Maintenance and break of immune tolerance against human factor VIII in a new transgenic hemophilic mouse model

Abstract

Replacement of the missing factor VIII (FVIII) is the current standard of care for patients with hemophilia A. However, the short half-life of FVIII makes frequent treatment necessary. Current efforts focus on the development of longer-acting FVIII concentrates by introducing chemical and genetic modifications to the protein. Any modification of the FVIII protein, however, risks increasing its immunogenic potential to induce neutralizing antibodies (FVIII inhibitors), and this is one of the major complications in current therapy. It would be highly desirable to identify candidates with a high risk for increased immunogenicity before entering clinical development to minimize the risk of exposing patients to such altered FVIII proteins. In the present study, we describe a transgenic mouse line that expresses a human F8 cDNA. This mouse is immunologically tolerant to therapeutic doses of native human FVIII but is able to mount an antibody response when challenged with a modified FVIII protein that possesses altered immunogenic properties. In this situation, immunologic tolerance breaks down and antibodies develop that recognize both the modified and the native human FVIII. The applicability of this new model for preclinical immunogenicity assessment of new FVIII molecules and its potential use for basic research are discussed. (Blood. 2011; 118(13):3698-3707) IntroductionHemophilia A is an X-linked bleeding disorder that is caused by reduced function or lack of clotting factor VIII (FVIII). 1 Replacement of the missing protein is the current standard of care for patients. However, the short half-life of FVIII, ϳ 7-17 hours, 2 makes frequent treatment necessary. Current efforts focus on the development of longer-acting FVIII concentrates, which should decrease the required treatment frequency and therefore improve the quality of life for patients. Recently described approaches in the development of longer-acting concentrates are based on strategies that have been successfully applied to other therapeutic proteins: chemical modifications such as the addition of polyethylene glycol (PEG) polymers, polysialic acids, or hydroxyethyl starch 3,4 ; alternative formulations with PEG-modified liposomes 3 ; and fusion to the Fc part of human IgG. 5 In addition, molecular modifications of the FVIII protein aimed at increasing the duration of its cofactor activity or reducing its clearance in vivo have been reported. 3 Any chemical or molecular modification of the FVIII protein can potentially increase its immunogenic potential. Modifications could generate neo-epitopes for both B and T cells or may induce altered structures that could bind and trigger receptors expressed on cells of the innate immune system, thereby amplifying potential anti-FVIII antibody responses. 6 Finally, modifications could generate repetitive epitopes for B cells that might cause the activation and differentiation of B cells and the subsequent production of antibodies without the requirement for T-cell help. 7,8 Therefore, the potential impact that any...

Dangerous liaisons: how the immune system deals with factor VIII

“…Moreover, studies performed in hemophilic mice, rats and rabbits showed a lower incidence of anti-FVIII antibodies in animals infused with PEGylated FVIII than in those infused with its recombinant non-PEGylated counterpart. On the other hand, a recent report by van Helden et al [48] indicated that PEGylation of FVIII could also result in an FVIII protein that expresses higher immunogenicity in hemophilic mouse models. Also, incorporation of PEGylated lipids into complexes of FVIII and phosphatidylinositol resulted in increased immunogenicity following intravenous infusion in a murine hemophilia A model [49].…”

Animal Models of Hemophilia

“…34 The expression of hFVIII is liver specific since it is directed by the murine albumin enhancer/promoter. While a transgenic line had detectable hFVIII mRNA but no detectable hFVIII protein in the circulation, the mice were immunologically tolerant to hFVIII upon challenge with recombinant hFVIII.…”

Inhibitors – genetic and environmental factors

“…In the second humanized haemophilic mouse model, a human FVIII cDNA transgene, regulated by the liver‐specific albumin promoter, has been microinjected into fertilized oocytes, and founder mice were crossed with exon 17 knockout haemophilia A mice . Despite the fact that FVIII mRNA can be found in several tissues in these mice (including liver, brain and gonads), they do not express FVIII in their plasma.…”