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Immunology Letters 110 (2007) 23–28

Review

Immune response against therapeutic factor VIII in hemophilia Apatients—A survey of probable risk factors

Suryasarathi Dasgupta a,b,c, Ana-Maria Navarrete a,b,c, Sandrine Delignat a,b,c,Bharath Wootla a,b,c, Sebastien Andre a,b,c, Valakunja Nagaraja d,

Sebastien Lacroix-Desmazes a,b,c, Srinivas V. Kaveri a,b,c,∗a INSERM, UMR S 872, Les Cordeliers, Paris F-75006, France

b Universite Pierre et Marie Curie-Paris6, Paris F-75006, Francec Universite Paris Descartes, Paris F-75006, France

d Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India

Received 13 March 2007; accepted 22 March 2007Available online 13 April 2007

bstract

A number of diseases are treated by passive administration of human proteins. Human coagulation factor VIII (FVIII) is one such protein whichs adminsitered to hemophilia A patients in order to manage and treat hemorrhagic incidences. This mode of therapy suffers from the side effect ofenerating anti-FVIII antibodies (inhibitors) which neutralizes the function of the infused FVIII. At a time when efficient viral screening procedures

re at place, development of inhibitors poses the greatest threat to such a therapy. Various predisposing factors, both patient and product-related,re responsible for the development of inhibitory antibodies. A proper understanding of these “risk-factors” would eventually help to better designherapeutic regimen to tackle hemophilia A.

2007 Elsevier B.V. All rights reserved.

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eywords: Factor VIII; Hemophilia; Inhibitors; Risk factors

. Introduction

Treatment of hemophilia A patients with therapeutic factorIII (FVIII) results in 15–30% of the cases, in the emergencef anti-FVIII antibodies (inhibitors) that neutralize the procoag-lant activity of therapeutically administered FVIII [1,2]. Theccurrence of inhibitors is considered to reflect an allogeneicmmune response to the repeated administration of an exo-eneous FVIII protein. The development of FVIII inhibitorsepresents both a major medical hurdle and a critical soci-tal concern; patients with FVIII inhibitors become resistant

o conventional replacement therapy and hence their quality ofife is dramatically affected, while morbidity and mortality arencreased. Moreover, the occurrence of an inhibitor to therapeu-

∗ Corresponding author at: INSERM UMRS 872, Equipe 16, Centre deecherche des Cordeliers, 15 rue de l’Ecole de Meıdecine, 75006 Paris, France.el.: +33 1 55 42 82 64; fax: +33 1 55 42 82 62.

E-mail address: srini.kaveri@umrs681.jussieu.fr (S.V. Kaveri).

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165-2478/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.imlet.2007.03.006

ic FVIII in a patient increases by more than three-folds the costf the treatment [3]. Thus, in developed countries, the yearly costf care of patients with inhibitors reaches D 190,000. The naturef the risk factors that predispose a patient to develop a FVIIInhibitor are still debated and include various patient-related androduct-related factors (Fig. 1).

. Genetic make-up of the patient contributing tonti-FVIII immune response

.1. Alteration in the FVIII gene

Hemophilia A is a clinically heterogeneous disorder, arisingrom a large number of molecular defects in the factor viiiene. Broadly, these molecular defects result in three differentategories of hemophilia A patients: severe hemophilia A,

hose plasma has no detectable FVIII activity, moderateemophilia A (1–4% of the normal FVIII level) or mild5–25% of the normal level) hemophilia A [4]. Incidence ofnhibitor development in these categories of patients reflect the

24 S. Dasgupta et al. / Immunology Letters 110 (2007) 23–28

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ig. 1. Various risk factors associated with the development of anti-FVIII immactors can be broadly from three different categories: those related to the geneto the particular product utilised in the therapeutic intervention.

everity of the molecular defect. Thus, in patients with severeemophilia A, 20–35% cases are manifested with anti-FVIIInhibitory antibodies [5–7], while in case of mild/moderateatients, the number is restricted to 3–13% [8,9].

Various mutations which result in the absence or severe trun-ation of the FVIII proteins are associated with the highestisk for inhibitor formation. This indicates that a complete lackf endogenous circulating FVIII protein enables the patient’smmune system to treat the incoming therapeutic FVIII molecules a ‘non-self’ antigen. A correlation between the severity ofemophilia A, type of mutation and inhibitor formation haseen envisaged in several clinical trials. In one such study bychwaab et al. [6], the risk for developing anti-FVIII anti-odies was shown to be strongly related to stop mutations,arge deletions and intrachromosomal recombinations. In a sep-rate study [10], involving previously untreated patients (whoad all received the same clotting factor treatment and under-ent regular monitoring for inhibitor development), 35% of

atients with severe molecular defects, i.e. intron 22 inversions,arge deletions or stop mutations—developed inhibitors. Nonef the patients with small insertions or deletions developednhibitors.

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esponse in hemophilia A patients undergoing FVIII replacement therapy. Riskkground of the patient, those patient-specific, yet non-genetic and those related

The risk of inhibitor development in mild hemophilia patientsas been reportedly linked with mutations in the A2 and2 domains, especially the Arg593-Cys and the Trp2229-Cysutations. A stable conformational change, effected by theseutations in the FVIII molecule might have rendered the FVIIIolecule with distinct immugenic properties in comparison with

he wild-type molecule [8].

.2. Other associated genetic factors influencing theevelopment of FVIII inhibitors

The concept of a genetic predisposition to inhibitor develop-ent has been further emphasized by the Malmo Internationalrother Study (MIBS) which was initiated in 1996, in order

o set up an international registry of twins and non-twin broth-rs with hemophilia and to search for genetic and compoundactors predisposing for inhibitor development [11]. The studyeported a 31.7% of inhibitor incidence in all families with

evere hemophilia A. It further reported twins in the inhibitorositive families along with a correlation of inhibitor historynd type (i.e. either high- or low-responding). The general riskor inhibitor development in families with a previous inhibitor

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istory was found to be much higher than in families with norevious known inhibitor. The latter observation involving fam-ly history of inhibitor development has recently been supportedn literature [12]. The MIB study group reported that the typef mutation provides, to some extent, the basis for the associa-ion between inhibitor formation and the causative FVIII gene

utation in siblings. However, the mutation itself is not enougho predict the risk for therapy-induced inhibitor formation [13].

Ethnicity has also been reported to be an indicator fornhibitor formation. An increased risk of inhibitors in African-merican hemophiliacs has been reported [14]. Such a trendas been seconded with more recent observations where non-aucasoid patients were shown to be at a higher risk thanaucasians [11,12].

Despite the obviously strong evidence of a genetic predis-osition, discordant inhibitor status in monozygotic hemophilia

twins demonstrates that environmental factors, in additiono genetic factors, play a role in the development of inhibitors7]. Similar hypothesis, i.e. environmental factors may be moremportant than genetic factors in antibody response to FVIII,as suggested on other occasions [15].

.3. MHC and cytokine genes

The major histocompatibility complex (MHC) moleculesind specific peptide sequences and present those peptides toD4 + T lymphocytes to initiate the cellular cascade leading

o B-cell stimulation and differentiation and ultimately to anti-ody formation. Jacquemin et al. demonstrated that in a mildemophiliac with an Arg2150His substitution in the C1 domainf FVIII, CD4 + T-cells were responsive to the wild-type peptide16], indirectly highlighting the importance of the MHC boundVIII-derived peptide in engendering an immune response.

In a recent computer generated model, the number and loca-ion of potential consensus recognition motifs on FVIII forarious HLA-DR molecules have been described [17]. Inter-stingly HLA DRB1*1501, the MHC molecule, which haseen previously reported to be associated with higher inhibitorncidence [18,19], contains 13 potential recognition motifs inontrast to HLA DRB1*1101 which contains only 2 motifs.owever, the reported increased prevalence of specfic MHCaplotype in inhibitor patients as compared to non-inhibitoratients presented with a weak correlation.

The HLA class I/II alleles and the tumor necrosis factorlpha (TNF-�) locus are closely linked in the MHC complex.NF-� is an important cytokine with potent proinflammatorynd immunomodulatory functions and polymorphisms in theene have been associated with autoimmune antibody-mediatediseases [20–22]. Data from Astermark et al. implied that theNF-�-G308A polymorphism within Hap 2 can be a usefularker and potential modulator of the immune response to

eplacement therapy in patients with hemophilia [23]. In anothertudy from the same group, IL-10 was reported to be the first

ene located outside the causative FVIII gene mutation to bessociated with inhibitor development [24]. Recently, polymor-hism in the CTLA-4 gene has also been correlated to inhibitorevelopment [25].

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Letters 110 (2007) 23–28 25

. Patient-related non-genetic factors

.1. Age of the first treatment

Age of the patient at initial treatment might influence inhibitorormation. Lorenzo et al. reported, in a study involving 62 severeemophilia A patients out of which 15 (i.e. 24%) showed per-istent or high-titre inhibitors, a correlation between the aget which they were exposed to FVIII for the first time andnhibitor incidence. Accordingly, as reported by this group, theaplan–Meier cumulative incidence at 3 years from first FVIII

xposure was 41% in patients starting therapy before the age ofmonths, 29% in patients starting therapy between 6 and 12onths of age and 12% in those starting therapy beyond 1 year

f age (P = 0.03) [26].In a separate study to assess the relationship between age of

rst administration of FVIII and the risk of developing inhibitorsn severe hemophilia A patients (infants), van den Berg and co-orkers showed cumulative incidence at 100 exposure days was4% in patients starting therapy before the age of 6 months, 20%n patients starting therapy between 6 months and 1 year, 13% inhose starting therapy between 1 and 1.5 years and 0% in thoseho started therapy beyond 1.5 years of age [27].In the recently reported Canal cohort study, similar trend

etween age of first treatment and inhibitor development wasound [28].

.2. Intensity of treatment

Continuous infusion of FVIII has been reported to be a causalactor for inhibitor development in mild hemophilia A patientsFVIII:C > 5%), in whom usually the appearance of inhibitors israre phenomenon. In this study, 4 patients developed inhibitorsut of 29 patients who received any form of FVIII infusion.nterestingly, only seven patients received continuous infusionf FVIII and all the four patients belonged to this group, giving aery high incidence of inhibitor development to this group (57%,.e. 4/7) [29]. Several other studies indicate similar relation ofontinuous FVIII infusion with inhibitor development, includingffects of switching between different FVIII products on theevelopment of inhibitors [30,31].

. Product-related factors

.1. FVIII aggregation and immune response

The physical state of the therapeutic protein to be infused tohe patient, especially aggregate formation, play an importantole in the immunogenicity of the said proteins [32,33]. FVIIIas a tendency to aggregate. In an attempt to understand theffect of aggregation of FVIII on its immunogenicity, Purohit etl. have studied the effect of aggregation of FVIII produced byhermal stress in a murine model of hemophilia A [34]. Their

bservation suggests that aggregation per se does not enhancemmunogenicity of FVIII.

Interestingly, immunogenicity of FVIII in the sameurine model of hemophilia A was downmodulated when

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6 S. Dasgupta et al. / Immun

hey complexed O-phospho-l-serine, the head group ofhosphatidylserine, with the C2 domain of FVIII [35]. O-hospho-l-serine also prevented aggregation of the protein under

hermal stress. Several universal epitopes for CD4 + T-cells haveeen identified within the C2 domain of FVIII [36–38], aegion containing epitopes with phosphoplipid (especially phos-hatidyl serine, PS) affinity [39]. Similar reduction of FVIIImmunogenicity was observed when other PS oriented chemical

odifiers were used [40]. Thus, affinity of FVIII for phos-holipid structures, especially PS, can be a risk factor for theevelopment of inhibitors. In line with this argument, it haseen previously shown that indeed the increase in phospholipidffinity can be correlated with a higher frequency of inhibitorevelopment in patients. While, qualifying the effect of variousirus inactivation procedures, this study showed that the proce-ure which generated an increased phospholipid affinity resultedn inhibitors predominantly against the C2 domain [41].

.2. Type of FVIII used

Probably the most controversial risk factor for the develop-ent of inhibitors in hemophilia A patients comes from the type

f the therapeutic FVIII molecule used. In particular, the pres-nce/absence of von Willebrand factor (VWF), in connection tonhibitor development has been intensely debated [42,43].

A role for von Willebrand factor as a chaperone molecule forrocoagulant FVIII has been extensively documented [44–47].nder physiologic conditions, VWF binds to FVIII after its

elease in the circulation. VWF protects FVIII from proteoly-is by lipid-bound proteases, stabilizes the FVIII heterodimerictructure, modulates its activity by thrombin and further reg-lates its elimination by lipoprotein-related receptors [48,49].hus, the FVIII molecule derived from plasma contains vari-ble amounts of VWF, depending on the type of purification, inontrast to the recombinant FVIII molecule which is completelyevoid of VWF.

In the first international prospective studies for variousecombinant FVIII molecules, an inhibitor incidence of 20–33%as observed. In one of the first multicentre studies on the effi-

acy and inhibitory properties of recombinant FVIII, Lusher etl. documented, on previously untreated patients (PUPs), theevelopment of inhibitory antibodies in 16 out of 81 patientsfter a median of nine days of exposure to FVIII treatment.owever, inhibitor titres were found to be low with time in 9f the 16 patients despite continued episodic treatment with theoncentrate [50].

In a separate study with another recombinant full-lengthVIII molecule in previously untreated patients (PUPs) withemophilia A, 17 (23.9%) subjects were found to have devel-ped inhibitors [51]. Similar incidence of inhibitor developmentas also observed with a B-domain deleted recombinant FVIII

BDDrFVIII, this molecule was created since the B-domain wasound to be redundant for maintaining haemostasis). Thirty-two

reviously untreated patients (around 33%) developed inhibitorsfter a median duration of 12 exposure days. Sixteen of 32 (50%)atients had low levels (≤5 Bethesda units) and 16 had highevels of inhibitors [52].

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Letters 110 (2007) 23–28

In contrast to the recombinant FVIIIs, plasma-derived FVIIIroducts have been reported to yield lower incidence ofnhibitors. In a study involving 56 previously untreated patientsith severe hemophilia A, receiving only one brand of highlyurified FVIII concentrate (HPSD-VIII), five patients (9%)eveloped an inhibitor after 6–19 exposure days, out of whichnly one was a high responder (2%), showing a low incidence ofnhibitor [53]. In a separate study with another plasma-derivedVIII which contained well preserved von Willebrand factorultimers, inhibitor development was found to be absent when

reviously untreated patients with severe haemophilia A werereated with the product [54]. Similar incidence of low or nonhibitor development has been also reported with other plasma-erived FVIII products [55].

In recent retrospective studies, the issue of the presence ofWF in FVIII preparations has further been brought to light withne study indicating that plasma-derived FVIII product (whichontains VWF) induces lesser incidence of inhibitor formationhan recombinant FVIII [12], while another study shows thebsence of such an association [56]. The available informationarrants further investigation through prospective controlled

tudies and through the dissection of the molecular mechanismshat could be at play in the possible protective effect of VWF onVIII immunogenicity.

. Conclusion

Recent advances in the field of cellular immunology havereated a wide spectrum of opportunities for further investi-ations and betterment of our understanding of the genesis ofVIII immune response. This area of knowledge might becomextremely pertinent to the way hemophilia A patients are treatedith various FVIII products. The nature of the FVIII-specific B

nd T lymphocytes involved in the anti-FVIII immune responseas been elucidated [16,57–62]. Little is known, however, on thentry of FVIII in professional antigen-presenting cells (APCs),n event upstream from the activation of immune effectors andandatory to the elicitation of the primary immune response

gainst FVIII. In addition to the MHC peptide signal given by thePCs to the T-cells, various co-stimulatory molecules on APCs

lso influence the development of an immune response. Accord-ngly, by targeting one such APC-target T-cell co-stimulatoryathway (B7-CD28), FVIII immune response has been showno be kept in leash in the murine model of hemophilia A [63].n a similar model of hemophilia A, the addition of VWF toVIII have shown to reduce anti-FVIII titres [64]. Our recenttudy have highlighted the role of VWF as an immunoprotec-ive chaperone for FVIII, that is, by preventing, upstream fromhe activation of immune effectors, the entry of FVIII in profes-ional APCs. Further, we demonstrate in vitro that increasinghe VWF/FVIII ratio reduces FVIII endocytosis by dendriticells in a dose-dependent manner [65]. However, similar den-ritic cells do not undergo a maturation program in presence

f FVIII and/or VWF [66], a phenomenon required for mount-ng a suitable immune response. Thus, proper delineation ofow different types of FVIII interact with various types ofPCs in initializing an anti-FVIII immune response, would

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nable us to understand the potential risk factors in a betteright.

cknowledgements

This work was supported by Institut National de la Santet de la Recherche Medicale (INSERM), by Centre Nationale la Recherche Scientifique (CNRS), by Universiteı Pierre etarie Curie-Paris 6, by Indo-French Center for the Promotion ofdvanced Research (CEFIPRA) and by Agence Nationale de laecherche (ANR-05-MRAR-030). SuD and BW are the recip-

ents of fellowships from Fondation de la Recherche Medicale,nd from LFB, France, respectively.

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