Successful immune tolerance induction by FVIII in hemophilia A patients with inhibitor may occur without deletion of FVIII‐specific T cells

Summary. Background: The development of an inhibitor is the major complication facing patients with hemophilia A treated by administration of factor (F) VIII concentrates. Restoration of tolerance to FVIII can be achieved by prolonged administration of FVIII (immune tolerance induction, ITI). Although ITI has been used for more than 30 years in patients with hemophilia A and inhibitor, its mechanism of action is still poorly understood. Objectives: As administration of high doses of antigen can induce the apoptosis of the T cells recognizing the antigen, a potential mechanism of action of ITI may be the deletion of FVIII‐specific T cells. Patients/Methods: We studied the CD4+ T‐cell response to FVIII in five (one mild, one moderate and three severe) patients successfully desensitized by administration of FVIII and in control subjects. Results: Following repeated stimulation with autologous dendritic cells loaded with FVIII, FVIII‐specific T oligoclonal cell lines were expanded from the blood of one of the successfully desensitized patients. The FVIII‐specific T cells produced IL‐5, IL‐13 and IL‐2. By contrast, FVIII‐specific T‐cell lines could not be derived from three patients with mild hemophilia A without inhibitor or from four normal control subjects. Conclusions: These data represent the first analysis of the cellular mechanisms regulating the induction of tolerance to FVIII. They demonstrate that successful tolerance induction may occur without deletion of FVIII‐specific T cells.     

Most factor VIII B domain missense mutations are unlikely to be causative mutations for severe hemophilia A: implications for genotyping

Summary. Background & Objective: The factor VIII (FVIII) B domain shares very little amino acid homology with other known proteins and is not directly necessary for procoagulant activity. Despite this, missense mutations within the B domain have been reported in patients with hemophilia A. Given that the B domain is dispensable for secretion and function of FVIII, we hypothesized that these mutations should not be causative of hemophilia A in these patients. Methods: Plasmid vectors containing B domain missense mutations that were reported to be associated with moderate/severe hemophilia A (T751S, D826E, V993L, H1047Y, T1353A, N1441K, L1462P, E1579D, A1591S, P1641L and S1669L) were analyzed for their effect on synthesis and secretion compared with FVIII wild‐type (WT) following transient transfection into COS‐1 and CHO cells in vitro. Further, H1047Y, N1441K and E1579D mutants were expressed in vivo in a hemophilia A mouse model by hydrodynamic tail‐vein injection. Results: FVIII activity and antigen levels for all mutants expressed into the conditioned media of COS‐1 and CHO cells were similar to FVIII WT. Also, plasma expression of these mutants was similar to FVIII WT in hemophilia A mice. An in vivo tail clip bleeding assay also demonstrated that blood loss from hemophilia A mice expressing FVIII WT, H1047Y, N1441K and E1579D was similar. Conclusions: We conclude that most missense mutations within the FVIII B domain would be unlikely to lead to severe hemophilia A and that the majority of such missense mutations represent polymorphisms or non‐pathologic mutations.     

Clinical prediction models for inhibitor development in severe hemophilia A

Summary.  Clinical prediction models for factor VIII inhibitor development may potentially facilitate the identification of patients at high risk of this complication. In particular, recognition of early intensive FVIII replacement therapy as a significant risk factor for inhibitor development has defined a clear clinical decision point that influences patient care. To assess the effect and potential acceptance of a prediction tool for FVIII inhibitor development in clinical practice, a 15-item survey was developed to assess whether these included items were accepted as potential risk factors by the health care providers taking care of these patients. The rating of these items was on a 5-point linear scale, with '1' representing it to be very unlikely and '5' very likely. Responses from 42 centers were available for analysis. These centers were responsible for taking care of 2642 children <18 years with severe hemophilia A. In addition to genetic factors (mean score ∼4.5), early intensive treatment was considered important for inhibitor development (mean score 4.07). Other factors likely to reduce inhibitor development were early onset of prophylaxis (mean score 3.68) and avoidance of early surgery (mean score 4.05). Physicians also agreed that institution of early prophylaxis and avoidance of elective surgery are important management strategies to reduce inhibitor development (mean scores 3.54 and 4.32, respectively).     

Analysis of mRNA in hemophilia A patients with undetectable mutations reveals normal splicing in the factor VIII gene

BACKGROUND: haemophilia A (HA) is characterized by partial or total deficiency of factor VIII (FVIII) protein activity. It is caused by a broad spectrum of mutations in the FVIII gene. Despite tremendous improvements in mutation screening methods, in about 2% of HA patients no DNA change could be found, even after sequencing the whole coding part of the FVIII gene including the flanking splice sites, as well as the promotor and the 3' UTR regions. OBJECTIVES, PATIENTS AND METHODS: In the present study we performed a detailed RNA analysis of three groups of patients. The first included control patients with known splicing defects, the second included two patients with already identified nucleotide changes close to splicing sites, that could potentially alter the normal splicing process, and a third group of 11 unrelated patients whose genomic DNA have already been screened for mutations by DHPLC and direct sequencing with no mutation being identified. RESULTS: Both candidate splice site mutations were shown to result in either skipping or alternative splicing of at least one exon, therefore these DNA changes must be considered as causal for the patients' HA phenotype. In contrast, no abnormalities on the RNA level were observed in any of 11 unrelated patients without mutations in the FVIII gene. CONCLUSIONS: These findings exclude mutations that could be located deep in the introns and affecting either normal splicing or lead to mechanisms causing some unknown rearrangements of the FVIII gene. In fact, our results point to the presence of still unknown factor(s) causing HA, which might be either allelic or in the close proximity of the FVIII gene or non-allelic associated with other genetic loci that are involved in the processing of the FVIII protein.     

The humoral response to human factor VIII in hemophilia A mice

BACKGROUND: Inhibitory antibodies (Abs) to factor VIII (FVIII inhibitors) constitute the most significant complication in the management of hemophilia A. The analysis of FVIII inhibitors is confounded by polyclonality and the size of FVIII. OBJECTIVES: The goal of this study was to dissect the polyclonal response to human FVIII in hemophilia A mice undergoing a dosage schedule that mimics human use. METHODS: Splenic B-cell hybridomas were obtained following serial i.v. injections of submicrogram doses of FVIII. Results of a novel, anti-FVIII domain-specific enzyme-linked immunosorbent assay were compared to Ab isotype and anti-FVIII inhibitory activity. RESULTS: The robust immune response resulted in the production of approximately 300 hybridomas per spleen. We characterized Abs from 506 hybridomas, representing the most comprehensive analysis of a protein antigen to date. Similar to the human response to FVIII, anti-A2 and anti-C2 Abs constituted the majority of inhibitors. A novel epitope was identified in the A2 domain by competition ELISA. Anti-A2 and anti-C2 Abs were significantly associated with IgG(1) and IgG(2a) isotypes, respectively. Because the IgG(2a) isotype is associated with enhanced Fc receptor-mediated effector mechanisms, this result suggests that anti-C2 Abs and inflammation may be linked. Additionally, we identified a novel class of Abs with dual specificity for the A1 and A3 domains. Forty per cent of the Abs had no detectable inhibitory activity, indicating that they are prominent and potentially pathologically significant. CONCLUSION: The expanded delineation of the humoral response to FVIII may lead to improved management of hemophilia A through mutagenesis of FVIII B-cell epitopes.     

血友病A患者凝血因子Ⅷ抑制物筛查及分析

本研究通过检测重型血友病A(hemophilia A,HA)患者凝血因子Ⅷ(FⅧ)抑制物,探讨抑制物阳性患者基因突变情况.选取58例一期法检测FⅧ:C均小于1% HA患者,以APTT法为基础进行FⅧ抑制物筛查,筛查阳性者用Bethesda法进行FⅧ抑制物定量分析.以基因组DNA为模版,对抑制物阳性患者FⅧ12、14、16外显子进行基因扩增,PCR产物通过直接测序检测突变情况.结果表明:58例HA患者中4例(6.9%)抑制物检测为阳性,FⅧ12、14、16外显子基因均未发现基因突变.结论:本组病例HA患者抑制物阳性率低于国外文献报道,抑制物产生的原因有待于进一步研究.     

Rate of inhibitor development in previously untreated hemophilia A patients treated with plasma‐derived or recombinant factor VIII concentrates: a systematic review

Summary. Background: Different rates of inhibitor development after either plasma‐derived (pdFVIII) or recombinant (rFVIII) FVIII have been suggested. However, conflicting results are reported in the literature. Objectives: To systematically review the incidence rates of inhibitor development in previously untreated patients (PUPs) with hemophilia A treated with either pdFVIII or rFVIII and to explore the influence of both study and patient characteristics. Methods: Summary incidence rates (95% confidence interval) from all included studies for both pdFVIII and rFVIII results were recalculated and pooled. Sensitivity analysis was used to investigate the effect of study design, severity of disease and inhibitor characteristics. Meta‐regression and analysis‐of‐variance were used to investigate the effect of covariates (testing frequency, follow‐up duration and intensity of treatment). Results: Two thousand and ninety‐four patients (1167 treated with pdFVIII, 927 with rFVIII; median age, 9.6 months) from 24 studies were investigated and 420 patients were observed to develop inhibitors. Pooled incidence rate was 14.3% (10.4–19.4) for pdFVIII and 27.4% (23.6–31.5) for rFVIII; high responding inhibitor incidence rate was 9.3% (6.2–13.7) for pdFVIII and 17.4% (14.2–21.2) for rFVIII. In the multi‐way anova study design, study period, testing frequency and median follow‐up explained most of the variability, while the source of concentrate lost statistical significance. It was not possible to analyse the effect of intensity of treatment or trigger events such as surgery, and to completely exclude multiple reports of the same patient or changes of concentrate. Conclusions: These findings underscore the need for randomized controlled trials to address whether or not the risk of inhibitor in PUPs with hemophilia A differs between rFVIII and pdFVIII.     

Intensive exposure to factor VIII is a risk factor for inhibitor development in mild hemophilia A

Summary.  Background : Inhibitors are rare in boys with mild hemophilia A (MHA; factor (F)VIII:C > 5%) but may arise following intense FVIII exposure, e.g. continuous infusion (CI). Objectives : To determine the impact of intense FVIII exposure in inhibitor formation in MHA at our institution and to compare this with previous reports. Patients and methods : We reviewed FVIII exposure and inhibitor development in boys (ages 0–18 years) with MHA followed at our institution from 1996 to 2001 and conducted a Medline search (1966–2002) on the experience of inhibitor development following intensive/CI exposure to FVIII. Results : We identified 54 boys with MHA. Twenty-nine (54%) had been exposed to FVIII. Seven had received FVIII by CI. Four developed inhibitors; three high titer (at ages 10 years, 16 years and 17 years) and one low titer (at 1 month old). All four had received a CI of recombinant (r) FVIII of at least 6 days within 6 weeks of developing inhibitors. Baseline FVIII levels fell to < 1% in all cases and the three with high-titer inhibitors developed severe bleeding. Immune tolerance therapy (ITT) was attempted in two boys and was successful in one. Our literature search identified 35 cases (only four children) with MHA developing inhibitors following intense FVIII exposure often in the context of surgery. Conclusions : The incidence of inhibitors in our MHA population was 7.4%. If expressed according to exposure the incidence was significantly higher: 14% (4/29) for any exposure to FVIII and 57% (4/7) for exposure by CI. A prospective study to address whether CI is associated with an increased incidence of inhibitor development in MHA is warranted.     

T-cell responses over time in a mild hemophilia A inhibitor subject: epitope identification and transient immunogenicity of the corresponding self-peptide

BACKGROUND: Antibodies that neutralize factor (F) VIII activity, clinically referred to as 'inhibitors', complicate the treatment of hemophilia A patients; current tolerance and bypass strategies are extremely costly and sometimes ineffective. The development of inhibitors requires T-cell help. OBJECTIVES: We characterized T-cell responses of a subject with mild hemophilia A with missense genotype A2201P for one year following his initial inhibitor response, with the goals of defining the primary epitope(s) and its (their) MHC Class II restriction. We investigated the possible involvement of regulatory T cells in modulating immune responses. PATIENTS/METHODS: The subject developed high-titer FVIII-neutralizing antibodies (250 BU mL(-1)) that declined over time to 8 BU ml(-1). His clotting activity was initially impaired (3%) but returned to baseline (8-10%) within four weeks. MHC Class II tetramers were used to analyze his CD4 T cells, which were stimulated with peptides spanning the C2 domain. Responses of total and CD25-depleted CD4 cells to sequences containing A2201 (native), P2201 (hemophilic), and other predicted T-cell epitopes were evaluated. RESULTS AND CONCLUSIONS: An HLA-DRA-DRB1*0101 restricted T-cell epitope containing the wild-type A2201 sequence was identified. Interestingly, peptides containing A2201 were recognized by CD4 T cells at all time points, whereas a P2201 peptide was recognized only near the initial peak response. The responsiveness of CD25-depleted CD4 cells to an A2201 peptide was enhanced 11 and 19 weeks following inhibitor detection, suggesting the possible involvement of CD4+CD25+ regulatory T cells in modulating immune responses. Patient-derived T-cell clones proliferated in response to C2 protein and to peptides containing A2201 but not P2201.     

Harmonization of clinical trial guidelines for assessing the risk of inhibitor development in hemophilia A treatment

Summary. At the present time, the most significant complication of hemophilia therapy is the development of neutralizing antibodies (inhibitors) to factor (F) VIII, which adds greatly to the difficulty and expense of preventing and treating bleeding episodes. Both patient‐related and therapy‐related variables contribute to the development of inhibitors. The multifactorial nature of inhibitor development and the relatively small numbers of patients that participate in clinical trials make it difficult to accurately assess the risk of inhibitor development. Adding to that challenge is the lack of a uniform standard of design for conducting clinical trials to evaluate the safety of FVIII products. This hinders the comparison of products and is an obstacle to the meta‐analysis necessary to make statistically valid assessments of inhibitor risk. This article reviews similarities and differences in clinical trial guidelines of European and US regulatory agencies and discusses the need for their harmonization to facilitate the assessment of FVIII products.     

Comparative immunogenicity of recombinant B domain‐deleted porcine factor VIII and Hyate:C in hemophilia A mice presensitized to human factor VIII

Summary. Hyate is a commercial plasma‐derived porcine factor (F)VIII concentrate that is used in the treatment of patients with inhibitory antibodies to FVIII. OBI‐1 is a recombinant B domain‐deleted form of porcine FVIII that is in clinical development for the same indication. Hemophilia A mice were presensitized with human FVIII to simulate clinical inhibitory antibody formation and then were randomized to receive OBI‐1 or Hyate:C in a comparative immunogenicity trial. OBI‐1 or Hyate:C were given in a series of four intravenous injections at weekly intervals at doses of 1, 10, or 100 U kg^?1. Inhibitory antibodies to porcine FVIII were not detected by Bethesda assay in most of the mice given OBI‐1 or Hyate:C at doses of 1 or 10 U kg^?1, but were identified in 81% and 94% of mice given 100 U kg^?1 of OBI‐1 or Hyate:C, respectively. There was no significant difference between OBI‐1 and Hyate:C in inhibitory antibody formation at any dose, although there was a trend toward a lower Bethesda titer in OBI‐1‐treated mice at 10 U kg^?1 (P = 0.09). Total anti‐FVIII antibodies to Hyate:C and OBI‐1 were also measured by ELISA using immobilized purified plasma‐derived porcine FVIII and OBI‐1, respectively, as antigens. At the 10 and 100 U kg^?1 doses, the mean anti‐FVIII response was higher in Hyate:C‐treated‐mice than in OBI‐1‐treated mice (P = 0.02 and P = 0.004, respectively). The results using this model suggest that OBI‐1 may be less immunogenic and safer than Hyate:C in FVIII inhibitor patients.     

Low detection rate of antibodies to non-functional epitopes on factor VIII in patients with hemophilia A and negative for inhibitors by Bethesda assay

Summary.  In patients with hemophilia A who have an inhibitor to factor (F)VIII measured by Bethesda assay, enzyme-linked immunosorbent assay (ELISA) can also be used to detect the inhibitor. In some studies non-inhibitory antibodies were also detected by ELISA in many patients who were negative by Bethesda assay. Our aim was to investigate whether there is a higher detection rate of FVIII antibodies by ELISA compared with Bethesda assay. We also compared outcomes using three different preparations of recombinant FVIII (rFVIII) to coat the microtiter plates for ELISA. Inhibitor detection by ELISA generally agreed with the Bethesda method. Only four of 26 patients with no clinical suspicion of an inhibitor and with no detectable inhibitor by Bethesda assay showed a non-inhibitory antibody by ELISA, and three of these were only weakly positive. Patients with severe hemophilia A and the intron 22 inversion ( n  = 21) did not show a higher incidence of non-inhibitory antibodies compared with those without that mutation. Finally, we found that the formulation of rFVIII has a small effect on ELISA performance, mainly in detection of low-level antibody. The results of the present study are in contrast to and fail to confirm previously published reports showing a higher incidence of non-inhibitory antibodies in hemophilia A.     

T‐cell responses in two unrelated hemophilia A inhibitor subjects include an epitope at the factor VIII R593C missense site

Summary. Background: Development of neutralizing anti‐factor (F)VIII antibodies (‘inhibitors’) is a serious clinical problem in hemophilia A. Increased inhibitor risk has been associated with certain FVIII missense substitutions, including R593C in the A2 domain. Objectives: The aim of the present study was to identify T‐cell epitopes in FVIII and characterize T‐cell responses in two unrelated hemophilia A subjects sharing F8‐R593C and HLA‐DRB1*1101 genotypes. We hypothesized that the hemophilic substitution site coincides with an important T‐cell epitope. Patients/methods: The binding affinities of peptides for recombinant HLA‐DR proteins were measured and compared with epitope prediction results. CD4+ T cells were stimulated using peptides and stained with fluorescent, peptide‐loaded tetramers. Results: The inhibitor subjects, but not HLA‐matched controls, had high‐avidity HLA‐DRB1*1101‐restricted T‐cell responses against FVIII_589–608, which contains the hemophilic missense site. Antigen‐specific T cells secreted Th1 and Th2 cytokines and proliferated in response to FVIII and FVIII_592–603. FVIII_589–608 bound with physiologically relevant (micromolar) IC₅₀ values to recombinant DR0101, DR1101 and DR1501 proteins. Conclusions: Hemophilia A patients with R593C missense substitutions and these HLA haplotypes had an increased incidence of inhibitors in our cohorts, supporting a paradigm in which presentation of FVIII epitopes containing the wild‐type R593 influences inhibitor risk in this hemophilia A sub‐population.     

Integrated analysis of safety and efficacy of a plasma- and albumin-free recombinant factor VIII (rAHF-PFM) from six clinical studies in patients with hemophilia A

Background: Hemophilia A is an X-linked bleeding disorder that results from insufficient levels of factor VIII (FVIII) coagulant activity. Objective: To evaluate the efficacy and safety of ADVATE^® rAHF–PFM (Baxter Healthcare Corporation), a recombinant FVIII concentrate manufactured without human or bovine blood-derived additives, and to assess the effect of compliance with prophylactic use in preventing bleeding episodes (BEs). Methods: Clinical data were integrated from six prospective studies. Two hundred thirty-four hemophilia A subjects (FVIII levels ≤ 2%) (median age 14.7 (range: 0.02 – 72.7) years) were included. Results: BEs were managed with one or two infusions and nearly all (1953/1956) responded to treatment. Compliance with a prophylactic treatment regimen significantly reduced the incidence of BEs (p = 0.0061) and prevented non-traumatic joint BEs (median annualized BE rate was 0). One previously treated subject developed an inhibitor; no other safety concerns were observed. Conclusions: These results reinforce the efficacy and safety of rAHF-PFM and suggest that compliance is an essential contributor to the effectiveness of prophylaxis in the treatment of hemophilia A.     

A single adeno-associated virus (AAV)-murine factor VIII vector partially corrects the hemophilia A phenotype

Summary.  A major obstacle for delivery of factor (F)VIII using adeno-associated virus (AAV) vectors is the large size of FVIII cDNA, which is well above the 5 kb packaging limit for AAV. Here we construct a < 5 kb FVIII-AAV vector using murine FVIII cDNA and a strong liver-specific albumin promoter. We assessed the efficacy of this vector using three different routes of administration, intraportal, intrasplenic and tail vein injection, in FVIII knockout (FVIII KO) mice. The peak level of FVIII observed was about 8% of normal mouse FVIII activity. Even at 9 months, post vector injection, 14 of 19 mice receiving FVIII-AAV demonstrated phenotypic correction and roughly 2% FVIII activity. The transgene copy number ranged from 0.001 to 0.1 copies per cell, depending upon the somatic tissue. The potential for germline transmission of AAV was assayed in 34 pups obtained from five pairs of treated, phenotypically corrected adult hemophilic mice. Although the parents harbored the transgene in liver, spleen, and gonads, none of the 34 offspring was positive for the transgene, suggesting that the risk of inadvertent germline transmission is low.     

Body weight and fat mass index as strong predictors of factor VIII in vivo recovery in adults with hemophilia A

Summary. Background: The treatment of hemophilia A requires infusions of factor VIII (FVIII) concentrates. The number of units to be given in order to obtain the target level is calculated using the formula: [body weight (BW) × desired FVIII increase]/2, which assumes that each unit infused per kg of BW increases the FVIII level by 2%. Objectives: The present observational study evaluated the dependence of FVIII recovery on different morphometrical variables: BW, fat mass index (FMI), body mass index, and the difference between actual and ideal BW. Patients and methods: FVIII recovery was measured in 46 non‐actively bleeding hemophilia A patients, being treated with a recombinant FVIII concentrate. Regression trees were used to identify morphometrical predictors of recovery. Results: The median recovery was 2.08 for all patients, 2.63 for those with a BW ≥ 81.0 kg and 1.87 for others (P < 0.001). The recovery was significantly higher when FMI was ≥ 20% compared with FMI < 15% (median recovery: 2.35 vs. 1.74; P = 0.007). Using regression trees, three groups were created: BW < 80.5 kg and FMI < 22.3%, BW < 80.5 kg and FMI ≥ 22.3% and BW ≥ 80.5 kg. Median recovery in these groups was 1.80, 2.16 and 2.63, respectively (P < 0.001). Conclusions: The dose calculation of FVIII should take into account both BW and FMI, and be adapted to underweight or overweight patients. Comparison of the average recovery after different FVIII concentrates should keep in mind morphometrical patient characteristics.