CT (Table 1). Incubation with BT200 concentrations of ≥0.6 μg/mL significantly prolonged CADP-CT (P < .001) when compared to vehicle control. No sexdependent differences were seen (analysis not shown). BT200 also inhibited platelet plug formation in human whole blood in a concentration-dependent manner (Table 1). In human blood, significant prolongation of CADP-CT was seen at concentrations ≥ 0.1 μg/mL.

| Pharmacokinetics of BT200 in cynomolgus monkeys
The PK and ratio of exposure parameters following a single dose of BT200 are presented in Table 2. In summary, plasma exposures as  bbreviations: AUC ∞-OBS , area under the concentration versus time curve from time 0 to the last quantifiable timepoint; AUC last , area under the first moment curve from time 0 to time of last measurable concentration; C 0 , initial concentration at time 0 following bolus intravenous injection; Cl/F obs , apparent total clearance of the drug from plasma after drug administration; Cl obs , clearance of drug; C max , maximum observed concentration; F, bioavailability; ~estimated after dose normalization of AUC values; MRT last , mean residence time; ND, not determined; T 1/2z , elimination half-life; PK, pharmacokinetics; T max , time to maximum concentration after dosing; Vss obs , volume of distribution of drug at steady state; Vz/F obs , apparent volume of distribution in terminal phase after drug administration; Vz obs , volume of distribution following intravenous administration.

| D ISCUSS I ON
von Willebrand factor is an interesting target for primary or secondary prevention of cardiovascular diseases, 26 particularly in high-risk patients. Numerous epidemiologic studies have established that an excess of VWF predicts an increased risk for both stroke 9 and stroke mortality. 27 Conversely, deficiency of VWF may protect against cardiovascular disease or stroke. 28,29 For example, von Willebrand disease patients with average VWF levels of 24% had a 35% to 67% reduced risk for ischemic stroke when compared to controls, suggesting that partial inhibition of VWF could be protective in the stroke-prone population. 28 The underlying pathophysiologic mechanism of large artery atherosclerosis stroke is likely shear-and VWF dependent platelet thrombus formation in the setting of atherosclerotic stenosis. 5,6,9 Importantly, VWF and platelet rich thrombi are more resistant to thrombolysis and are associated with poorer outcome after revascularization in stroke patients. 30,31 At one end extracellular VWF binds to exposed collagen, for example that found in ruptured plaques, via its A3 domain, and at the other end VWF uses its A1 domain to bind to platelet GPIb, thus serving as a bridge between collagen and platelets. 32 VWF, therefore, is a key player in mouse models of acute stroke. 33 Due to its binding to the A1 domain, BT200 pharmacologically blocks the bridge between collagen and platelets. This property confers a degree of anatomic specificity to the pharmacology of BT200, favoring F I G U R E 5 Effect of BT200 on FeCl 3 -induced thrombus formation time in monkeys. Time to reduction of blood flow to <20% of baseline or no blood flow. Time to thrombus formation exceeded the maximum observation period of 2 hours (3600 s) after subcutaneous injection of 1mg/kg BT200. **P = .008, ***P = .005, Compared with PBS control (Kruskal-Walis analysis of variance followed by U test). SEM, standard error BT200 is an agent offering an innovative approach to secondary stroke prevention with a focus on VWF. BT200 is a pegylated synthetic RNA oligonucleotide drug candidate belonging to the medicinal chemical family known as aptamers, 34 which offers the opportunity to rapidly develop a reversal agent. Based on the mechanism of action of BT200, mucosal bleeding will likely be the predominating type of bleeding, such as epistaxis observed under caplacizumab. 13 Consistent with the effect of caplacizumab in baboons, BT200 also prolonged the bleeding time in cynomolgus monkeys. This class effect was associated with increased surgical blood loss under therapeutic doses of caplacizumab (Cablivi European Assessment report). Thus, availability of a reversal agent is a desirable asset in case emergency reversal of VWF inhibition is needed.
In vivo stability of BT200 is enhanced by its methylated oligonucleotide backbone, while it is also pegylated to improve pharmacokinetics and provide a convenient subcutaneous clinical dosing schedule based on the ~3.5 day T 1/2 in non-human primates. This half-life represents a substantial improvement when compared to the 2-hour half-life of the intravenously infused ARC1779, an anti-VWF aptamer coupled to a 20 kD polyethylene (PEG) residue, 35 which successfully established proof of concept in various patient populations. [36][37][38][39] Whereas drugs conjugated with 20 kD PEG are usually filtered renally, molecules with larger PEG sizes (>30 kD) are cleared by the reticulo-endothelial system, 40 which is also true for pegylated aptamers. 34 Despite having a similar PEG size to ARC15105, 19 the stem stabilization found in BT-200 further improved its half-life up to 103 hours after 10mg/kg compared with ARC15105 (~66 hours after 20 mg/kg) or >3-fold compared with a 8 mg/kg dose of the nanobody caplacizumab in cynomolgus monkeys. 41 However, the affinity of BT200 for VWF is in the same low nanomolar range as caplacizumab. The co-crystallography studies showed that BT200 specifically binds to the A1 domain of primate VWF and thereby inhibits VWF binding to platelet GPIb, the first step in the cascade of platelet-mediated thrombogenesis. Functional inhibition of platelet thrombogenesis by BT200 has been demonstrated by in vitro human pharmacology studies and in vivo studies in nonhuman primates described in this study. The reduction of VWF antigen levels by <20% at 24 hours after BT200 injection indicates no major effect on VWF clearance, which is in contrast to the 46% reduction induced by caplacizumab. 42 As VWF antigen levels still fell into the high normal range after BT200 injection, this minor effect is unlikely to play a major role in the effective prevention of arterial thrombosis seen in the Folts model. Preclinical testing of novel anti-platelet drugs in a monkey model is a rarity similar to use of the FeCl 3 model in primates. This was not only necessary due to the limited cross-reactivity of aptamers between species, but it also represents a particular strength of this study. This is because of interspecies differences in hemostasis 43 as well as other differences in important physiological variables between small rodents and larger non-rodents. 44 However, the protective effect of BT200 on arterial occlusion in primates matches the beneficial effects of VWF deficiency or blockade seen in mice, dogs, or pigs using FeCl 3 induced arterial occlusion. 7,45 A further strength of the monkey study was that it included both a placebo and the fibrinogen receptor inhibitor tirofiban as an active control, which was given at a dose that exceeded the loading dose in humans 2.5-fold.

| Limitations
While even potent platelet inhibitors like tirofiban or prasugrel do not optimally inhibit shear-dependent platelet plug formation, 46,47 we cannot exclude that expanding the limited sample size would have resulted in a significant effect of tirofiban. However, the 3R  TA B L E 3 Effect of BT200 on collagen/ adenosine diphosphate-induced closure time following subcutaneous injection to cynomolgus monkeys principle (replacement, refinement, and reduction of animals in research) mandates the use of minimal sample sizes, particularly in non-human primate trials. Whereas proof of concept has previously been established for VWF inhibitors in various animal models of stroke, 6 stroke models for stroke recurrence are unfortunately lacking, as are comorbidities or atherosclerosis in rodent models. 44 However, a human trial has already provided proof of concept that VWF inhibition by ARC1779 markedly reduces the onset and numbers of microembolic signals in patients after carotid endarterectomy, 36 which are strong predictors of stroke. 48 This is encouraging, because other antiplatelet drugs including clopidogrel did not reduce the number of microembolic signals after endarterectomy. 49 In conclusion, BT200 has shown promising inhibition of human VWF in vitro and prevented arterial occlusion in non-human primates. These data including a long half-life after subcutaneous injections provide a strong rationale for ongoing clinical development of BT200.

ACK N OWLED G M ENTS
The authors thank Dr Paul Tarantino and Dr Robert Schaub for their editorial contributions and review of the manuscript. The authors would like to dedicate this work to Prof. Evan Sadler. 50

CO N FLI C T S O F I NTE R E S T
SZ and JG are employees of Guardian Therapeutics, Inc, PH, WH and BJ consultants. Drs SZ, JG, and BJ also have an equity position with Guardian. SG and DW are employees and ZL is the chairman of Suzhou Ribo Life Science Co.

AUTH O R CO NTR I B UTI O N S
All authors contributed to the preparation of the article and met the required conditions for authorship. All authors participated in the design, analysis, and interpretation of the data. All authors revised the article for critical content. All the authors revised the article for critical content.