PB0252 - NXT007, a novel bispecific antibody, in FVIII chromogenic substrate assays

Background: NXT007 is a bispecific antibody that mimics the cofactor function of activated factor (F) VIII and is currently in early clinical development (Ph1/2) for hemophilia A. Chromogenic substrate assays (CSAs) measuring FVIII activity (FVIII:C) are manufactured using purified human or bovine coagulation factors (Table 1). NXT007 was engineered and optimized based on emicizumab, which shows FVIII-like activity in CSAs containing human, but not bovine coagulation factors.

Aims: To test if NXT007 has FVIII-like activity in CSAs that are manufactured using human, mixed human-bovine or bovine coagulation factors.

Methods: NXT007 was spiked into pooled FVIII-deficient plasma (HRF Inc.) at 0, 5, 10, 15, and 30 µg/mL. Samples were measured using eight CSA kits (Table 1) calibrated with human plasma standards (Hyphen Biophen): Biophen FVIII:C (Hyphen Biophen), Biophen FVIII:C variant (Hyphen Biophen), Technochrom® FVIII:C (Technoclone), Cryocheck (Precision Biologics), ROX FVIII (Rossix), FVIII chromogenic (Siemens), Coamatic FVIII (Chromogenix), and Coatest SP FVIII (Chromogenix). The interference of NXT007 (30 µg/mL) on chromogenic Bethesda assay (CBA) with bovine reagents was tested.

Results: NXT007 concentration dependent increase in FVIII-like activity was seen in CSA with human reagents and using the human-bovine Technoclone kit, which gave the highest FVIII-like activities (137 IU/dL at 30 µg/mL of NXT007) (Table 1). Some FVIII-like activity (9-13 IU/dL at 30 µg/ml NXT007) was observed in the other human-bovine CSAs. No FVIII-like activity of NXT007 was detected in purely bovine CSAs. NXT007 did not interfere with FVIII inhibitor measurements using CBA with bovine CSA.

Conclusion(s): CSAs containing human FX and/or FIXa are sensitive to NXT007 generating surrogate FVIII-like activities, interfering with clinical interpretation if these kits are used to measure FVIII:C or FVIII inhibitors. CSAs with only bovine reagents are insensitive to NXT007 and can be used for measuring FVIII:C and/or in the CBA for inhibitor screening and surveillance in the presence of NXT007.