2003; baxter - Supplements - Haematologica

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[Immunological Aspects]review paperAnalysis of factor VIII inhibitorsusing phage displayhaematologica 2003; 88(suppl. n. 12):18-25http://www.haematologica.org/free/immunotolerance2001.pdfJAN VOORBERG, WENDY S. BRIL, EDWARD N. VAN DEN BRINK *Department of Plasma Proteins, Amsterdam, the Netherlands*Current address: Crucell BV, Leiden, the NetherlandsInhibitory antibodies that develop in patients withhemophilia A bind to restricted regions in the A2, A3and C2 domain of factor VIII. Functional studies haveshown that anti-A2 and anti-A3 antibodies interferewith assembly of the factor VIIIa-factor IXa complex.Binding of inhibitors to the C2 domain precludesbinding of factor VIII to phospholipids. We have usedphage display to isolate a large number of humanmonoclonal antibodies from the immunoglobulinrepertoire of hemophilia A patients with an inhibitor.Epitope mapping studies suggest that the majorityof human monoclonal antibodies bind to previouslyidentified epitopes on factor VIII. Inspection of theamino acid sequence of the variable heavy chain(VH) domains of human anti-factor VIII antibodiesreveals some striking features. Anti-A2 and anti-A3-C1 antibodies incorporate VH gene segments thatare frequently used for assembly of human IgG molecules.This may explain the presence of antibodieswith this specificity in a large number of inhibitorpatients. Anti-C2 antibodies are derived from twoclasses of VH gene segments, both belonging to theVH1 family, that bind to two distinct antigenic sitesin the C2 domain. Our findings suggest that B-cellsexpressing immunoglobulin molecules that compriseVH gene segments with the above-mentioned characteristicsare selectively amplified from the totalrepertoire following exposure to antigenic determinantsin the C2 domain of factor VIII.Correspondence: Jan Voorberg, Department of Plasma Proteins,Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.Phone: international +33.20.5123120. Fax: international+33.20.5123680. E-mail: j_voorberg@clb.nlFactor VIII participates in the intrinsic pathwayof blood coagulation. The physiologicimportance of factor VIII is illustrated by theX-linked bleeding disorder hemophilia A whichis due to functional absence of factor VIII. Thebleeding tendency in patients with hemophilia Acan be corrected by the administration of factorVIII concentrates. In approximately 20-40% ofpatients with severe hemophilia A inhibitory antibodiesdevelop usually after 5-12 exposure days. 1Inhibitor formation hampers further treatmentof patients with factor VIII. Activated prothrombincomplex concentrates and activated factor VIIare used for treatment of bleeding episodes ininhibitor patients. 1 Based on internal sequencehomology factor VIII is divided into a series ofhomologous domains which are interspersed byshort spacer regions that are rich in acidic aminoacids. 2 In plasma factor VIII circulates as a metal-ionlinked heterodimer. The heavy chain consistsof the domains A1-a1-A2-a2-B whereas thelight chain is composed of the domains a3-A3-C1-C2. Due to proteolysis at various sites in theB domain the size of factor VIII heavy chainranges between 90 and 220 kDa. In plasma factorVIII is bound to von Willebrand factor (VWF)which protects factor VIII from proteolytic degradation.2 Upon activation by thrombin or factorXa, factor VIII dissociates from VWF and assemblyof the factor VIIIa-factor IXa complex onphospholipid surfaces can occur. 2 Activation offactor X by factor IXa is significantly enhanced bythe non-enzymatic cofactor factor VIII. Functionalsites on factor VIII involved in binding tofactor IXa and phospholipids have been defined inconsiderable detail. 3 Concomitantly, knowledgeon the binding sites for factor VIII inhibitors onfactor VIII has grown rapidly during the last fiveyears. 4 Based on different experimental approachesthree major epitopes have been defined on factorVIII. In the A3 domain of factor VIII a bindingsite for factor VIII inhibitors has been localizedto residues Gln 1778 -Met 1823 . 5,6 Binding ofinhibitory antibodies to this site in the A3 domaininterferes with complex assembly of the factorIXa-factor VIIIa which is mediated by this part ofthe factor VIII light chain. 5-7 Residues Arg 484 -Ile 508comprise a major determinant of a binding sitefor factor VIII inhibitors in the A2 domain. 8 Singlealanine replacements in this part of the A2domain suggested that Tyr 487 is a residue criticalfor binding of inhibitory antibodies to this part ofhaematologica vol. 88(supplement n. 12):september 2003
IV International Workshop on Immune Tolerance in Hemophilia 19the A2 domain of factor VIII. 9 Antibodies directedtoward residues Arg 484 -Ile 508 prevent the stimulatoryeffect of isolated A2 domain on the catalyticactivity of factor IXa. 10 The inhibitory propertiesof anti-A3 and anti-A2 antibodies are inagreement with our current model of the assemblyof the factor VIIIa-IXa complex. Initial highaffinity binding is mediated by interaction of thefactor VIII light chain with residues present in theEGF1-and EGF2 domains of factor IXa whereasstimulation of factor VIII cofactor activity resultsfrom interaction of the A2 domain with the proteasemoiety of factor IXa. 3 Apparently, factor VIIIinhibitory antibodies can block the interaction offactor VIIIa with factor IXa via two distinct mechanisms.Conflicting data have been reported on the epitopefor factor VIII inhibitors in the C2 domain.Recombinant factor VIII fragments expressed inEscherischia coli revealed the presence of a bindingsite within region Val 2248 -Ser 2312 . 11 Evaluationof the functional inhibition of a panel of anti-C2inhibitors against a series of human/porcinehybrids suggested that residues Glu 2181 -Val 2243 areinvolved in binding of factor VIII inhibitors. 12 Theapparent discrepancies between the two studiesmay be explained by the different approachesthat have been used to characterize factor VIIIinhibitors. Antibodies directed toward the C2domain interfere with binding of factor VIII tophospholipids. 11,13 Current data suggest that arestricted number of 3-4 major binding sites forinhibitory antibodies are present on factor VIII.At present it is not clear why only a limited numberof sites on factor VIII is targeted by theimmune system. This may be explained by theuse of only a selected proportion of the totalavailable immunoglobulin repertoire for the generationof antibodies that interact with immunodominantregions on factor VIII. To explore thisissue we analyzed the anti-factor VIII repertoireof a number of well-characterized inhibitorpatients by phage display.Assembly of human immunoglobulin repertoiresImmunoglobulins are composed of a light anda heavy chain that contain both variable andconstant regions. The variable or V-regions areencoded by two and three gene segments for thelight and heavy chain, respectively. The heavychain locus is present on chromosome 14 whereasloci encoding κ and λ light chains are presenton chromosome 2 and 22, respectively. The individualloci contain large numbers of differentgene segments that are assembled into functionalantibody molecules during maturation of Bcells. The heavy chain locus consists of over 120variable heavy chain gene segments, 27 diversity(D) segments and 6 joining (J) segments 14,15which are followed by gene segments that encodeconstant parts of immunoglobulin heavy chains(µ, δ, γ3, γ1, α1, γ2, γ4, ε, α2). The κ light chainlocus on chromosome 2 comprises 91 variablelight (VL) chain gene segments and 5 J segments.The λ light chain on chromosome 22 contains>45 variable light chain and four J segments. Notall variable heavy and light chain segments areused for assembly of human antibodies. Approximately50-75% of VH and VL segments are nonfunctional.This may be due to the absence of anopen reading frame or lack of recombinationsites flanking the variable segments. 16 Assemblyof immunoglobulin molecules proceeds in ahighly regulated manner. The first event thatoccurs is the joining of a D segment with a J segmentin the variable region of the immunoglobulinlocus. Subsequently, a VH gene segmentand a µ constant region is added giving riseto a functional heavy chain which is co-expressedtogether with a surrogate light chain on a B-cellprecursor in the bone marrow. 17 Next, a VL segmentis fused with a JL segment resulting in afunctional light chain segment that replaces thesurrogate light chain. Following negative selectionof self-reactive B-cells in the bone marrow,immature B-cells are transported to the peripherywhere they express both IgM and IgD. 17 Internalizationof antigen via surface immunoglobulinson B-cells is followed by proteolytic processingof antigens into peptides that are amenablefor presentation by MHC class II molecules onthe surface of B-cells. T helper cells specificallyrecognizing the presented peptide then help B-cells to proliferate. Proliferating B-cells migrate tothe lymph nodes where germinal centers arisethat provide an environment for adequate T-cellhelp for the generation of high affinity antibodies.Affinity maturation proceeds via amino acidreplacements in the variable parts of the antibody,a process termed somatic hypermutation.Additionally, class switching from IgM to IgA,IgG or IgE occurs at this stage. Following affinitymaturation, B-cells develop into plasma cellsthat produce antibodies that are present in plasma.Alternatively, B-cells develop into memoryB-cells, which are present for extended periods inthe periphery thereby facilitating secondaryresponses to incoming antigens.Analysis of factor VIII inhibitors by phagedisplay: general outlineUsing peripheral B lymphocytes as a source ofRNA we isolated immunoglobulin repertoires ofpatients with an inhibitor using a series of polymerasechain recations (PCR) that targeted thevariable domains of the immunoglobulin heavychain. 18 Several restrictions were applied to preferentiallyamplify the anti-factor VIII repertoireof these patients. The majority of factor VIIIinhibitors are of subclass IgG4: therefore, anIgG4 specific amplification step was introducedin our experimental protocol. The concentrationof IgG4 is approximately 5% of the total quantityof IgG in human serum. Although this numberdoes not necessarily correspond to the percentageof IgG4 positive B cells in the peripheralblood, it is anticipated that the resultinghaematologica vol. 88(supplement n. 12):september 2003
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Index of authorsAhmad, R.U., 56Albe
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