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Novel DNA aptamer targeting VWF A1 domain
Discussion
VWF plays a pivotal role in the initial phase of platelet thrombus formation under high shear stress through the interaction between its A1 domain and platelet GPIb.1,2 The binding site of the VWF A1 domain is usually cryptic and prevents spontaneous binding to platelets. High shear stress of blood flow induces conformational changes in VWF and exposes the VWF A1 domain. Therefore, the association between VWF A1 and platelet GPIb usually occurs under high shear stress.2
Inhibition of VWF A1 domain binding to platelet GPIb can potentially prevent the development of platelet thrombus formation that causes cardiac infarction, cere- bral infarction, and TTP. TTP results from the formation of platelet thrombi in the microvasculature due to a deficien- cy of ADAMTS13, a VWF-cleaving protease.22,23 Therefore, preventing VWF A1 binding to platelet GPIb could be a promising therapeutic target for TTP. For this purpose, caplacizumab was evaluated for its anti-throm- botic effects in a phase II clinical trial (the TITAN study)11 and a phase III clinical trial (the HERCULES study).12 These studies reported a therapeutic effect of caplacizumab on aTTP, including faster recovery of platelet counts, fewer plasma exchange sessions, and shorter hospital stays. A bleeding event was reported as a common adverse event in patients treated with caplacizumab compared to patients without it.12 In the TITAN and HERCULES study, 36 and 72 patients of aTTP were treated with caplacizum- ab and 19 (54%) and 46 (65%) patients had bleeding events, respectively. The most common adverse events were epistaxis and gingival bleeding, neither of which generally required treatment.
Nucleic acid aptamers are single-strand DNA or RNA molecules that can form 3D structures capable of specifi- cally binding to proteins or other cellular targets. They are superior to existing antibody products in terms of their specificity, manufacturing cost, relatively small size, and non-immunogenicity. Pegaptanib (Macugen®, Pfizer) is the only aptamer approved by the Food and Drug Adminstration in 2004 for the treatment of wet age-relat-
ed macular degeneration blocking vascular endothelial growth factor.24 As of June 2019, no aptamer for the patients with coagulation or thrombotic disorder has been approved. However, some aptamers targeting coagulation factors, ARC 1779 against VWF A1, NU172 (ARCA Biopharma) against factor IIa, and REG1/REG2 (Regado Biosciences) against factor IXa, stepped into clinical tri- als.13,25-27 ARC1779 was developed to target the VWF A1 domain and was evaluated in a phase II clinical trial in patients with aTTP.13 Unfortunately, the recruitment of patients in this trial was terminated without completing the study due to sponsor-related financial issues, but seven patients with aTTP received combined therapy with intra- venous ARC1779 injections and plasma exchange.13 The trial showed no severe adverse events such as bleeding, even in patients with aTTP who had severe thrombocy- topenia.13 A study of healthy volunteers confirmed that this aptamer had an antithrombotic effect and did not cause severe bleeding events.28
TAGX-0004 is a novel DNA aptamer that targets the VWF A1 domain and contains Ds, an artificial nucleic acid. TAGX-0004 was obtained using modified SELEX (sys- temic evolution of ligands by exponential enrichment) methods incorporating the Ds base.14 Since the Ds base has no complementary base in nature, Ds-containing DNA aptamers can have unique 3D structures. The high hydrophobicity of the Ds base may contribute to signifi- cantly high binding affinity to target proteins. In fact, the results of EMSA showed that the TAGX-0004 binding affinity for VWF (KD=2.2±0.9 nM) is 16-fold higher than that of ARC1779 (KD=35.5±1.5 nM) (Figure 3). However, the KD values in this study might be underestimated in comparison with previous reports; the KD value of TAGX- 0004 was shown to be 61.3 pM by a SPR assay,14 and the KD value of ARC1779 was 2 nM in a RI-labeled assay.18 Previous results also indicated that the binding affinity of TAGX-0004 was much higher than that of ARC1779.
The binding affinity of aptamers to VWF A1 domain might affect the interaction between VWF and platelets. Although both TAGX-0004 and ARC1779 inhibited the platelet aggregations which were induced by ristocetin
AB
Figure 3. Biophysical interaction analysis of TAGX-0004 and ARC1779 with electrophoresis mobility shift assay (EMSA). The white arrowhead indicates the complex of wild-type (WT) recombinant von Willebrand factor (VWF) A1 with an aptamer (TAGX-0004 or ARC1779). The black arrowhead indicates unbound aptamer. Increasing the concentration of WT VWF A1 increased the density of complex bands and decreased that of unbound aptamer bands. These experiments were per- formed four times for each aptamer. The dissociation rates (KD) of TAGX-0004 and ARC1779 were 2.2±0.9 nM and 35.5±1.5 nM, respectively.
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