After blood vessel damage, effective blood clotting is essential to halt bleeding. However, this process is inefficient in some individuals due to hereditary factors. Hemophilia B, for example, results from a deficiency in coagulation factor IX (FIX), which can lead to prolonged bleeding after injuries or surgery. Thus, patients with hemophilia B are often treated with recombinant FIX as a replacement therapy, and while this approach has improved treatment for hemophilia B, the plasma half-lives of the approved products are only about 3-4 days, and frequent injections are needed. Consequently, there is a pressing need for recombinant FIX products with enhanced plasma half-life that allows less frequent dosing.
In the current study, the laboratories of Professor Jan Terje Andersen at the University of Oslo and Alessio Branchini/Mirko Pinotti at the University of Ferrara (Italy) present design and characterization of long-acting human albumin-fused FIX variants, each exhibiting unique pharmacokinetic properties. The advancement was made possible by the use of an engineered human albumin variant with three amino acid substitutions, E505Q/T527M/K573P (QMP), with increased pH-dependent binding to the neonatal Fc receptor (FcRn), a cellular receptor critical for albumin homeostasis. This albumin variant was fused to engineered FIX variants, and the resulting molecules were shown to have extended but distinct plasma half-lives.
The hyperactive FIX R338L (Padua) amino acid substitution was incorporated alongside an additional modification that affects the interaction of FIX with the extravascular collagen IV reservoir. Specifically, a lysine (K5) was replaced with either alanine (K5A) or arginine (K5R). This modulates the binding affinity between FIX and collagen IV, in that K5A reduces and K5R enhances the interaction. Notably, the Padua substitution counteracts the reduced activity of the K5A variant, as hyperactivity was observed in all albumin-fused FIX fusions that contain FIX Padua. Importantly, the engineered FIX variants were efficiently cleaved from albumin upon activation by FXIa, which is necessary for optimal coagulant activity of FIX.
Furthermore, while the efficacy of replacement therapy is typically assessed based on FIX activity in plasma, it is important to consider that FIX also binds to extravascular collagen IV, which contributes to the overall efficiency of blood clotting. Studies in a hemophilia B mouse model showed that the FIX K5R amino acid substitution enhanced extravascular distribution of Padua-fused albumin QMP. The modification actually improved both distribution and the functional half-life by threefold. Consequently, this engineering resulted in a therapeutic candidate characterized by improved biodistribution and a favorable functional profile. In contrast, the Padua FIX variant with the K5A amino acid substitution demonstrated negligible extravascular distribution, while exhibiting the highest plasma levels at early time points followed by a rapid decline.
Taken together, these findings endorse the utilization of engineered albumin (QMP)-fused Padua K5A and Padua K5R as hyperactive options for short- or long-term therapy, respectively, providing opportunities for personalized hemophilia B replacement therapy.
"Before advancements in treatment, patients with hemophilia B - particularly those with severe cases - faced significantly reduced life expectancy, often succumbing to complications such as uncontrolled intracranial bleeding or joint hemorrhages before reaching adulthood. However, major biotechnological breakthroughs over decades have led to the development of improved replacement therapies capable of effectively managing the disease. Despite these advancements, there remains a substantial opportunity to enhance the design of FIX products, particularly to improve adherence to prophylactic regimens that currently require frequent infusions. In our latest paper, we discuss how engineered human albumin, optimized for enhanced FcRn engagement, can serve as a carrier for FIX variants that are specifically designed to either or not target the extracellular space while maintaining prolonged circulation in the bloodstream. We firmly believe that such biology-guided protein designs can pave the way for more personalized treatment options", says Professor Jan Terje Andersen.
Funding
The study was financed through the Research Council of Norway, the South-Eastern Norway Regional Health Authority, the Novo Nordisk Haemostasis Grants Initiative and by the Fondo di Ateneo per la Ricerca from the University of Ferrara.
Publication
Aaen, K.H., Testa, M.F., Nilsen, J., Tarantino, R., Canepari, C., Benedusi, M., Benjakul, S., Nyquist-Andersen, M., Herigstad, M.L., Cantore, A., Valacchi, G., Sandlie, I., Bernardi, F., Pinotti, M., Branchini, A., and Andersen, J.T. Tailored collagen binding of albumin-fused hyperactive coagulation factor IX dictates in vivo distribution and functional properties. Nat Commun 16, 8433 (2025). https://doi.org/10.1038/s41467-025-62955-9
