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Molecular determinants of FVIII immunogenicity in hemophilia A
Wróblewska, A.
Publication date
2013
Document Version
Final published version
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Citation for published version (APA):
Wróblewska, A. (2013). Molecular determinants of FVIII immunogenicity in hemophilia A.
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Molecular determinants of FVIII immunogenicity
in hemophilia A
Molecular determinants of FVIII immunogenicity
in hemophilia A
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam
op gezag van de Rector Magnificus prof. dr. D.C. van den Boom
ten overstaan van een door het college voor promoties ingestelde commissie, in het openbaar te verdedigen in de Aula der Universiteit
op vrijdag 24 mei 2013, te 11.00 uur
door
Aleksandra Wróblewska
Promotiecommissie
Promotores: Prof. Dr. J.J. Voorberg Prof. Dr. K. Mertens Co-promotores: Dr. A. ten Brinke
Dr. A.B. Meijer
Overige leden: Prof. Dr. J.C.M. Meijers Prof. Dr. R.A.W. van Lier Prof. Dr. C.J.M. de Vries Prof. Dr. S. Middeldorp Prof. Dr. H.C.J. Eikenboom Faculteit der Geneeskunde
The work described in this thesis was performed at the department of Plasma Proteins, Sanquin Blood Supply, as a part of Sanquin-AMC Landsteiner and Van Creveld Laboratory, Amsterdam, The Netherlands.
Financial support by the Netherlands Heart Foundation and Stichting Haemophilia for the printing of this thesis is gratefully acknowledged.
Additional financial support was kindly provided by: Sanquin Research, University of Amsterdam, Lonza, CellGenix and eBioscience.
ISBN 978-94-6182-263-5
Copyright © 2013 by Aleksandra Wróblewska Printed by Offpage, Amsterdam, The Netherlands
Cover: interaction between dendritic cell (green) and T lymphocyte (pink), posterized scanning electron micrograph
Table of contents
Chapter 1General introduction 9
Chapter 2 Dangerous liaisons: how the immune system deals with factor VIII 29
J Thromb Haemost. 2013;11(1):47-55 Chapter 3 Uptake of blood coagulation factor VIII by dendritic cells is mediated via its C1 domain 47
J Allergy Clin Immunol. 2012;129(2):501-509, 509.e1-5 Chapter 3+ Reply to: Mannose-sensitive receptors mediate the uptake of factor VIII therapeutics by human dendritic cells 71
J Allergy Clin Immunol. 2012;129(4):1172-5 Chapter 4 Modification of an exposed loop in the C1 domain reduces immune responses to factor VIII in hemophilia A mice 79
Blood. 2012;119(22):5294-300 Chapter 5 Immune complexes enhance uptake of blood coagulation factor VIII by antigen presenting cells 103
Chapter 6 Pharmacological modulation of the uptake of blood coagulation FVIII by antigen presenting cells 119
Chapter 7 General discussion 135 Chapter 8 Summary 151 Samenvatting 153 Podsumowanie 155 Curriculum vitae 157 List of publications 159 Acknowledgments 161
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1
Hemophilia AHemophilia A is hereditary X-linked disorder caused by dysfunction or absence of blood coagulation factor VIII. The bleeding syndrome has been recognized as early as in the fifth century, but only since 1937 was linked to a deficiency of a plasma component, named in 1962 “factor VIII”. 1 Depending on the
severity of the disease, it can be divided into three categories: mild (5-25% of normalplasmalevelsof FVIII),moderate (1-5%)and severe(<1%)hemophilia A. In its severe form it can lead to spontaneous joint and muscle bleeds, which consequently can cause deformation of joints requiring surgical intervention. Trauma can cause life-threatening internal bleedings and hemorrhages. Treating hemophilia became possible in 1964, when Judith Graham Pool, working at Stanford University, described how to obtain cryoprecipitate from human plasma. 2 Since then, so-called replacement therapy comprising regular injections
of FVIII of either recombinant or plasma-derived origin became commonly used in hemophilia care. However, such treatment is hampered by high costs of the FVIII products – it is estimated that more than 75% of the hemophilia communityworldwidereceiveeitherinadequateornotreatmentwhatsoever.3
In response to frequent FVIII infusions, a subset of patients develops anti-FVIII antibodies. In severe hemophilia A, inhibitors develop after a median of 10 to 15 days of treatment with FVIII. 4 After 50 exposure days, the cumulative
incidence of inhibitors reaches a plateau, after which the occurrence of inhibitors is exceptional. 5 Anti-FVIII antibodies can rapidly inhibit FVIII function,
rendering FVIII infusion therapy ineffective. Inhibitory antibodies interfere with binding of FVIII to other coagulation factors such as factor IIa, IXa and X or to phospholipids.6,7 Non-inhibitory antibodies can also compromise hemophilia
treatment by influencing FVIII stability and/or its pharmacokinetics by interfering with binding to von Willebrand factor (VWF). 8,9 The majority of inhibitory
antibodies directed towards FVIII are of subclass IgG1 and IgG4. 10-12 Factor VIII: structure and function
FVIII is synthesized as a polypeptide chain comprising a signal peptide of 19 amino acids and a mature protein of 2332 residues. FVIII consists of three A, two C domains and one unique B domain that are arranged in the following order:
A1-a1-A2-a2-B-a3-A3-C1-C2 (Figure 1). 13,14 The a1 (residues 337-372), a2 (residues
711-740) and a3 (residues 1649-1689) regions bordering the A domains are enriched for aspartic and glutamic acid residues. 13 In plasma, FVIII circulates as a
hetero-dimer consisting of a 90-220 kDa heavy chain that is non-covalently linked to a 80 kDa light chain (Figure 1). 15 Cleavage at Arg1648 by as yet unidentified
protease releases the light chain (a3-A3-C1-C2); additional processing at various positions (including Arg740 and Arg1312) within the B domain yields a heavy chain (A1-a1-A2-a2-B) that is heterogeneous in size. In the circulation, FVIII is tightly associated with its carrier protein – von Willebrand factor (VWF), which protects it from proteolytic degradation and premature clearance. 16-20 Sulfation
of Tyr1680 within the acidic a3 region is required for high affinity binding of FVIII to VWF. 21-23 Also residues in the C1 and C2 domain have been implicated
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in binding of FVIII to VWF. 16,21-25 FVIII circulates as an inactive precursor that
can only act as a cofactor for the FIXa-dependent conversion of FX following its activation by thrombin or FXa. 26 Cleavage by thrombin at Arg1689 releases the
acidic a3 region which promotes rapid disassembly of the FVIII-VWF complex 27,28;
cleavage at Arg372 and Arg740 results in a hetero-trimeric molecule comprising the A3-C1-C2, A1-a1 and A2-a2 domains. 27 The resulting hetero-trimer can
efficiently catalyze the conversion of factor X to Xa by factor IXa on phospholipid surfaces. 14 Dissociation of the A2 domain from hetero-trimeric FVIII results in
a rapid decline of FVIII cofactor activity. 29-31 Also cleavage of FVIII by activated
protein C at positions Arg336 and Arg562 abolishes cofactor activity of activated FVIII. 32,33 Residues 1811-1818 in the A3 domain and residues 558-565 in the
A2 domain have been shown to contribute to the binding of factor IXa. 34-37
Limited information is available with respect to the binding site for factor X on activated FVIII; binding sites for factor X in the C2 domain and the a1 region have been proposed. 38,39 It is now well-established that both the C1 and C2 domain
contribute to binding of FVIII to negatively charged phospholipids. 40-44 Analysis of
the three-dimensional structure of the C2 domain and site-directed mutagenesis have firmly implicated a role for Met2199, Phe2200, Leu2251 and Leu2252 in the binding of FVIII to negatively charged phospholipids. 45-48 More recent
studies have identified an exposed surface loop in the C1 domain harbouring Arg2090, Gln2091, Lys2092 and Phe2093 that modulates binding of FVIII to surfaces containing a low percentage of negatively charged, phosphatidylserine-containing phospholipids. 42,43 Figure 1 provides an overview of interactive sites
on FVIII for its major binding partners.
FVIII precursor form
FVIII circulating in plasma
Heavy chain Light chain
Me2+ A1 A2 A3 C1 C2
Figure 1. Structure of blood coagulation factor VIII (FVIII). Upper left panel shows
schematic domain organization of FVIII; lower left panel – schematic representation of circulating FVIII, where the heavy (A1-A2-B) and the light (A3-C1-C2) chain are non-covalently linked via a metal ion-dependent interaction between the A1 and A3 domain. In activated FVIII, the A1 and A3 domains remain non-covalently bound, while the A2 domain is weakly associated with the A1/A3-C1-C2 dimer. Right panel provides a crystal structure of B domain-deleted FVIII (pdb code 3cdz). Interactive sites for phospholipids, VWF, FIXa and FX are indicated in the model.
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Epitope mapping studies have revealed that inhibitory antibodies that develop in patients with hemophilia A bind to the A2 and C2 domain of FVIII. 7,49-52 More
detailed binding studies have shown that residues 484-508 provide a major binding site for anti-A2 domain antibodies. 52 The mode of action of anti-A2 inhibitors is not
yet entirely clear; an early study by Lollar and co-workers suggested that anti-A2 antibodies act as non-competitive inhibitors of intrinsic Factor X activation complex by blocking the conversion of FXase/FX complex to the transition state. 53 Follow-up
studies using isolated A2 domain suggested that anti-A2 antibodies directly inhibit the interaction of A2 subunit with factor IXa, thus abrogating the contribution of this subunit to cofactor activity. 54 Anti-C2 domain antibodies have been shown to bind to
exposed residues overlapping with the phospholipid binding site. 55 Co-crystalization
of a human monoclonal anti-C2 domain antibody, BO2C11, revealed that Arg2215, Arg2220, Met2199, Phe2200, Val2223, Leu2251 and Leu2252 comprise contact residues for this inhibitor. 56 Inhibitory antibodies directed towards residues
1811-1818 in the A3 domain have also been detected in patients with hemophilia A. 34,57
Antibodies binding to these sites have been shown to limit the binding of FIXa to FVIII. 34,57 Moreover, in a small number of patients antibodies directed towards the
acidic a1-region have been identified. 24,41,58
Replacement therapy and inhibitor development in hemophilia A
The development of inhibitors occurs in approximately 5% of mild or moderate hemophilia A patients, and in 25% of severe hemophilia A patients. 59
The low prevalence of inhibitor development in patients with mild and moderate hemophilia A is most likely caused by the presence of endogenous levels of circulating FVIII which render this group of patients tolerant to subsequent replacement therapy. 60 The lack of endogenous levels of circulating FVIII most
likely underlies the increased frequency of FVIII inhibitors in patients with severe hemophilia A. A large number of studies have addressed the correlation between FVIII genotype and inhibitor development. 5,61,62 Patients carrying intron
22 inversions, nonsense mutations or large deletions are more prone to develop inhibitors than those with small deletions and missense mutations. 63,64 Nonsense
mutations that affect the light chain of FVIII are more frequently associated with inhibitor development than those present in the FVIII heavy chain. 65,66 Formation
of inhibitors in mild hemophilia associates with a limited number of high-risk FVIII mutations within the A2, C1 or C2 domain, namely Arg593Cys, Arg2150His or Trp2229Cys. 67,68 Why only a fraction of patients generate antibodies against
FVIII is still poorly understood. Both treatment-related and genetic risk factors have been shown to contribute to inhibitor development in hemophilia A. Intensity of FVIII treatment is an acknowledged risk factor, as FVIII administered to treat bleeding episodes or to support surgery delivers high concentrations of FVIII that could promote antibody development. 69-72 Apart from FVIII gene
mutations 73,74, polymorphisms within the IL-10 75 and TNFA gene 76 have been
associated with inhibitor development. Interestingly, a C/T polymorphism in the promoter region of the CTLA-4 gene 77 was overrepresented in hemophilia A
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formation. Several studies have explored whether inhibitor development is linked to type of FVIII product. There are conflicting findings about the immunogenicity of recombinant versus plasma derived FVIII. 78-81 Results from a large
multi-center cohort study suggest that the risk of inhibitor formation is similar for recombinant and plasma derived FVIII products. 78-81 Also the von Willebrand
factor content of therapeutic FVIII products seems not to be associated with the risk of inhibitor development. 81
FVIII and the immune system
It is now well-established that the formation of high-affinity IgG molecules requires FVIII-specific CD4+ T-cell help. 10,82 Initial evidence for involvement of T
cells in the development of inhibitors in hemophilia A patients came from studies involving HIV-positive individuals, that due to diminished number of CD4+ T cells
showed also decline in anti-FVIII antibody responses. 83 The first step leading
to activation of specific T- and B-cell responses is recognition of antigen by specialized antigen-presenting cells (APCs). Subsequently, antigen is processed into small peptides which are loaded on MHC class II. Presentation of derived peptides on the surface of APCs triggers the activation of antigen-specific CD4+ T cells (Figure 2). In the past years, several studies have addressed
how FVIII is processed by APCs. 84-87 Although a role for several receptors has
been suggested (for a review see Chapter 2) 84,85, the exact mechanism of FVIII
endocytosis by APCs remains unclear. APCs, equipped with receptors recognizing foreign, pathogen-derived molecules (so-called pathogen-recognition receptors, PRRs), are able to sense “danger”, which prompts maturation of these cells. Upon maturation, they upregulate a number of co-stimulatory molecules such as CD40, CTLA-4, CD80 and CD86 and due to their simultaneous ability to release cytokines APCs can activate and modulate antigen-specific T cell responses (Figure 2). The importance of co-stimulatory mechanism for FVIII-specific T-cell activation has been illustrated by several studies on the blockade of CD40/CD40L interactions in vivo. 88,89 CD40/CD40L ligation provides a key event to induce
humoral responses against antigens 90; furthermore, blockage of CD40/CD40L
interaction leads to long-lasting tolerance in mice. 91,92 However, even though
the disruption of CD40/CD40L interaction by pre-administration of a monoclonal antibody targeting CD40L resulted in deficient immune responses against FVIII
in vivo, it failed to induce long-lasting tolerance. 89 Clinical trials suggested that
administration of a humanized anti-CD40 ligand antibody (hu5c8) can block anamnestic responses to factor VIII; however it remained unclear whether that effect would persist and result in long-lasting tolerance. 93 Moreover, due to
tromboembolic complications in patients treated with hu5c8, this approach for treatment has been discontinued.
During TCR activation in a particular cytokine milieu, naive CD4+ T cells may
differentiate into one of several lineages of T helper (Th) cells including Th1, Th2, Th17 and regulatory T cells (Tregs), as defined by their pattern of cytokine production and function. Generally, Th1 cells promote cellular immunity; Th2 cells mediate humoral immunity; Th17 cells play an important role in clearing pathogens during
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host defense reactions and in inducing tissue inflammation in autoimmune disease
94; Tregs are capable of inducing tolerance by suppressing T- and B-cell responses.
T-cell reponses against FVIII are of a polyclonal origin and directed against multiple epitopes present in different domains of FVIII. 95 Th1 10, Th2 10 as well as Th17 96 cells
have been shown to contribute to FVIII-specific immune responses in hemophilia subjects. Eventually, FVIII-specific T cells are able to activate FVIII-specific B cells and subsequently induce affinity maturation and class-switching of immunoglobulin genes. As a consequence, FVIII-specific long-living plasma cells and memory B cells are generated, which are able to rapidly respond to re-exposure to FVIII. 97,98
FVIII-specific B cell responses are also, similar to T-cell responses, of a polyclonal origin and directed against various epitopes. Determination of classes and subclasses of anti-FVIII antibodies revealed dominant contribution of IgGs and their subclasses – IgG1, IgG2 and IgG4. 8,10,12 Generated antibodies can inhibit FVIII function by
interfering with its interaction with other coagulation factors (mainly A2 and A3-C1 antibodies) and/or phospholipids (C1/C2-directed antibodies), as described earlier. It has been reported, that a subset of anti-factor VIII IgG hydrolyzes FVIII. These so-called catalytic antibodies were found in over 50% of inhibitor-positive patients with severe hemophilia A, but not in inhibitor-negative individuals. 100 FVIII-hydrolyzing IgG
from each patient tested exhibit multiple cleavage sites on FVIII and the specificity of cleavage varied from one patient to another. 100 Circulating antibodies that do not
inhibit FVIII function can influence FVIII half-life, either by interfering with binding of FVIII to VWF or by formation of immune complexes that can be efficiently cleared via Fc receptors. Antigen Dendritic cell T helper cell B cell Plasma cells Cytokines MHC II TCR CD28 B7.2 Antibodies
Figure 2. Simplified overview of the development of humoral immune responses. Antigen is endocytosed by
antigen-presenting cell (such as dendritic cell), processed and presented on MHC class II. For efficient activation, co-stimulatory molecules present on APCs and T cell receptors need to interact, while cytokines released by APCs determine the future direction of ongoing T-cell responses. Once primed, T cells can activate B cells in an antigen-specific manner, which leads to formation of long-living plasma cells producing high-affinity antibodies.
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Immune tolerance induction (ITI) therapy – eradication of inhibitors
Development of inhibitors is a serious complication in hemophilia care. The magnitude of antibody responses is quantified by a functional assay and expressed in Bethesda units (BU). Patients with low titer of inhibitors (< 5 BU/ ml) can be treated with higher and/or more frequent doses of FVIII, which leads to saturation of pre-existing inhibitors and still provides enough FVIII to restore hemostasis and normal coagulation. For patients with higher inhibitor titers (>5 BU/ml), simple infusion therapy becomes ineffective, therefore bypassing agents such as activated factor VII or activated prothrombin-complex concentrates need to be used. 101,102 Simultaneously, eradication of inhibitors and
immune tolerance induction (ITI) therapy is introduced. ITI comprises regular injections of FVIII for a period varying from several weeks up to two years and since its first description in 1977 103 remains the only strategy that proved to both
eradicate FVIII inhibitors as well as lead to induction of FVIII-specific immune tolerance. The most commonly used protocols, known as the Bonn 104, the Van
Creveld 105 and the Malmö 106-108 protocols, although considerably different 65,
result in comparable success rates (up to 87%). A recent study showed that high-dose ITI leads to faster recovery and tolerance induction, accompanied by fewer bleeding episodes as compared to low-dose ITI. 109 Van Helden and co-workers
showed a correlation between distribution of IgG-subclasses of anti-FVIII antibodies and outcome of ITI therapy. A predominance of IgG4 antibodies was observed in patients who needed prolonged ITI treatment. 11 Also complications,
such as venous catheter infection, can prolong the course of ITI or even lead to its failure. 110 The immunological mechanisms underlying success of ITI therapy
remain unclear. In a naïve, non-primed environment, chronic exposure to high doses of antigen would activate regulatory T cells able to suppress antigen-specific (in this case FVIII-antigen-specific) effector T cells, resulting in tolerance induction. 111,112 Consequently, no T cell help would be provided to FVIII-specific
B cells that could not differentiate into long-living antibody producing plasma cells and, as a result, would be depleted. This scenario is however unlikely to happen in patients with pre-existing antibodies that have enriched populations of FVIII-specific memory T and B cells. In this case, memory B cells serve as highly efficient antigen-presenting cells, able to effectively re-stimulate FVIII-specific memory T cells. Moreover, upon encounter with antigen, memory B cells rapidly differentiate into specific antibody producing plasma cells, enriching already pre-existing pool of such cells. However, the amount of antigen seems to be crucial factor for an optimal stimulation of memory B cells. 113 Studies using
FVIII-deficient mice showed that concentrations of FVIII below the physiologic plasma concentration of 0.1 μg/ml (1 U/mL) restimulate FVIII-specific memory B cells and induce their differentiation into antibody-secreting cells (ASCs). 113 However,
high concentrations (above 0.1 μg/ml), inhibit memory B cell restimulation and prevent the formation of ASCs. 113 The inhibition of FVIII-specific memory B-cell
responses seems to be irreversible and not mediated by FVIII-specific T cells. Such depletion of memory B-cell can be an early event in the inhibitors eradication in patients undergoing ITI therapy, who receive high doses of FVIII. Gilles and
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colleagues 114 followed two patients with FVIII inhibitors during their course of
ITI and suggested that induction of anti-idiotypic antibodies, neutralizing the existing anti-FVIII antibodies, might be one of the reasons underlying success of ITI.
Animal models of hemophilia A
In1995Biandco-workers115,116 introducedtwomousemodelsforhemophilia A,
which over the years proved to be very useful for broadening our knowledge on induction and modulation of immune responses against FVIII. Targeted disruption of exon 16 (E16-KO mice) or exon 17 (E17-KO) resulted in the absence of functional FVIII in the circulation (<1% normal FVIII activity). Both strains displayed a bleeding tendency 117, which could be corrected by infusions with
human FVIII. 118 Similar to hemophilia A patients, FVIII-deficient mice develop
an immune response following repetitive intravenous injections of therapeutic doses of FVIII. 113,119,120 Generated antibodies are directed both to the light as
wellastotheheavychainofFVIII120,persistinthecirculationforalongtime 98 and
their development is strictly dependent on CD4+ T helper cells. 121,122 Cytokine
profiles of factor VIII-specific T cells indicate that the regulation of the anti-FVIII antibody response in hemophilic mice involves both Th1- and Th2-type cells. 92,122 Over the years, studies involving FVIII-deficient mice enabled us to
gain more knowledge regarding modulation of immune response to FVIII and tolerance induction. Furthermore, it allowed for evaluation of a number of novel therapeutic approaches to prevent or eradicate inhibitor development in hemophilia A. Administration of complexes of FVIII and phosphatidylserine (PS) liposomes resulted in reduced antibody formation against FVIII in hemophilia A mice. 123 PS associated with apoptotic cells is known to induce anti-inflammatory
responses in APCs 124, moreover, PS liposomes reduce the maturation,
pro-inflammatory cytokine production and T cell priming of APCs. 124 Formation of
complex between FVIII and phospholipid molecules is mediated by residues located in the C1 and C2 domains of FVIII. 42,44 Therefore, a possible alternative
explanation of the observed inhibitory effect is that PS occupies residues of FVIII that are crucial for its endocytosis by APCs. CTLA4-IgG, blocking the co-stimulatory interaction between B7 and CD28, has been shown to transiently inhibit anti-FVIII antibody formation in hemophilia A mice. 88 An independent study revealed
that simultaneous blockage of CD40-CD40L and B7-CD28 pathways abolishes development of inhibitors and promotes long-term immune tolerance specific for FVIII. 125 CD3 antibody has been studied as a tolerance-inducing agent for
several autoimmune and inflammatory diseases. 126-128 It modulates the
CD3–T-cell receptor (TCR) complex and leads to anergy or apoptosis of effector T CD3–T-cells, or to the expansion of regulatory CD4+CD25+ T cells. 128 Consequently, anti-CD3
treatment proved to successfully prevent inhibitor formation in hemophilia A mice with both BALB/c and C57BL/6 background. 129,130 Rituximab, a therapeutic
anti-CD20 antibody, has been used for several years to treat patients with inhibitors, although with various success rates. 131,132 In hemophilia A mice,
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titers in FVIII-challenged animals. 133 However, hyporesponsiveness to FVIII was
sustained over time only when an anti-CD20 antibody of subclass IgG1 was used, which, in contrast to the other isotype tested – IgG2a, did not deplete marginal zone B cells. 133 Moreover, treatment with anti-CD20 IgG1, but not IgG2a, was
accompanied by substantial increase of splenic regulatory T cells, implicating a possible tolerogenic role for the remaining B cell population. 133 A recent study
by Sack and colleagues, where anti-CD20 treatment was combined with AAV-based gene therapy, showed that B cell depletion can render FVIII-deficient mice hyporesponsive to FVIII, but results were dependent on the genetic background of the strain used. 134 Transduction with retroviral constructs encoding FVIII
A2- or C2-domain in frame with an IgG heavy chain backbone of B cell blasts has been shown to induce immune tolerance to FVIII in hemophilic mice. 135
Mice treated with transduced B cells showed reduced inhibitor titers, which were persistently low after additional challenges with FVIII. Furthermore, the lower antibody titers correlated with an increased frequency of FVIII-specific regulatory T cells. 135,136 Factor VIII deficient mice have also been successfully
used to study the presence and persistence of memory B cells, as in detailed described in the previous paragraph. 113
Bril and colleagues 137 generated transgenic mice expressing human FVIII with the
Arg593 to Cys mutation, which is associated with mild hemophilia phenotype. 68,138,139
Unlike E16-KO or E17-KO mice, these animals did not develop inhibitor titers upon repetitive intravenous injections of human FVIII, however induction of an immune response took place after subcutaneous FVIII administration in the presence of a strong adjuvant. 137 A similar model was described by van Helden and colleagues who
constructed a transgenic mouse expressing full length human FVIII. 140 In accordance
with findings by Bril and co-workers no inhibitor formation was observed in this model following the intravenous administration of human FVIII. Interestingly, infusion of PEGylated FVIII evoked an immune response in transgenic mice expressing human FVIII. These results suggest that transgenic mouse models that express human FVIII are useful models for assessing the potential immunogenicity of genetically or chemically modified FVIII variants. More recently, the development of a humanized hemophilic E17 HLA-DRB1*1501 mouse model has been described. 89,141 Humanized
mice have been utilized to study the regulation of HLA class II-restricted immune responses to various antigens and they proved to be highly suitable for in vivo research into the mechanistic basis of human diseases associated with activation of CD4+ T cells. 142 HLA-DRB1*1501 was selected due to a strong connection between
this haplotype and many immunologic diseases 143, as well as a previously noted
linkbetweeninhibitorincidenceandDRB1*1501inpatientswithseverehemophilia A.144 Despite some obvious limitations, such new models can be used to analyze the
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Scope of the thesisUnderstanding the recognition of FVIII by antigen-presenting cells and subsequent activation of specific T and B cells is essential for development of new strategies for treatment and/or prevention of inhibitor formation to FVIII in hemophilia A patients. In this thesis we explored the mechanism of FVIII endocytosis by both human and mouse dendritic cells (Chapter 3 and Chapter 6). A search for structural determinants important for immune recognition of FVIII revealed that C1 domain-targeting monoclonal antibody KM33 prevents the uptake of FVIII by APCs and delays the formation of anti-FVIII antibodies in hemophilia A mice.
Chapter 4 follows up on this finding and shows that modification of C1 domain
residues crucial for KM33 binding diminishes FVIII uptake by dendritic cells. Upon in vivo administration in FVIII-deficient mice these C1 domain variants developed significantly lower anti-FVIII antibody titers and reduced CD4+ T cell
responses. In Chapter 5 we address the role of immune complex formation in FVIII endocytosis by APCs and its influence on subsequent FVIII-specific T cell responses. Chapter 6 elaborates in more detail on the possible mechanism of FVIII endocytosis by human dendritic cells and the potential role of the C2 domain in this process. Together our findings provide more insight into immune recognition of FVIII that can be utilized to develop novel strategies for treatment or prevention of inhibitor formation in hemophilia A.
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2
Dangerous liaisons:
how the immune system deals with
factor VIII
Aleksandra Wroblewska, Birgit M. Reipert, Kathleen P. Pratt and Jan Voorberg
Fa
ct
or VIII and the immune s
ys
tem
2
Abstract
Only a fraction of hemophilia A patients develops a neutralizing antibody (inhibitor) response to therapeutic infusions of factor VIII (FVIII). Our present understanding of the underlying causes of the immunogenicity of this protein is limited. In the past few years, insights into the uptake and processing of FVIII by antigen presenting cells (APCs) have expanded significantly. While the mechanism of endocytosis remains unclear, current data indicate that FVIII enters APCs via its C1 domain. Its subsequent processing within endolysosomes allows for presentation of a heterogeneous collection of FVIII peptides on MHC class II, and this peptide-MHC class II complex may then be recognized by cognate effector CD4+ T cells, leading to anti-FVIII antibody production. Here we aim to summarize
recent knowledge gained on FVIII processing and presentation by APCs, as well as the diversity of the FVIII-specific T-cell repertoire in mice and men. Moreover, we discuss possible factors that can drive FVIII immunogenicity. We believe that increasing understanding of immune recognition of FVIII and the cellular mechanisms of anti-FVIII antibody production will lead to novel therapeutic approaches to prevent inhibitor formation in patients with hemophilia A.
Fa
ctor VIII and the immune s
ystem
2
IntroductionProfessional APCs such as dendritic cells (DCs), macrophages and B cells endocytose proteins that are degraded along the endocytic pathway and then presented on MHC class II molecules to CD4+ T cells. Apart from internalized
foreign antigens, many peptides presented on MHC class II are derived from proteins residing in intracellular compartments that are sampled by autophagy. Peptides from proteins degraded in the endosome are loaded on MHC class II in the so-called MHC class II peptide-loading compartment and subsequently transported to the plasma membrane (Figure 1). In DCs, MHC class II expression at the cell surface increases following their maturation, which also results in up-regulation of co-stimulatory molecules required for activation of naive CD4+
T cells. The complex mechanisms involved in antigen presentation and their coordinated interplay with pattern-recognition receptors of the innate immune system have been exploited by modern medicine, e.g. by harnessing individuals with pre-existing immunity (neutralizing antibodies) against incoming microbial challenges. 1,2 A growing appreciation of the skewing of immune responses
by pattern recognition receptors of the innate immune system has advanced the field of rational design of vaccines. 1,2 Undesired immune responses have
been observed following repeated administration of a number of therapeutic proteins, including anti-TNF-alpha antibodies such as adalimumab or infliximab, coagulation factors VIII and IX and erythropoietin. 3 In view of the ubiquitous
presentation of self- and non-self peptides on MHC class II, one can easily appreciate that peptides derived from biotherapeutic proteins are also presented on MHC class II. However, it is more difficult to envision how biotherapeutics, in the absence of immunologic “danger signals”, would induce activation of APCs thereby promoting the up-regulation of co-stimulatory molecules required for activation of naïve CD4+ T cells.
This review will explore the issue of neutralizing anti-drug antibodies in the context of current knowledge of blood coagulation factor VIII (FVIII) immunogenicity. Approximately 25% of patients with the severe form of the X-linked bleeding disorder hemophilia A (defined by FVIII pro-coagulant activity < 1% normal) develop an immune response resulting in the formation of neutralizing anti-FVIII antibodies. A number of recent reviews have summarized genetic and non-genetic treatment-related risk factors that contribute to inhibitor formation in hemophilia A. 4-7 Guidelines for treatment of hemophilia A patients with inhibitors have been
provided in a recent paper by Kempton and White. 8 Inhibitor formation as well
as somatic hypermutations and subclass switching of anti-FVIII antibodies are considered to be CD4+ T-cell dependent processes in both hemophilic mice 9-11
and hemophilia A patients. 12-17 Activation of FVIII-specific T cells is preceded
by the uptake of FVIII by antigen-presenting cells (APCs) and the subsequent presentation of FVIII peptides on MHC class II molecules on the surface of these APCs. 18 HLA alleles DRB1*15 and DQB1*0602 have been suggested to correlate
with increased risk for inhibitor development in hemophilia patients. 5,19 However,
the association between MHC class II molecules and FVIII antibody formation is not strong, and this reflects a central and intriguing aspect of anti-FVIII immune
Fa
ct
or VIII and the immune s
ys
tem
2
responses: a large majority of hemophilia A patients achieve functional immune tolerance to FVIII, either following their initial infusions or after eradicating an inhibitor response via immune tolerance induction (intensive FVIII therapy) or immunosuppression. The promiscuity of FVIII peptides in terms of binding to different HLA alleles 20, and the huge diversity in HLA types when compared
to the number of hemophilia patients included in earlier studies investigating the role of HLA in inhibitor risks, also make the establishment of statistically significant associations with individual HLA types challenging.
This review focuses on the most recent data available on FVIII endocytosis and processing by APCs as well as presentation of FVIII-specific peptides to T cells. We discuss new insights into mechanisms of FVIII endocytosis by DCs in humans and mice and describe recent investigations into what determines the immunogenicity of FVIII. We also summarize efforts made to interfere with immune recognition of FVIII and with events subsequent to FVIII uptake by APCs that influence anti-FVIII antibody formation. Finally, we discuss investigations into the presentation of FVIII-specific peptides and the diversity and functionality of the subsequent FVIII-specific T-cell repertoire.
Endocytosis and processing of FVIII by APCs
Factor VIII is internalized by APCs such as DCs and macrophages, where it is processed efficiently into peptides, some of which may then be presented on MHC class II molecules at the cell surface. 20 If a given MHC class II-peptide
is then recognized by a T-cell receptor on an effector T cell, as can happen in hemophilia A patients who do not have pre-existing immune tolerance to
Figure 1. MHC class II presentation pathway. DCs endocytose antigens
that subsequently undergo proteolytic degradation in endosomal compartments. Transport of newly synthesized MHC class II molecules from the ER is facilitated by the invariant chain (not shown) which is processed into a so-called CLIP peptide that occupies the binding groove of MHC class II. In the MHC class II compartment the CLIP-peptide is exchanged for antigen-derived peptides. Following peptide loading MHC class II molecules are transported to the cell surface and antigen-derived peptides are presented to CD4+ T cells.
