IgG subclasses of anti-FVIII antibodies during immune tolerance induction in patients with hemophilia A

The eradication of inhibitory antibodies in patients with haemophilia A can be accomplished by frequent administration of high or intermediate doses of factor VIII (FVIII), so-called immune tolerance induction (ITI). This study monitored the distribution of IgG subclasses of anti-FVIII antibodies during ITI. FVIII-specific antibodies of subclass IgG1 were detected in all inhibitor patients tested, anti-FVIII IgG4 in 16, IgG2 in 10 and IgG3 in one of 20 patients analysed. Levels of anti-FVIII IgG1 and IgG4 correlated well with inhibitor titres as measured by Bethesda assay. In low-titre inhibitor patients, anti-FVIII antibodies consisted primarily of subclass IgG1 whereas, anti-FVIII antibodies of subclass IgG4 were more prominent in patients with high titre inhibitors who needed prolonged treatment or who failed ITI. Longitudinal analysis of 14 patients undergoing ITI revealed that the relative contribution of IgG subclasses was constant for most of the patients analysed. In two patients, the relative contribution of IgG4 increased during ITI. Overall, our findings document the distribution and dynamics of anti-FVIII IgG subclasses during ITI. Future studies will need to address whether monitoring the relative contribution of anti-FVIII subclasses IgG1 and IgG4 may be useful for the identification of patients who are at risk of failing ITI.     

Alloantibodies in previously untreated hemophilia A patients: the role of environmental factors

Abstract

The therapy of hemophilia A has nowadays reached a high degree of quality, being probably the most efficacious and safe treatment available among other monogenic disorders. In this context, the most challenging complication of therapy has become the development of inhibitory alloantibodies against FVIII. These inhibitors, which develop in up to 30% of severe hemophilia A patients, render replacement therapies ineffective, limit patient access to a safe and effective standard of care, and predispose them to an increased risk of morbidity and mortality. Several possible risk factors, both genetic and environmental, for inhibitor development have been identified in previously untreated patients, confirming that the etiology of this phenomenon is multifactorial. In this narrative review, we will focus on acquired risk factors, discussing the pathogenic and clinical data available from the literature analysis.     

Late immune tolerance induction in haemophilia A patients

The incidence of inhibitor development in patients with severe haemophilia A is approximately 30%. Immune tolerance induction (ITI) is commonly utilized to eradicate these antibodies and is successful in 63–100% of cases. Potential predictors of a poor outcome in ITI include a high preinduction titre, high historical peak titre, older age at start of ITI and prolonged interval from diagnosis to start of ITI. The goal of this study was to characterize the outcomes of patients from our centre who have undergone late ITI, many of whom had poor prognostic features. Medical records of patients in our centre with severe/moderately severe haemophilia A (< 2% FVIII activity) and history of inhibitor were reviewed. Data were abstracted from all patients who attempted late ITI. Nine patients underwent late ITI between January 1999 and December 2011. Within this cohort, 7 (78%) patients were black, 6 (67%) were <21 years old and 4 (44%) had a family history of inhibitor. Three patients had previously received ITI unsuccessfully. To date, 4 (44%) patients are tolerized (persistently negative inhibitor titre, FVIII recovery >66% and successfully treated with FVIII products ±FVIII t^½ of > 6 h). Three patients are partially tolerized (have low responding inhibitor, variable FVIII recovery and successfully treated with FVIII products). Two patients are not tolerized. Some patients with haemophilia A and long‐standing inhibitors may benefit from ITI.     

Requirements for immune recognition and processing of factor VIII by antigen-presenting cells

Generation of inhibitory antibodies upon repeated FVIII infusion represents a major complication in hemophilia care. Professional antigen presenting cells (APCs) are crucial for orchestration of humoral immune responses. APCs are capable of internalizing soluble as well as particulate antigens through various mechanisms resulting in loading of antigen-derived peptides on MHC class I or II for presentation to T cells. This review highlights how FVIII is recognized and processed by APCs. The significance and contribution of candidate receptors involved in FVIII uptake by APC are discussed. Recent findings defining the repertoire of FVIII peptides presented on MHC class II are addressed. Studies in murine models of hemophilia A suggest that modulation of APC function can reduce inhibitor formation. Based on this we anticipate that modulation of FVIII uptake by APCs may yield novel therapeutic approaches for treatment or prevention of inhibitor formation in patients with hemophilia A.     

Antibodies to factor VIII in plasma of patients with hemophilia A and normal subjects

 Non-neutralizing factor VIII (FVIII) antibodies (FVIII-Ab) in hemophilia A may be associated with an abnormal clinical response to FVIII concentrates. Patients with FVIII inhibitors may develop noncoagulation FVIII-Ab after the induction of immunotolerance. Natural FVIII-Ab may be detected in the plasma of some healthy subjects. The aim of this study was to analyze the presence of FVIII-Ab in the plasma of 53 normal blood donors and 124 patients with hemophilia A (18 patients had a previous history of FVIII inhibitor, but only 12 had inhibitor at the moment this study was performed). FVIIII inhibitor was measured using the Bethesda method. FVIII-Ab were analyzed by a specific ELISA assay using purified FVIII from a monoclonal concentrate and a standard plasma containing 26 Bethesda units (BU) of FVIII inhibitor. Purified FVIII was used to coat wells of a microtiter plate and was incubated with dilutions of plasma to be tested. Bound human IgG FVIII-Ab were detected by incubation with polyclonal sheep anti.human IgG alkaline phosphatase conjugate, and the OD₄₀₅ was quantitated. A linear fit was obtained (by plotting FVIII-Ab positivity [OD 405nm] versus BU titer) when serial dilutions of this standard inhibitor plasma, containing titers of 0.5 BU or higher, were used. Four different levels of FVIII-Ab positivity [OD 405nm] were distinguished in this assay: Negative levels (–) were obtained with dilutions of the standard inhibitor containing <0.5 BU. Mild levels (+) were obtained with dilutions of 0.5–5 BU. Moderate levels (++) were obtained for dilutions ranging from 5–25 BU. Maximum positivity (+++) was obtained for dilutions of titers > 25 BU. FVIII-Ab positivity was detected in eight of the normal subjects (15%): three were found to be moderately positive (++) and five mildly positive (+). No inhibitory activity was detectable when whole plasma was used. All the hemophilic patients with a presence of FVIII inhibitor at the time of the study were found to be positive for FVIII-Ab. In addition, the level of positivity correlated with the corresponding BU. Four of the six patients who had a history of inhibitor were negative and two positive. Twenty additional patients (16.12%) in whom no inhibitory activity was detected were found to be positive for FVIII-Ab: 16 + and four ++. The mean age of patients with FVIII-Ab positivity was significantly higher than that of patients of the FVIII-Ab negative group (p<0.005). In conclusion, FVIII-Ab positivity in patients with hemophilia A was 17.7% higher than the level of positivity detected by an inhibitory assay. We propose that this method for FVIII-Ab analysis could be used for patients with hemophilia A, at least to complement the functional inhibitor assay. FVIII recovery or half-life should be assessed in patients who test positive for FVIII-Ab and who show no evidence of inhibitor. Received: 31 July 1995 / Accepted: 25 January 1996     

Frequency of inhibitor development in severe haemophilia A children treated with cryoprecipitate and low-dose immune tolerance induction.

The frequency of factor VIII inhibitor development was evaluated in a hundred severe haemophilia A patients < 18 years of age (mean 10.4 +/- 5.1 years); 25 were previously untreated patients (PUPs), with a mean age of 11.2 +/- 2.9 months. All were followed up for 3 years from December 1996. Immune tolerance (IT) was induced with low-dose factor VIII (FVIII); 25-50 IU kg(-1) every other day for the 10 haemophiliacs who developed persistent inhibitors. The incidence of inhibitors for PUPs was 3/25 (12%; 95% confidence interval [CI], 0. 7-24.7%) and were detected after 4, 15 and 20 exposure days (mean 13 +/- 8.2 days; 95% CI, 3.7-22.2%). Children with maximum inhibitor levels of > 40 Bethesda units (BU) per mL (n=4) received IT therapy as 25 U kg(-1) FVIII in the form of cryoprecipitate every other day for 1-4 months (mean 2.4 +/- 1.6 months; 95% CI, 0.8-3.9%), which was successful in all of them. FVIII (50 U kg(-1)) was given every other day for six patients with maximum inhibitor level > 40 BU mL(-1) for 3-9 months (mean 5.4 +/- 3.2 months; 95% CI, 2.9 -7.9%) with success in 4/6 (66.6%; 95% CI, 28.8-104.3%). Patients who showed a good IT response had an inhibitor level < or = 30 BU mL(-1), were < or = 9 years of age at inhibitor development with few exposure days to FVIII and had an early immune tolerance. In conclusion, inhibitor development in severe haemophilia A children exclusively treated with cryoprecipitate is low. Early low-dose IT induction for high responders may be achieved successfully if inhibitor level is < or = 50 BU mL(-1).     

International workshop on immune tolerance induction: consensus recommendations1

Summary. Although immune tolerance induction (ITI) has been used for 30 years to eliminate inhibitors and restore normal factor pharmacokinetics in patients with hemophilia, there is a paucity of scientific evidence to guide therapeutic decision‐making. In an effort to provide direction for physicians and hemophilia treatment center staff members, an international panel of hemophilia opinion leaders met to develop consensus recommendations for ITI in patients with severe and mild hemophilia A and hemophilia B. These recommendations draw on the available published literature and the collective clinical experience of the group and are rated based on the level of supporting evidence .     

Management of Factor VIII Inhibitors

The development of inhibitory alloantibodies to factor VIII (FVIII) is a major complication of clotting factor replacement therapy for hemophilia A. Inhibitor development compromises effective hemostasis management in affected individuals and results in higher morbidity and costs of care compared with hemophilic individuals without anti-FVIII antibodies. The therapeutic approach to the management of bleeding in the presence of low- and high-titer inhibitors is founded on the principles of either saturating antibody with excess FVIII or bypassing the FVIII requirement altogether. Although spontaneous antibody disappearance does occur, immune tolerance is often required for antibody eradication. Studies aimed at optimizing this treatment approach and developing newer strategies for inhibitor prevention are ongoing.     

Optimizing management of immune tolerance induction in patients with severe haemophilia A and inhibitors: towards evidence‐based approaches

Immune tolerance induction (ITI) is the only strategy proven to eradicate persistent inhibitors in severe haemophilia A patients. Thirty years experience has shown high success rates (60–80%) with heterogeneous dose regimens and has led to the identification of clinical features that define the patients’ prognostic profile. Children with recently diagnosed inhibitors are the best candidates for ITI and adequate management may further contribute to improve the short‐ and long‐term ITI outcome. In these patients inhibitor eradication represents a cost‐effective option because it enables the restoration of FVIII prophylaxis and consequently prevents arthropathy development. Adults with long‐standing inhibitors often show bad predictors of ITI outcome, however, ITI may be considered as a suitable and cost‐effective approach in cases with frequent bleeds that are not satisfactorily controlled by by‐passing treatment and/or when orthopaedic surgery is needed. Optimal ITI regimens should be established in these different settings and randomized trials are addressing these issues. This article reviews the available literature evidence and clinical implications with current recommendations on ITI management, and highlights the issues still unsolved.     

Use of Haemate® P as immune tolerance induction in patients with severe haemophilia A who failed previous induction attempts: a multicentre observational study

Immune tolerance induction (ITI) can eliminate factor VIII (FVIII) inhibitory antibodies that appear during FVIII replacement therapy. If first‐line ITI fails, switching to a different FVIII concentrate, especially one containing von Willebrand factor (VWF), has been advocated. The objective of the study was to assess the efficacy and safety of Haemate^® P, a plasma‐derived FVIII concentrate containing high levels of VWF, as ITI in severe haemophilia A patients who had failed at least one prior ITI attempt with a different FVIII concentrate. In this multicentre, observational study, Haemate^® P was administered at a starting dose of 83–308 IU kg^?1 day^?1 (1500–6000 IU day^?1). Efficacy was assessed by standard criteria (e.g. Bethesda titre, FVIII recovery and half‐life), and bleeding characteristics. Nine patients from six haemophilia centres were treated with Haemate^® P after failing one (n = 2), two (n = 5) or three (n = 2) prior ITI courses. The median time from inhibitor detection to Haemate^® P treatment was 5.4 years. The median Haemate^® P dose was 134 IU kg^?1, and the median treatment duration 32 months. During median of 47 months of follow‐up, complete response, partial response and treatment failure were observed in one, three and five patients respectively. Five patients experienced seven adverse events (AEs), including two serious AEs (sepsis). Haemate^® P was discontinued due to an AE in one patient with a partial response. Haemate^® P salvage ITI resulted in complete or partial tolerization in four of nine patients (44%) who had failed previous ITI attempts using different FVIII concentrates.     

HBV转基因小鼠免疫耐受状态的研究

目的研究HBV转基因小鼠免疫耐受状态机制。方法以正常ICR小鼠为对照,检测转基因小鼠脾树突状细胞(DC)表面la(MHC-Ⅱ)和CD_(80)(B7)表达水平及其免疫功能;脾及外周血T淋巴细胞表面CD_8和CD_(28)及肝细胞膜上MHC-I和腹腔巨噬细胞MHC-Ⅱ抗原表达情况。结果发现HBV转基因小鼠DC表面MHC-Ⅱ及CD_(80)表达水平(13.34±1.11及12.24±1.31)明显低于对照组(20.47±1.78及15.83±1.52,P<0.05);其DC体外诱发T淋巴细胞增殖能力(6409.67±445.71cpm)明显低于对照组(13870.67±1706.9cpm,P<0.01);而脾及外周血T淋巴细胞CD_8及CD_(28)表达水平与对照组差异不明显;肝组织冰冻切片免疫组化测定其MHC-Ⅰ抗原表达量低于正常对照组;腹腔巨噬细胞表面 MHC-Ⅱ抗原表达水平明显低于正常对照组。结论本研究表明 HBVH基因小鼠免疫耐受状态并非免疫系统单一环节障碍,在抗原提呈细胞、靶细胞抗原标志,特异性细胞免疫应答等方面均存在免疫功能低下及调节、识别障碍,其中DC功能低下,在免疫耐受状态中有重要作用。     

A review of immune tolerance induction with Haemate® P in haemophilia A

Immune tolerance induction (ITI) has been shown to successfully eliminate factor VIII (FVIII) inhibitors in haemophilia patients with inhibitors. We performed a literature search to identify reports from January 1980 to October 2012 on the use of the plasma‐derived, von Willebrand factor (VWF)‐containing FVIII concentrate Haemate^® P/Humate‐P^® in the setting of ITI. Six reports were identified that specifically evaluated the use of Haemate^® P/Humate‐P^® including 32 children and 9 adults. Dosing regimens ranged from 20 IU kg⁻¹ every 2–3 days in patients with low‐responding (LR; n = 5) inhibitors to 300 IU kg⁻¹ day⁻¹ in patients with high‐responding (HR; n = 36) inhibitors. Complete success was achieved in all five LR patients, in all three HR patients with good prognostic factors (age ≤7 years, pre‐ITI inhibitor titre <10 BU, historical inhibitor titre <200 BU, time between inhibitor detection and ITI start <2 years), and in 24 of 33 (73%) HR patients with poor prognostic factors. The time to complete success was 0.5–4 months in good‐prognosis patients and 0.5–42 months in poor‐prognosis patients. Few adverse events were observed during ITI, and no cases of inhibitor relapse were reported with follow‐up periods of up to 12 years. On the basis of this retrospective review of a diverse range of studies and case reports, we conclude that Haemate^® P/Humate‐P^® for ITI in patients with inhibitors is effective and produces high rates of ITI success.     

Immune tolerance induction in the treatment of paediatric haemophilia A patients with factor VIII inhibitors

The development of an inhibitor to transfused factor VIII (FVIII) is a serious treatment-related problem in haemophiliac children. The management of patients with high titre FVIII inhibitors is difficult, and immune tolerance induction (ITI) is the only method available for the eradication of these inhibitors. The results of the ITI regimen used at the Children's Hospital of Michigan Haemophilia Treatment Center are described and discussed. ITI was attempted in 14 children with severe haemophilia A (13 high responders, one low responder), with daily doses of FVIII alone. FVIII dosage was chosen according to the patient's historical peak inhibitor titre. ITI included three phases; induction phase, dose reduction phase and maintenance phase. During the first phase, the starting dose was 50 or 100 U kg-1 d-1; during the second phase the FVIII dosage was reduced gradually to 25 U kg-1 every other day according to the inhibitor titre, FVIII recovery and/or half-life study. In the third (maintenance) phase, the children received either prophylactic therapy or episodic therapy for 12 months. The inhibitor elimination was defined as the time taken to achieve a negative inhibitor assay with no anamnestic response and normal FVIII recovery and/or normal half-life. Immune tolerance was achieved in 11 of 14 patients (79%) patients within a median time of 6 months; two children are still on therapy, three failed ITI. We observed either failure or prolongation of immune tolerance if the historical peak titre or the maximum titre during ITI was >200 BU. The success rate of our low dose ITI regimen is not different from that reported by other investigators and the inhibitor elimination time is similar to some of the studies reported previously.     

Low-dose immune tolerance induction in hemophilia A patients with inhibitors

In patients with hemophilia A and inhibitory alloantibodies against factor VIII, various dosage schedules are used to obtain immune tolerance. In this study, we have evaluated the results of 13 years of low-dose immune tolerance induction and factors that are predictive of a positive result. The effect of immune tolerance induction in relation to age at inhibitor development, number of exposure days, age at start of therapy, maximum inhibitor titer, factor VIII products involved, and virologic status were determined. We evaluated 24 patients with severe hemophilia A and inhibitors who were treated with regular infusions with low-dose (25 U/kg every other day) factor VIII to obtain immune tolerance. In 21 of 24 patients (87%), immune tolerance induction was successful. The response time was determined by two factors: the highest inhibitor level and the age at inhibitor development. In patients with maximum inhibitor levels of less than 40 Bethesda units (BU)/mL, immune tolerance was obtained sooner than in patients with inhibitor levels exceeding 40 BU/mL (P = .005). Patients in whom an inhibitor developed before the age of 2.5 years also tended to have a quick immune response (P = .014). Immune tolerance with low-dose factor VIII is often successful in hemophilia A patients with inhibitors. Young children and patients with maximum inhibitors of less than 40 BU/mL show a relatively rapid response.     

Immune tolerance induction in patients with severe hemophilia with inhibitors: expert panel views and recommendations for clinical practice

For hemophilia patients with inhibitors, immune tolerance induction (ITI) may help to restore clinical response to factor (F) VIII or FIX concentrates. Several ITI regimens and protocols exist; however, despite 30 yr of progressive investigation, the ITI evidence base relies mainly on observational data. Expert opinion, experience, and interpretation of the available evidence are therefore valuable to support clinical decision-making. At the Sixth Zürich Haemophilia Forum, an expert panel considered recent data and consensus to distill key practice points relating to ITI. The panel supported current recommendations that, where feasible, ITI should be offered early to children and adults (ideally ≤ 5 yr of inhibitor detection) when inhibitor titers are <10 Bethesda units (BU) and should be stopped when successful tolerance is achieved. For hemophilia A inhibitor patients, ITI can be founded on recombinant FVIII at high doses. The panel considered that patients with a high bleeding frequency should be offered additional prophylaxis with a bypassing agent. For patients with hemophilia B, there may be a benefit of genetic testing to indicate the risk for inhibitors. ITI is often less effective and associated with a greater risk of side effects in these patients. For high-titer inhibitor (≥ 5 BU) hemophilia B patients, the panel advised that bypassing agents could be offered on demand in addition to ITI. Within future ITI regimens, there may be a role for additional immunosuppressant therapies. Participants agreed that research is needed to find alternatives to ITI therapy that offer durable and sustained effects and reduced rates of complications.     

Immune tolerance induction in patients with haemophilia A with inhibitors: a systematic review

Summary. In some patients with haemophilia A, therapeutically administered factor VIII (FVIII) comes to stimulate the production of antibodies (inhibitors) which react with FVIII to render it ineffective. As a result, FVIII cannot be used prophylactically and patients become liable to recurrent bleeds. There are two elements to the management of patients with inhibitors: the treatment of bleeding episodes, and attempts to abolish inhibitor production through the induction of immune tolerance. This paper reports a systematic review of the best available evidence of clinical effectiveness in relation to immune tolerance induction (ITI) in patients with haemophilia A with inhibitors. Owing to the lack of randomized controlled trials on this topic, broad inclusion criteria with regard to study design were applied in order to assess the best available evidence for each intervention. As a result of the clinical and methodological heterogeneity of the evidence, it was not appropriate to pool data across studies; instead, data were synthesized using tabulation and qualitative narrative assessment. The International Registry provides the most reliable estimate of the proportion of successful cases of ITI [48.7%, 95% confidence interval (CI) 42.6–52.7%]. The duration of effect is unclear, but relapses appear to be infrequent. The International Registry shows a rate of relapse of 15% at 15 years. The comparative effectiveness of different protocols is uncertain, as no trials have been undertaken which compare them directly. However, the evidence suggests that the Bonn protocol may be more effective than the Malmö or low‐dose protocols. There is no good evidence that immunosuppressive drug regimens are effective.