Effects and interferences of NXT007, a novel bispecific antibody, on coagulation assays Free

Background: NXT007 is a bispecific antibody that mimics the function of activated factor (F)VIII and is currently in clinical development for treatment of hemophilia A (HA). NXT007 was engineered and optimized based on emicizumab, which has been shown to interfere with some standard coagulation assays (Adamkewicz et al. Thromb Haemost 2019). The effects of NXT007 in various standard coagulation assays with clotting, chromogenic and immunologic reaction principles were evaluated.

Methods: NXT007 was spiked into healthy volunteer, HA, and FV Leiden thrombophilia plasmas at concentrations of 0, 10, 30 and 100 µg/mL. Plasma samples analyzed for activated partial thromboplastin time (aPTT), and those for the anti-Xa assay, were also spiked with unfractionated heparin to final concentrations of 0.5 and 1.0 IU/mL. Samples with known high international normalized ratio (INR) and D-dimer levels were also used for prothrombin time (PT) and D-dimer assays, respectively.aPTT (Siemens), thrombin time (TT; Stago), PT (Stago, Siemens, and Instrument Laboratories [IL]), derived fibrinogen (IL), Clauss fibrinogen (Stago), activated protein C resistance (APC-R) FV Leiden (Pentapharm), anti-Xa (Stago), antithrombin activity (Stago), plasminogen activity (Stago), plasminogen antigen (Abcam), protein C chromogenic (Stago), protein C aPTT-based (Stago), protein S aPTT-based (Stago), protein S PT-based (IL), D-dimer (Stago), von Willebrand factor (VWF) antigen (IL), VWF activity (IL), FXIII antigen (IL), aPTT-based single factor (Siemens), PT-based single factor (Stago), and chromogenic FIX (Hyphen BioMed) assays were all performed according to the manufacturers' instructions.

Results: NXT007 had a strong effect on aPTT, which decreased to below the limit of detection (<20s) at 10 µg/mL of NXT007 and above in all plasmas. In the presence of heparin, aPTT was prolonged when no NXT007 was present and decreased with increasing NXT007 concentration. Increased INR values were seen at 100 µg/mL NXT007, which is a supratherapeutic level, with all three PT assays tested. Increasing INR values with increasing NXT007 concentrations were observed in samples that had elevated INRs (INR 2–3) before spiking with NXT007. NXT007 had a strong effect on aPTT-based single factor assays (FVIII, FIX, FXI and FXII). In contrast, NXT007 did not interfere with PT-based single factor assays (FII, FV, FVII and FX). NXT007 had no effect on TT. A decrease in PT-derived fibrinogen concentration was seen with increasing NXT007 concentration, but there was no effect on fibrinogen concentration measured by Clauss fibrinogen assay. NXT007 had no effect on APC ratios by PT-based APC-R FV Leiden assay, heparin potency measurements with the anti-Xa assay, anti-thrombin activity, plasminogen activity or antigen concentration. In the aPTT-based protein C and protein S assays, protein C and protein S concentrations decreased with increasing NXT007 concentration. NXT007 had no impact on chromogenic protein C or PT-based protein S concentrations. NXT007 had no impact on D-dimer measurement, VWF antigen or activity, or FXIII antigen concentration. Chromogenic FIX activity decreased at 100 µg/mL NXT007.

Conclusions: As expected based on its mode of action, and similar to emicizumab, NXT007 had a very strong effect on aPTT that resulted in interference with all aPTT-based assays. NXT007 also affected PT at the supratherapeutic concentration of 100 µg/mL, potentially due to inhibition of FX. PT was also affected in samples with an already increased INR. NXT007 interfered with the PT-based derived fibrinogen assay, but not with the PT-based single-factor assays or other PT-based assays (APC-R or protein S). It has been previously shown that NXT007 has different effects on chromogenic FVIII activity assays depending on the species of origin of the FIX and FX in those assays (Kiialainen et al. ISTH 2024: PB0252). In this study, NXT007 affected chromogenic FIX activity at high concentration (100 µg/mL). NXT007 had no effect on other chromogenic assays or on assays with immunologic reaction principles. Coagulation assay interference should be considered when selecting assays for measuring samples from people with HA receiving NXT007, and when interpreting the results of those measurements.