FVIII-albumin fusion proteins enhance the induction of immune tolerance towards coagulation factor VIII in haemophilia A mice

Abstract

Haemophilia A (HemA) is an X-linked recessive disease caused by the lack of functional coagulation factor VIII (FVIII) causing life-threatening prolonged bleeding events due to a coagulation incapacity. HemA is treated by prophylactic or on-demand infusions of recombinant FVIII (rFVIII), according to the grade of disease severity. Individuals affected by severe haemophilia A are characterised by large deletions or intron inversions in the fviii gene and therefore present a significantly different portion of FVIII compared with the wildtype protein present in healthy individuals. Therefore, intravenously-injected exogenous rFVIII is recognised as a foreign antigen. FVIII-specific B cells are activated upon immunisation and trigger the production of anti-drug neutralising antibodies, known as inhibitors. Presently, the only known method to induce tolerance towards FVIII is the Immune Tolerance Induction regime, known as ITI, which consist of repetitive high-dose FVIII infusions pursued until the inhibitor levels are abrogated. The limitations and poor accessibility of the ITI therapy open the door to alternative strategies to enhance tolerance induction and improve the quality of the individuals’ life. <br /> This work aimed to induce immune tolerance towards exogenous FVIII by the administration of FVIII-albumin conjugates in a haemophilia A mouse model. Here, FVIII-albumin fusion proteins were generated by chemical cross-linking of FVIII to albumin in a 1:1 or 1:5 molar ratio, referred to as FP(1:1) and FP(1:5), respectively. Unlike the ITI models, the fusion proteins were injected intravenously in singular weekly intervals and aligned with FVIII control group. The mechanisms underlying the FVIII-specific immune response were elucidated in vivo by flow cytometry, confocal microscopy and ELISA and by the establishment of an in vitro HemA splenocytes cultures. <br /> Collectively, the data indicated that FVIII-albumin impairs the development of FVIII-specific B cells into activated subtypes reducing dramatically the inhibitor production compared with FVIII alone, while being coagulative active. Fusion protein is transported to the liver where it could be internalised by professional antigen-processing cells, including dendritic cells and potentially by macrophages. Nevertheless, fusion protein treatments affect the co-stimulation of APCs causing the disruption of follicular CD4+ T helper cell activation. CD25+ regulatory T cells may contribute to the suppression of epitope-specific T helper cells. In vitro co-cultures indicated that the Fas/FasL and PD-1/PD-L1 axes may be employed as a potential FVIII-specific B cell suppressive Treg-mediate mechanism. Overall, FVIII-albumin stimulation in vitro failed to stimulate FVIIIspecific B cells and TFH in comparison with FVIII stimulation. <br /> To explore the membrane trafficking of albumin and FVIII-albumin, an uptake assay was established in RAW264.7 macrophages. Here, I evaluated the compartmentalisation of albumin within the recycling or degrading endosomes and its segregation into recycling tubules by confocal microscopy and live cell imaging. FVIII-albumin fusion protein, similar to albumin alone, segregated from FVIII-positive endosomes into tubular structures which may undergo membrane trafficking and recycling. <br /> Overall, FVIII-albumin fusion proteins seem to be a promising therapeutic tool to enhance induction of immune tolerance protocols towards FVIII.