FVIII Inhibitors can Accurately be Measured in the Presence of Mim8 in Hemophilia A Plasma

Abstract

Introduction: Mim8 (Denecimig; Novo Nordisk) is a next generation bispecific antibody being developed for prophylactic treatment of hemophilia A with or without inhibitors. Mim8 complexes with FIXa and FX to generate FXa in the absence of FVIII. There is a clinical need to monitor FVIII inhibitors in hemophilia A patients, however measuring FVIII inhibitors in the laboratory can be challenging. Using aPTT-based Modified Nijmegen-Bethesda Assays in the presence of FVIIIa Mimetics such as Mim8 is not recommended due to interference. While FVIII chromogenic substrate assays (CSA) containing human or human/bovine proteins vary in their sensitivity to FVIII Mimetics. This study investigated if bovine FIXa/FX FVIII CSA can accurately measure FVIII inhibitors in the presence of Mim8 in hemophilia A plasma. Methods: Two FVIII CSA kits using bovine FIXa/bovine FX reagents, Factor VIII Chromogenic Assay (Siemens Healthineers) and Chromogenix Coatest ® SP4 FVIII (Diapharma Group, Inc.) were evaluated to investigate whether Mim8 at 5, 10, 20 and 40 ug/mL (final concentration) interferes with the measurement of FVIII inhibitor (Affinity BioLogicals, Inc.) spiked into pooled congenital FVIII deficient plasma (George-King Bio-Medical, Inc.), at 0.2, 1.0 and 4.8 Bethesda Units/mL (BU/mL, final concentration). CSA were analyzed on BCS ®XP analyzers (Siemens Healthineers) using manufacturer or laboratory developed protocols. Inhibitor samples were prepared from a FVIII inhibitor working solution that was assigned a potency of 11.8 BU/mL (Siemens CSA). Mim8 was added to the FVIII inhibitor samples at 20x the final concentration so that the plasma matrix was 95% inhibitor plasma. CRYOcheck™ Factor VIII Inhibitor kit (Precision BioLogic, Inc.) was used to measure the FVIII inhibitor levels. Plasma containing FVIII inhibitor alone or in combination with Mim8 was heat inactivated at 56 ± 2°C for 30 minutes, followed by centrifugation at 2700xg for 5 minutes. The inactivated plasma was incubated at 37 ± 1°C for 2 hours with Imidazole buffered Pooled Normal Plasma (PNP). By standardizing the amount of FVIII in the PNP (⁓100%), the inhibitor levels were determined according to how much of the added FVIII was inactivated relative to a control tube mix with no inhibitor present. Mim8 interference was defined as a negative result (<0.6 BU/mL) becoming positive for samples containing 0.2 BU/mL of FVIII inhibitor, and a result outside of ±25% (%RE) of the corresponding un-spiked sample for the samples containing 1.0 or 4.8 BU/mL of FVIII inhibitor. Results: The data presented for each CSA is from 3 separate aliquots tested on 3 independent occasions. No Mim8 interference was observed at any FVIII inhibitor levels whichever CSA kit, using bovine FIXa or FX reagent, was used with all results for Mim8 spiked samples falling within ±25.0% of the target value. Using the Siemens CSA inhibitor samples at 0.2 BU/mL remained negative (<0.6 BU/mL) with no results reporting >0.4 BU/mL. Inhibitor samples with target values of 1.0 or 4.8 BU/mL, recovered 0.96, 0.99, 0.98 and 0.97 BU/mL and 5.66, 5.69, 5.66 and 5.62 BU/mL, as Mim8 concentrations increased, compared to the un-spiked samples at 1.01 and 5.74 BU/mL, respectively. The spiked samples at 1.0 and 4.8 BU/mL recovered within -2.0 to -5.0%, and -2.1 to -0.9%, of the un-spiked sample, whilst the un-spiked 1.0 and 4.8 BU/mL samples recovered within 1.0% and 19.6% of target, respectively. The Chromogenix CSA recovered 0.90, 0.95, 0.94 and 0.84, as Mim8 concentrations increased, compared to the un-spiked sample at 0.88 at 1.0 BU/mL. At 4.8 BU/mL the results were 5.22, 5.50, 5.57 and 5.72 BU/mL compared to the un-spiked sample of 5.07 BU/mL. The spiked sample at 1.0 BU/mL and 4.8 BU/mL recovered within -4.5 to 8.0%, and 3.0 to 12.8%, of the un-spiked sample. The un-spiked 1.0 and 4.8 BU/mL samples recovered within -12.0% and 5.6% of target, respectively. Conclusion: This study showed that by using FVIII chromogenic assays containing bovine reagents, FVIII inhibitors levels up to at least ⁓5.0 BU/mL can be accurately determined in severe hemophilia A plasma in the presence of up to 40 µg/mL Mim8.