Inhibitor Formation in the Era of Novel Haemophilia Treatment—A Humanized Mouse Model to Answer Our Questions?

In this issue of Thrombosis and Haemostasis, a mouse model of severe haemophilia A with a humanized immune system is described by Oda et al.[1] With this mouse model, the antibody formation directed against factor VIII can be better investigated compared to current factor VIII knock-out models that lack a humanized immune system. This new model opens the opportunity to investigate human-specific aspects of anti-factor VIII antibody formation which remains an important challenge in the treatment of congenital haemophilia A.

Since the 1980s haemophilia treatment has improved substantially.[2] After viral inactivation and generation of fully recombinant clotting factor products, extended half-life products were engineered to achieve higher trough levels and decrease infusion frequency. However, with these products the risk of antibody formation, which occurs in up to one-third of the patients with severe haemophilia A, is still present. Non-factor replacement therapy mitigates this risk, but as it is not capable to fully replace factor substitution therapy, factor VIII replacement therapy is still necessary in case of bleeding, trauma, or surgery. Therewith, the risk of anti-factor VIII antibody development remains. Moreover, new questions arise:

1. How does non-factor replacement therapy affect the risk of inhibitor formation?

   As non-factor replacement therapy replaces prophylactic factor infusions, people with haemophilia will be exposed less to factor replacement therapy, which might decrease the number of patients developing an inhibitor, or maybe only postpone it.[3] In theory, as factor replacement therapy will mainly be necessary at times of tissue damage, the concomitant presence of damage-associated molecular patterns (DAMPs) due to tissue damage at time of factor replacement therapy might even increase inhibitor formation.[4] Will inhibitor development risk still be highest in the first 50 exposure days, or will there be a continued risk every time people are exposed to factor VIII like patients with mild haemophilia?

2. How should we treat patients developing an inhibitor? Is there still a role for immune tolerance induction (ITI)?

   ITI is an effective but burdensome and expensive treatment. Is this still necessary if proper prophylaxis with non-factor replacement therapy is possible? In whom can we await the fall in inhibitor titer in the absence of factor VIII exposure? Is continued factor VIII exposure indicated after successful ITI?

3. What is the effect of the substantially modified factor VIII products, like the new ultra-long half-life (UHL) efanesoctocog alfa, on inhibitor formation?

   Efanesoctocog alfa consists of an Fc fusion protein molecule coupled with the FVIII binding D'D3 domain of von Willebrand factor (VWF), as well as two XTEN® linkers. These engineering steps effectively increase its half-life, and in the clinical trial inhibitor formation was not detected.[5] But the effect of these modifications on inhibitor formation in previously untreated patients needs to be awaited.

In the era of new haemophilia treatment, as the heterogeneity of treatment increases, number of exposure days to factor VIII decreases, hampering the investigation of the effect of these new treatments on inhibitor formation. The mouse model developed by Oda et al provides a tool to study the immune response to new factor VIII products, response after re-exposure at different intervals and with costimulatory signals, immune modulating treatment etc. (see [Fig. 1]).

Fig. 1 Possibilities to investigate the human immune response to factor VIII in humanized factor VIII deficient mice. EHL, extended half-life; ITI, immune tolerance induction; IVIG, intravenous immunoglobulins; LPS, lipopolysaccharide; SHL, standard half-life; UHL, ultra-long half-life.

Oda et al (2025) present a humanized NOG haemophilia A mouse model which is a significant advancement in haemophilia A research, integrating severe factor VIII deficiency with a fully humanized immune system. Unlike traditional factor VIII knock-out mice, which develop inhibitors but lack human immune characteristics, the NOG haemophilia A model enables human-specific immune studies.[6] However, it requires LPS stimulation to induce non-neutralizing anti-factor VIII antibodies, limiting its ability to mimic spontaneous inhibitor formation. HLA-transgenic factor VIII-KO mice improve HLA-restricted factor VIII responses but still lack full human B- and T-cell maturation. Similarly, humanized models via bone marrow transplantation or injection of human peripheral blood mononuclear cells allow for human immune development but face short-lived immune responses and inconsistent inhibitor formation.[7]

The low chimerism in the bone marrow transplanted mouse model by Oda maintains a severe haemophilia A phenotype; thus, it risks compromising immune response reliability. As such it is important to keep a balance maintaining the disease phenotype while ensuring a robust human immune system for accurate factor VIII inhibitor studies.

As a next step, a promising future model is the NOG W41 mouse, which supports functional lymphoid structures and improved B-cell maturation, potentially allowing spontaneous factor VIII inhibitor development. To enhance Oda et al's model, future research could explore next-gen NOG models, optimize factor VIII administration, and test alternative adjuvants to promote more natural immune responses. Irrespective of these future steps, the presented model provides a valuable tool for studying factor VIII immunogenicity, tolerance, and inhibitor formation mechanisms and as such potential applications in preclinical drug testing for new FVIII tolerance induction therapies.

Publikationsverlauf

Eingereicht: 05. März 2025

Angenommen: 05. März 2025

Accepted Manuscript online:
06. März 2025

Artikel online veröffentlicht:
28. März 2025

• References

• 1 Oda A, Furukawa S, Kitabatake M. et al. Generation of a severe hemophilia A humanized mouse model capable of inducing an anti-FVIII immune response. Thromb Haemost 2025
• 2 Mannucci PM. Hemophilia treatment innovation: 50 years of progress and more to come. J Thromb Haemost 2023; 21 (03) 403-412
• 3 Meeks SL, Zimowski KL. Haemophilia in the era of novel therapies: Where do inhibitors feature in the new landscape?. Haemophilia 2024; 30 (Suppl. 03) 95-102
• 4 Schep SJ, Boes M, Schutgens REG, van Vulpen LFD. An update on the “danger theory” in inhibitor development in hemophilia A. Expert Rev Hematol 2019; 12 (05) 335-344
• 5 von Drygalski A, Chowdary P, Kulkarni R. et al; XTEND-1 Trial Group. Efanesoctocog alfa prophylaxis for patients with severe hemophilia A. N Engl J Med 2023; 388 (04) 310-318
• 6 Navarrete A, Dasgupta S, Delignat S. et al. Splenic marginal zone antigen-presenting cells are critical for the primary allo-immune response to therapeutic factor VIII in hemophilia A. J Thromb Haemost 2009; 7 (11) 1816-1823
• 7 Chou SC, Yen CT, Yang YL. et al. Recapitulating the immune system of hemophilia A patients with inhibitors using immunodeficient mice. Thromb Res 2024; 235: 155-163