T he development of inhibitory antibodies to therapeutic factor VIII is the major complication of replacement therapy in patients with hemophilia A. The first step in the initiation of the anti-factor VIII immune response is factor VIII interaction with receptor(s) on antigenpresenting cells, followed by endocytosis and presentation to naïve CD4 + T cells. Recent studies indicate a role for the C1 domain in factor VIII uptake. We investigated whether charged residues in the C2 domain participate in immunogenic factor VIII uptake. Co-incubation of factor VIII with BO2C11, a monoclonal C2-specific immunoglobulin G, reduced factor VIII endocytosis by dendritic cells and presentation to CD4 + T cells, and diminished factor VIII immunogenicity in factor VIII-deficient mice. The mutation of basic residues within the BO2C11 epitope of C2 replicated reduced in vitro immunogenic uptake, but failed to prevent factor VIII immunogenicity in mice. BO2C11 prevents factor VIII binding to von Willebrand factor, thus potentially biasing factor VIII immunogenicity by perturbing its half-life. Interestingly, a factor VIII Y1680C mutant, that does not bind von Willebrand factor, demonstrated unaltered endocytosis by dendritic cells as well as immunogenicity in factor VIII-deficient mice. Co-incubation of factor VIII Y1680C with BO2C11, however, resulted in decreased factor VIII immunogenicity in vivo. In addition, a previously described triple C1 mutant showed decreased uptake in vitro, and reduced immunogenicity in vivo, but only in the absence of endogenous von Willebrand factor. Taken together, the results indicate that residues in the C1 and/or C2 domains of factor VIII are implicated in immunogenic factor VIII uptake, at least in vitro. Conversely, in vivo, the binding to endogenous von Willebrand factor masks the reducing effect of mutations in the C domains on factor VIII immunogenicity.

The bispecific antibody Emicizumab is increasingly used for hemophilia A-treatment. However, its specificity for human factors IX and X (FIX, FX) has limited its in vivo functional analysis to primate models of acquired hemophilia. Here we describe a novel mouse model allowing to examine Emicizumab function. Briefly, FVIII-deficient mice receive Emicizumab intravenously 24h before performing a tail clip-bleeding model. A second infusion with human FIX and FX is administered 5 min before bleeding. This approach generates consistent levels of Emicizumab (0.7-19 mg/dL for 0.5-10 mg/kg dose) and of both FIX and FX (85 and 101 U/dL respectively, after dosing 100 U/kg). Plasmas from these mice display FVIII-like activity in a diluted aPTT and in thrombin generation assays, similar to human samples containing Emicizumab. Emicizumab doses of 1.5 mg/kg and higher significantly reduced blood loss in a tail clip-bleeding model using FVIII-deficient mice. However, reduction was incomplete compared to mice treated with human FVIII concentrate, and no difference in efficacy between doses was observed. From this model, we deducted a FVIII-like activity of Emicizumab that corresponds to a dose of 4.5 U FVIII/kg (i.e. 9.0 U/dL). Interestingly, combined with a low FVIII dose (5 U/kg), Emicizumab provided additive activity to allow a complete bleeding arrest. This model could be useful for further in vivo analysis of Emicizumab.

Induction of heme oxygenase-1, a stress-inducible enzyme with anti-inflammatory activity, reduces the immunogenicity of therapeutic factor VIII in experimental hemophilia A. In humans, heme oxygenase-1 expression is modulated by polymorphisms in the promoter of the heme oxygenase-1-encoding gene (HMOX1). We investigated the relationship between polymorphisms in the HMOX1 promoter and factor VIII inhibitor development in severe hemophilia A. We performed a case-control study on 99 inhibitor-positive patients and 263 patients who did not develop inhibitors within the first 150 cumulative days of exposure to therapeutic factor VIII. Direct sequencing and DNA fragment analysis were used to study (GT) n polymorphism and single nucleotide polymorphisms located at -1135 and -413 in the promoter of HMOX1. We assessed associations between the individual allele frequencies or genotypes, and inhibitor development. Our results demonstrate that inhibitor-positive patients had a higher frequency of alleles with large (GT) n repeats (L: n≥30), which are associated with lesser heme oxygenase-1 expression (odds ratio 2.31; 95% confidence interval 1. 46-3.66; P<0.001]. Six genotypes (L/L, L/M, L/S, M/M, M/S and S/S) of (GT) n repeats were identified (S: n<21; M: 21≤n<30). The genotype group including L alleles (L/L, L/M and L/S) was statistically more frequent among inhibitor-positive than inhibitor-negative patients, as compared to the other genotypes (33.3% versus 17.1%) (odds ratio 2.21, 95% confidence interval 1.30-3.76; P<0.01). To our knowledge, this is the first association identified between HMOX1 promoter polymorphism and development of anti-drug antibodies. Our study paves the way towards modulation of the endogenous anti-inflammatory machinery of hemophilia patients to reduce the risk of inhibitor development Development of inhibitory antibodies to therapeutic factor VIII in severe hemophilia A is associated with microsatellite polymorphisms in the HMOX1 promoter

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The development of anti-factor VIII antibodies is a major complication of the treatment of patients with hemophilia A. Generation of high affinity anti-factor VIII antibodies is dependent on help provided by CD4+ T cells that recognize factor VIII-derived peptides presented on class II major histocompatibility complex on the surface of antigen-presenting cells. In order to identify the immune-dominant epitopes that can be presented to CD4+ T cells, we previously developed a mass spectrometry-based method to identify factor VIII-derived peptides that are presented on human leukocyte antigen (HLA)-DR. In the present work, we compared the repertoire of FVIII-derived peptide presented on HLA-DR and HLA-DQ. Monocyte-derived dendritic cells from nine HLA-typed healthy donors were pulsed with recombinant factor VIII. HLA-DR and HLA-DQ molecules were purified using monoclonal antibodies. Our data show that HLA-DQ and HLA-DR present a similar repertoire of factor VIII-derived peptides. However, the number of peptides associated with HLA-DQ was lower than that with HLA-DR. We also identified a peptide, within the acidic a3 domains of factor VIII, which is presented with higher frequency on HLA-DQ. Interestingly, this peptide was found to have a higher predicted affinity for HLA-DQ than for HLA-DR. Taken together, our data suggest that HLA-DQ participates in the presentation of factor VIII peptides, thereby contributing to the development of inhibitory antibodies in a proportion of patients with severe hemophilia A.

Summary.  Background:  Heme is a redox active macrocyclic compound that is released upon tissue damage or hemorrhages. The extracellular release of large amounts of heme saturates scavenging heme‐binding proteins. Free heme has been proposed to affect coagulation and has been co‐purified with the factor VIII (FVIII)‐von Willebrand factor (VWF) complex. The sites from which heme is released upon injury overlap with the sites to which FVIII is targeted for performing its hemostatic functions. Objectives:  To investigate the interaction of heme with FVIII and the consequence for the procoagulant activity of FVIII in vitro. Methods and results:  Heme bound to several sites on FVIII with high apparent affinity. Heme‐binding inhibited FVIII procoagulant activity in a dose‐dependent manner. FVIII inactivation in the presence of saturating amounts of heme implicated a reduced interaction of FVIII with activated FIX, as shown by ELISA, surface plasmon resonance and fluorescence quenching. Heme‐mediated inactivation of FVIII was prevented by VWF, but not by human serum albumin, a heme‐binding protein known for its protective activity in hemolytic conditions. Conclusions:  Our data identify FVIII as a novel heme‐binding protein. Occupation of high affinity heme‐binding sites on FVIII at low concentrations of free heme did not inactivate FVIII. Conversely, large molar excesses of heme over FVIII, which correspond to conditions of extensive heme release, inhibited FVIII activity in vitro. It remains to be demonstrated whether, under such conditions, heme‐mediated modulation of the activity of FVIII plays some role in the regulation of coagulation.

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