Abstract
Background
Activated factor VIII (FVIIIa) mimetic antibodies restore hemostasis in hemophilia A by bridging FIXa and FX. Next-generation mimetics such as denecimig and zemocimig have been optimized for improved hemostatic activity following the launch of emicizumab. However, direct comparisons of available mechanistic data are limited due to heterogeneous experimental conditions.
Objectives
This study employed FX activation and thrombin generation assays to assess how the distinct mechanistic properties of the 3 FVIIIa mimetics translate into differences in hemostatic potential.
Methods
Sequence-identical analogs (SIAs) of zemocimig and emicizumab were produced recombinantly and purified. FX activation kinetics in the presence of FVIIIa mimetics were assessed across increasing FIXa concentrations, and apparent dissociation constants and specific activity were derived. Thrombin generation assays were performed in hemophilia A platelet-poor plasma triggered with tissue factor, FXIa, or both.
Results
Complex assembly affinity was highest for zemocimig-SIA, intermediate for emicizumab-SIA, and lowest for denecimig. In contrast, denecimig demonstrated markedly higher specific activity than both comparators, indicating distinct kinetic behavior. However, the FX activation assay represents a simplified system, and its artificial nature limits broader conclusions regarding physiological coagulation. Thrombin generation assays showed comparable thrombin generation profiles for denecimig and zemocimig-SIA, both exceeding emicizumab-SIA in potency.
Conclusion
These data indicate distinct mechanistic strategies among the 3 FVIIIa mimetics, with efficient complex assembly for emicizumab-SIA and zemocimig-SIA versus lower-affinity assembly but higher catalytic activity for denecimig. Despite these differences, zemocimig-SIA and denecimig exhibit comparable thrombin generation potential, supporting similar improvement in hemostatic potential relative to emicizumab-SIA.
Keywords: biomimetics, bispecific antibodies, drug therapy, hemophilia A, humans, therapeutic use, thrombin
1. Introduction
After the launch and clinical experience with emicizumab (Hemlibra), the first activated factor (F)VIII (FVIIIa) mimetic for the treatment of hemophilia A (HA), next-generation FVIIIa mimetic antibodies such as denecimig (Mim8) and zemocimig (NXT007) are currently in clinical development. These antibodies replace the role of FVIIIa in promoting FX activation by mimicking the FIXa–FX bridging and cofactor activity of FVIII. Although FVIIIa mimetics share a common mode of action, they exhibit substantial differences in their interaction with FIXa and FX, which impacts their hemostatic potential [1]. Denecimig and zemocimig have both been optimized for better hemostatic protection than emicizumab [[2], [3], [4]]. The optimization of denecimig focused on enhancing cofactor activity by refining its interaction with FIXa and reducing target binding affinity [2]. The epitopes of the anti-FIXa arm are located within the serine protease domain of FIXa, whereas the epitope of the anti-FX arm overlaps between the epidermal growth factor (EGF)-like domain 2 and serine protease domain of FX. Denecimig stimulated the activity of FIXa to a greater extent than an emicizumab sequence-identical analog (SIA) and showed higher in vitro potency across functional hemostasis assays: approximately 13-fold higher potency for peak thrombin generation in FXIa-triggered severe HA platelet-poor plasma (PPP), based on the 50% effective concentration, and 18-fold higher potency for shortening clot time in tissue factor (TF)-triggered FVIII-neutralized whole blood measured by thromboelastography [2]. The data generated in vitro using an SIA were corroborated in the clinical trial FRONTIER1, in which denecimig demonstrated ∼15-fold higher potency in thrombin generation than emicizumab ex vivo [5]. In contrast to denecimig, which was optimized through altered FIXa–FX engagement, zemocimig was developed from the emicizumab scaffold and was optimized for potency, higher affinity, and lower clearance. The in vivo clearance of zemocimig has been reduced by introducing mutations into the fragment crystallizable (Fc) region [4]. Zemocimig showed substantially higher binding affinity for FX/FXa than emicizumab, while binding overlapping epitopes in the FIXa-EGF1 and FX-EGF2 domains [3,4]. In functional assays, zemocimig increased TF-triggered peak-height thrombin generation in FVIII-neutralized plasma at lower concentrations and with a higher maximum effect than emicizumab. In FVIII-neutralized whole blood (including platelets), zemocimig showed approximately 23-fold higher potency than emicizumab for correction of clotting times and kinetics measured by rotational thromboelastometry [3].
Increased in vitro potency may support lower effective concentrations and potentially increased administration convenience. Even though higher potency can lead to better clinical efficacy, it should not be directly interpreted as a surrogate for superior clinical benefit, as the latter also depends on the exposure and activity level maintained in vivo. While restoration of coagulation potential within the normal range is desirable, increases beyond this range may narrow the therapeutic window and increase the risk of thromboembolic events and thrombotic microangiopathy [6]. Therefore, potency differences among FVIIIa mimetics should be interpreted in the context of clinical safety, thrombogenicity, and dosing.
As these mechanistic data on FVIIIa mimetics were generated using different assays and experimental setups, direct comparison is not possible. We therefore evaluated all 3 FVIIIa mimetics using the same global coagulation assays to assess how their distinct mechanistic properties translate into differences in hemostatic potential.
2. Methods
2.1. Human material
Plasma pools from patients with congenital HA (FVIII level <1%) and normal healthy subjects were obtained from George King Bio-Medical Inc.
2.2. Reagents
Denecimig was produced by Novo Nordisk A/S. Relipidated TF reagent (PPP-low reagent) and phospholipid vesicles (microparticle [MP] reagent) were obtained from Stago. FXIa obtained from Enzyme Research Laboratories had a specific activity of 43.6 U/mg. Human proteins (FX and FIXa) were obtained from Prolytix.
2.3. Production of zemocimig-SIA and emicizumab-SIA
An SIA of zemocimig (zemocimig-SIA) was produced in human embryonic kidney cells based on the published sequence in the World Health Organization drug information [7]. An SIA of emicizumab (emicizumab-SIA) was expressed in Chinese hamster ovary cells based on the published sequence in the World Health Organization drug information [8]. Clarified zemocimig-SIA and emicizumab-SIA harvests were purified by protein A affinity chromatography (MabSelectSure; Cytiva) and eluted with formic acid (pH 3.5), followed by size-exclusion chromatography on a Superdex 200 column (Cytiva). Protein integrity and purity were assessed using size-exclusion ultra-performance liquid chromatography on an ACQUITY system (Waters) equipped with a BIOSEP-SEC-S3000 column (Phenomenex) and a running buffer containing 200 mM sodium phosphate (pH 6.9), 300 mM NaCl, and 10% isopropanol. Molecular masses were determined by electrospray ionization time-of-flight mass spectrometry on a BioAccord instrument (Waters) after desalting on a MassPREP column (Waters). Intact mass analysis of reduced and peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase (PNGase-F)-treated samples was performed. Final protein concentrations were measured on a NanoDrop spectrophotometer (Thermo Fisher Scientific) using theoretical extinction coefficients. Emicizumab-SIA produced with this method has previously been shown to be comparable with emicizumab in functional thrombin generation assays (TGAs) [2].
2.4. Biochemical analysis
FX activation was assessed in the presence of a high concentration of phospholipid vesicles composed of 25% phosphatidylserine and 75% phosphatidylcholine (Prolytix), varying concentrations of FIXa (Prolytix), and 0.1 nM of FVIIIa mimetic (either denecimig, zemocimig-SIA, or emicizumab-SIA) in reaction buffer (50 mM HEPES [N-{2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid}], 100 mM NaCl, 5 mM CaCl2, 0.1% PEG-8000, and 1 mg/mL bovine serum albumin at pH 7.3), as described in Østergaard et al. [2].
2.5. Thrombin generation in human plasma
Thrombin generation was assessed using the calibrated automated thrombography method as described by Hemker et al. [9]. The assay matrix was human HA PPP using either 1 pM relipidated TF (PPP-low reagent), 0.1 mU/mL FXIa (assay concentration, corresponding to ∼1.4 pM) supplemented with 4 μM phospholipid vesicles (MP reagent), or a combination containing 1 pM TF and 0.1 mU/mL FXIa (∼1.38 pM FXIa) in 4 μM phospholipid vesicles (MP reagent) as trigger. The thrombin calibrator was from Stago, and thrombin generation was measured at 37 °C in a Fluoroskan Microplate Fluorometer (Thermo Fisher Scientific), using software from Stago. All samples in the assays were spiked with test compound and prepared in a 1:10 ratio; 90 μL plasma was supplemented with 10 μL test compound. For baseline controls and calibration, a buffer was used instead of the test compound to ensure similar dilution of the plasma samples and controls. Concentrations of individual coagulation factors and test compounds refer to their final concentrations in plasma. Trigger concentrations refer to their final concentrations in the assay well.
3. Results and Discussion
Upon production of zemocimig-SIA and emicizumab-SIA, intact mass analysis showed reduced, deglycosylated light (LC) and heavy chain (HC) masses consistent with the expected architectures of zemocimig (LC1: 23,335 Da, LC2: 22,714 Da, HC1: 49,321 Da, and HC2: 49,008 Da, ± 3-4 Da from theoretical mass) and emicizumab (LC: 23,603 Da, HC1: 49,473 Da, and HC2: 48,988 Da, ± 3-4 Da from theoretical mass).
To activate FX to FXa, a FVIIIa mimetic needs to (1) assemble a complex consisting of FIXa and FX on a procoagulant phospholipid surface; (2) facilitate the FIXa-mediated activation of FX; and (3) enable the release of the generated FXa (Figure 1). To gain insights into the kinetics of the above steps, a specific assay was designed. FX activation rates were measured across increasing FIXa concentrations in the presence of a high concentration of synthetic phospholipid vesicles and a limiting concentration of FVIIIa mimetic (Figure 2A). The high phospholipid concentration drives membrane association of FIXa and FX, favoring complex assembly on the phospholipid surface. The limiting concentration of FVIIIa mimetic allows a saturable increase in FX activation with increasing FIXa. The total FX activation rate reflects the combined contributions of the FIXa–FVIIIa mimetic complex and free FIXa. From this relationship, the apparent dissociation constant (KD) for complex assembly and the specific catalytic activity of the FIXa–FVIIIa mimetic complex can be estimated.
Figure 1.
Kinetic steps of FVIIIa mimetic-mediated FX activation. FIXa, activated factor IX; FVIIIa, activated factor VIII; FX, factor X; FXa, activated factor X.
Figure 2.
Biochemical characterization of FVIIIa mimetic antibodies. (A) FXa generation rate of 0.1 nM denecimig, zemocimig-SIA, or emicizumab-SIA as a function of FIXa assay concentration was assessed in the presence of a high concentration of phospholipid vesicles. (B) Estimated fitted apparent dissociation constants (KD) based on FX activation rates. (C) Estimated specific activity of the FIXa–FVIIIa mimetic complex based on FX activation rates. FIXa, activated factor IX; FVIIIa, activated factor VIII; FX, factor X; FXa, activated factor X; SIA, sequence-identical analog.
3.1. Complex assembly
As denecimig and emicizumab/zemocimig are known to bind different epitopes with varying binding affinity, the kinetics of the individual steps may differ as well. Emicizumab-SIA assembled the FIXa–FVIIIa mimetic–FX complex with an apparent KD of 1.06 ± 0.13 nM (mean ± SD, n = 3), in line with previous data (Figure 2B) [2]. Compared with the emicizumab-SIA, zemocimig-SIA assembled the complex with higher affinity (apparent KD = 0.31 ± 0.06 nM), whereas denecimig assembled the complex with lower affinity (KD = 16.40 ± 11.23 nM). Again, the results for denecimig are consistent with previously published data reporting an assembly KD of 16.0 ± 2.9 nM [2]. The comparatively lower assembly affinity of denecimig is part of its design strategy, aiming to ensure efficient release of FXa and rebinding of new, zymogen FX.
3.2. FIX-mediated FX activation
Contrary to the apparent assembly KD, the specific activity of emicizumab-SIA and zemocimig-SIA were comparable (0.11 ± 0.005 and 0.14 ± 0.013 min−1, respectively; mean ± SD, n = 3), whereas the specific activity of denecimig was much higher (29.6 ± 4.427 min−1) (Figure 2C). The specific activity of FIXa alone in these conditions was (8.4 ± 2.2) × 10−4 min−1. It should be noted that the FX activation assay represents a simplified system designed to isolate specific kinetic steps under controlled conditions. While it allows comparison of relative FX activation kinetics, its artificial nature limits broader conclusions regarding physiological tenase assembly or in vivo hemostatic activity.
3.3. Thrombin generation
Biochemical data showed differences in FX activation kinetics among the tested FVIIIa mimetic antibodies. Despite the apparent differences in the FX activation kinetics, the overall mechanism of the FVIIIa mimetics is the same, and it involves restoration of FX activation and subsequent thrombin generation in the absence of functional FVIII. Thus, the thrombin generation potential of an FVIIIa mimetic can be assessed and compared using TGAs with congenital HA PPP, where coagulation is initiated through either the extrinsic or the intrinsic coagulation pathway using TF and, for example, FXIa, respectively. As such, TGAs provide a comprehensive assessment of the effects of the FVIIIa mimetic on coagulation. Zemocimig-SIA and denecimig exhibited highly similar concentration-responses across peak thrombin height, endogenous thrombin potential, and time to peak in TGAs triggered with TF, FXIa, or a combination thereof (Figure 3). Both zemocimig-SIA and denecimig were more potent and increased thrombin generation compared with emicizumab-SIA, in accordance with previous publications (Figure 3) [2,4,10]. Full thrombograms at different FVIIIa mimetic concentrations are shown in Figure 4.
Figure 3.
Thrombin generation parameters as a function of denecimig, zemocimig-SIA, or emicizumab-SIA plasma concentration. TGAs were conducted in the presence of 4 μM microparticle reagent and triggered with 1 pM TF, 0.1 mU/mL FXIa (∼1.38 pM FXIa) or their combination. TGA values are provided as mean ± SD (n = 3). The light blue area shows the thrombin generation response in a normal human plasma pool (mean ± SD, n = 3). ETP, endogenous thrombin potential; FXIa, activated factor XI; PPP, platelet-poor plasma; SIA, sequence-identical analog; TF, tissue factor; TGA, thrombin generation assay; ttPeak, time to peak.
Figure 4.
Thrombin generation in severe HA plasma spiked with FVIIIa mimetics under different triggering conditions. Representative thrombograms of severe HA PPP spiked with denecimig (left column), zemocimig-SIA (middle column), or emicizumab-SIA (right column). Thrombin generation was triggered using 1 pM TF (top row), a combination of 0.1 mU/mL FXIa and 1 pM TF (middle row), or 0.1 mU/mL FXIa alone (bottom row) as described by Østergaard et al. [2]. The black curve represents NHP analyzed under the same conditions. FVIIIa, activated factor VIII; FXIa, activated factor XI; HA, hemophilia A; NHP, normal human plasma; PPP, platelet-poor plasma; SIA, sequence identical analog; TF, tissue factor.
The general peak thrombin levels reported in this study are slightly higher than those reported by Teranishi-Ikawa et al. [4] for emicizumab, zemocimig, and recombinant human FVIII, despite both studies using PPP-low reagent and similar assay protocols. This difference is most likely explained by subtle variations in assay conditions and reagent lots. Importantly, however, the relative differences between emicizumab, zemocimig, and recombinant human FVIII reported by Teranishi-Ikawa et al. [4] appear to be preserved in the present study using emicizumab-SIA and zemocimig-SIA (Supplementary Material).
3.4. Conclusion
Although all tested FVIIIa mimetic antibodies increase the FX activation rate of FIXa, data presented here indicate that emicizumab-SIA and zemocimig-SIA may exert their activity primarily through efficient assembly of the FIXa–FVIIIa mimetic–FX complex. In contrast, denecimig forms a ternary complex with comparatively lower affinity but compensates through faster complex turnover and markedly increased catalytic efficiency, potentially mediated by allosteric modulation of the FIXa active site. TGAs were used to compare the overall procoagulant potential of the FVIIIa mimetics. Denecimig and zemocimig-SIA demonstrated increased thrombin generation potential compared with emicizumab-SIA. For denecimig, this observation is supported by data from the clinical study FRONTIER5, in which patients previously using emicizumab were switched to denecimig, resulting in an increase in thrombin peak levels to within the normal range [11]. Despite the use of different optimization strategies for denecimig and zemocimig-SIA, these next-generation, ultrapotent FVIIIa mimetics seem to have a highly comparable thrombin generation potential, suggesting a similar improvement in hemostatic potential.
Acknowledgments
Medical writing support for the development of this manuscript, under the direction of the authors, was provided by Mona Blatter, PhD, of Ashfield MedComms GmbH (Mannheim, Germany), an Inizio company, and funded by Novo Nordisk.
Funding
This study was funded by Novo Nordisk A/S.
Author contributions
All authors have substantially contributed to concept and design, analysis and/or interpretation of data; critical writing or revising the intellectual content, and final approval of the version to be published.
Relationship Disclosure
J.L., T.E., and J.R.B. are employees of Novo Nordisk A/S and may hold stock or stock options in the company. M.E. is employed as a consultant to Novo Nordisk A/S and holds stock.
Footnotes
Handling Editor: Professor Michael Makris
The online version contains supplementary material available at https://doi.org/10.1016/j.rpth.2026.106816.
Supplementary material
References
- 1.Lenting P.J., Denis C.V., Christophe O.D. Emicizumab, a bispecific antibody recognizing coagulation factors IX and X: how does it actually compare to factor VIII? Blood. 2017;130:2463–2468. doi: 10.1182/blood-2017-08-801662. [DOI] [PubMed] [Google Scholar]
- 2.Østergaard H., Lund J., Greisen P.J., Kjellev S., Henriksen A., Lorenzen N., et al. A factor VIIIa-mimetic bispecific antibody, Mim8, ameliorates bleeding upon severe vascular challenge in hemophilia A mice. Blood. 2021;138:1258–1268. doi: 10.1182/blood.2020010331. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Locke M., Albiez J., Receveur N., Marbach S., Frey N., Girotra M., et al. Next-generation FVIIIa-mimetic bispecific antibody NXT007: evaluation in preclinical models of hemostasis and thrombosis. Blood Adv. 2026;10:1058–1067. doi: 10.1182/bloodadvances.2025018215. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Teranishi-Ikawa Y., Soeda T., Koga H., Yamaguchi K., Kato K., Esaki K., et al. A bispecific antibody NXT007 exerts a hemostatic activity in hemophilia A monkeys enough to keep a nonhemophilic state. J Thromb Haemost. 2024;22:430–440. doi: 10.1016/j.jtha.2023.09.034. [DOI] [PubMed] [Google Scholar]
- 5.Lentz S.R., Chowdary P., Gil L., Lopez-Jaime F.J., Mahlangu J., Matytsina I., et al. FRONTIER1: a partially randomized phase 2 study assessing the safety, pharmacokinetics, and pharmacodynamics of Mim8, a factor VIIIa mimetic. J Thromb Haemost. 2024;22:990–1000. doi: 10.1016/j.jtha.2023.12.016. [DOI] [PubMed] [Google Scholar]
- 6.Oldenburg J., Mahlangu J.N., Kim B., Schmitt C., Callaghan M.U., Young G., et al. Emicizumab prophylaxis in hemophilia A with inhibitors. N Engl J Med. 2017;377:809–818. doi: 10.1056/NEJMoa1703068. [DOI] [PubMed] [Google Scholar]
- 7.World Health Organization Zemocimig. Recommended INN: List 94. WHO Drug Information. 2025;39:1039–1041. [Google Scholar]
- 8.World Health Organization Emicizumab. Proposed INN: List 113. WHO Drug Information. 2015;29:217–218. [Google Scholar]
- 9.Hemker H.C., Giesen P., AlDieri R., Regnault V., de Smed E., Wagenvoord R., et al. The calibrated automated thrombogram (CAT): a universal routine test for hyper- and hypocoagulability. Pathophysiol Haemost Thromb. 2002;32:249–253. doi: 10.1159/000073575. [DOI] [PubMed] [Google Scholar]
- 10.Lund J., Ezban M., Jensen K., Lillicrap D. Factor VIII in vitro bioequivalence of denecimig (Mim8) hemostatic effect by thrombin generation assays. Res Pract Thromb Haemost. 2026;10 doi: 10.1016/j.rpth.2025.103333. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Oldenburg J., Benson G., Chowdary P., Klamroth R., Lienhart A., Matino D., et al. International Society on Thrombosis and Haemostasis 2025 Congress; Washington, DC, USA: June 21-25, 2025. FRONTIER5 direct switch study: safety of initiating Mim8 prophylaxis without washout of emicizumab. [abstract OC 20.4] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.