Introduction/Aim: LR769 is a new second-generation recombinant human Factor VIIa (rhFVIIa) developed for haemophilia treatment. We determined enzymatic properties of LR769 and its interaction with antithrombin, tissue factor, platelets and endothelial protein C receptor (EPCR), compared with NovoSevenRT. Methods: Kinetic enzyme assays and active site titration were used for enzymatic studies. Surface Plasmon Resonance (SPR) was used for determination of binding constants. Cellular binding was determined for platelets and cultured human umbilical vein endothelial cells (HUVEC). Results: The dissociation constant (K d ) for activated platelet binding was in the 1 lM range for both products. At saturation, more LR769 than NovoSevenRT was bound to the platelets. Binding to HUVEC was 25-50% higher for LR769 than for NovoSevenRT. Protein C, soluble EPCR, and anti-EPCR antibody all reduced the binding, indicating specificity for EPCR. . The K d values for binding of LR769 to soluble tissue factor and full-length tissue factor were 8.1 nM and 0.9 nM, respectively, and the K d for binding to soluble EPCR was 41 nM. Conclusion: Overall, LR769 exhibited characteristics similar to NovoSevenRT, but bound EPCR on HUVEC with somewhat higher affinity than NovoSevenRT.
Nucleic acid aptamers are promising ligands for analytical and preparative-scale affinity chromatography applications. However, a full industrial exploitation requires that aptamer-grafted chromatography media provide a number of high technical standards that remained largely untested. Ideally, they should exhibit relatively high binding capacity associated to a very high degree of specificity. In addition, they must be highly resistant to harsh cleaning/sanitization conditions, as well as to prolonged and repeated exposure to biological environment. Here, we present practical examples of aptamer affinity chromatography for the purification of three human therapeutic proteins from various sources: Factor VII, Factor H and Factor IX. In a single chromatographic step, three DNA aptamer ligands enabled the efficient purification of their target protein, with an unprecedented degree of selectivity (from 0.5% to 98% of purity in one step). Furthermore, these aptamers demonstrated a high stability under harsh sanitization conditions (100h soaking in 1M NaOH). These results pave the way toward a wider adoption of aptamer-based affinity ligands in the industrial-scale purification of not only plasma-derived proteins but also of any other protein in general.
Micelle-forming amphiphilic epipodophyllotoxin conjugates were synthesized and evaluated in vivo for their tumor targeting properties.
Congenital Hemophilia A and B treatment may be complicated by the development of inhibitors to the coagulation factors used in the replacement therapies. In such cases factor replacement becomes ineffective, and use of a bypassing agent is needed to treat or prevent bleedings. A recombinant form of activated Factor VII has been available for this purpose. In this study, a new recombinant human Factor VIIa (rhFVIIa, LR769) produced by LFB/rEVO Biologics was tested. The goal is to provide patients with Hemophilia A and B who develop inhibitors a cost-effective alternative treatment option. Recombinant Human Factor VII was activated during the purification process to yield a highly homogenous rhFVIIa product (LR769). Kinetic enzyme assays and binding studies were used to characterize LR769. Active site titration demonstrated approximately 1 mole of active site per mole of protein. The Km and kcat for activation of FX and FIX were determined using an assay containing recombinant human tissue factor and phospholipid. The Kd for binding to soluble tissue factor was 22.3 ± 1.7 nM as measured using a FX activation assay. The apparent second order rate constant for inactivation by human plasma antithrombin was 5.9 ± 0.4 x103 M-1 sec-1. In all kinetic assays, LR769 behaved as expected for rhFVIIa. Binding studies with isolated human platelets were performed by FACS and indicated that binding was dependent on Ca+2. Activation of the platelets with thrombin and convulxin increased the binding of LR769. Binding of LR769 to Endothelial Protein C Receptor (EPCR) on the surface of cultured HEK 293 cells was determined by FACS analysis and confirmed that LR769 binds to EPCR. This was further demonstrated with a surface plasmon resonance assay using purified soluble EPCR. Binding to EPCR was dependent on Ca+2, and could be stimulated by Mg+2. Specificity of the binding was confirmed by the fact that it was inhibited by excess Protein C. Disclosures: No relevant conflicts of interest to declare.
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