2003; baxter - Supplements - Haematologica

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14P. Lollarvation, inhibition of vWf binding would not bedetected by the Bethesda assay. Inhibitors havebeen described that block the binding of FVIII tovWf, and this may be a common property of anti-C2 inhibitors. 36 However, the relative contributionto the bleeding diathesis of inhibition ofbinding of FVIII to vWf versus binding to phospholipidis unknown.High resolution mapping of FVIII inhibitorepitopesHomolog scanning mutagenesis using hybridhuman/porcine, human/canine, or human/murineFVIII molecules has been successful inobtaining higher resolution maps of the B-cellepitopes. 37,38 This strategy is based on the observationthat inhibitors usually have limited crossreactivitywith non-human FVIII. A hybrid FVIIImolecule that is less reactive with an inhibitor tohuman FVIII localizes the inhibitor to the regionin which the non-human substitution has beenmade. Because the hybrids are active, reduction inantigenicity is unlikely to be due to improper foldingof the protein. In contrast, epitope localizationby deletion mapping is complicated by thepossibility that loss of antigenicity can occur bydenaturation of the polypeptide chain.Using this method, the A2 epitope recognizedby most A2-specific antibodies has been localizedto residues Arg 484 -Ile 508 . Analysis of several hybridscontaining A2 substitutions indicates that the A2epitope appears to be confined with the Arg 484 -Ile 508 linear segment. This is consistent with thefact that this segment protrudes as a large loopbetween β-strands in a homology model of the A2domain based on the X-ray structure of ceruloplasmin.The A2 epitope has been mapped further by alanine-scanningmutagenesis. Human A2-specificpolyclonal antibodies recognize several aminoacids in this segment, including Tyr 487 and to alesser extent Ser 488 , Arg 489 , Pro 492 , Val 495 , Phe 501 ,and Ile 508 . 39 There is variability between the antibodies,indicating that although the antibodyfootprint may be relatively invariant, individualamino acid residues recognized by the antibodiesdiffer.The region recognized by several C2-specificinhibitors has been mapped using a panel ofhybrid human/porcine FVIII molecules to residuesGlu 2181 -Val 2243 . 37 Recently, an X-ray structure ofthe human FVIII C2 domain has been solved, providinga more detailed approach to the analysis ofC2 inhibitors. The structure reveals a putativehydrophobic three-prong phospholipid membrane-bindingsite consisting of Met 2199 /Phe 2200 ,Val 2223 , and Leu 2251 /Leu 2252 . 40 Additionally, severalbasic residues contribute to a ring of positivelycharged residues that may contribute to an electrostaticinteraction of FVIII with negativelycharged phosphatidylserine. Several active humanFVIII mutants were constructed based on differencesbetween human, porcine, murine, andcanine FVIII at proposed phospholipid-bindingsites. The antigenicity of the mutants toward C2-specific polyclonal human antibodies, a humanmonoclonal anti-C2 antibody, BO2C11, 41 and amurine C2-specific monoclonal antibody, NMCVIII-5, 42 was measured. The results suggest thatC2 inhibitors frequently target the Met 2199 /Phe 2200and Leu 2251 /Leu 2252 β-hairpins, which is consistentwith the hypothesis that these residues participatein binding to phospholipid membranes. Incontrast, mutations at Val 2223 increased antibodybinding, indicating that Val 2223 may oppose antibodybinding sterically or through stabilization ofa low-affinity membrane-binding conformationof the C2 domain. Subsequently, the X-ray structureof BO2C11 in complex with the FVIII C2domain was solved and revealed contact with theMet 2199 /Phe 2200 and Leu 2251 /Leu 2252 . 43Delineation of the boundaries of an epitopeusing hybrid FVIII molecules can be limited bysequence identity. For example, human andporcine FVIII are identical from residues 2243(the end of the sequence mapped using human/porcinehybrids) through 2252. Thus, identificationof the epitope extending to residue 2252was not possible using hybrid human/porcineFVIII.Subsequently, we identified four C2-specificplasmas that do not appear to recognize the phospholipid-bindingregion (Barrow RT and Lollar P,personal observations). The functional propertiesof these antibodies have not been characterized.Overall, the analysis of C2 inhibitors appearsconsiderably more complex than that of A2inhibitors both with respect to epitope structureand mechanism of inhibition.Regions outside the A2 and C2 domains appearto be minor targets for inhibitory antibodiesbecause the inhibitory activity in most plasmascan be neutralized by the A2 and C2 domains. 28However, three inhibitor IgGs have been identifiedthat are neutralized by a synthetic peptidecorresponding to residues 1804-1819 in the A3domain. 44 This region encompasses a regiondefined by residues 1811-1819 that constitutespart of the factor IXa binding site. 45 Additionally,several inhibitor plasmas have been identified inwhich substitution of the human ap region withthe corresponding porcine segment results in areduction of antigenicity. This indicates that theap segment also contains an inhibitory epitope. 46Unusual FVIII inhibitorsInhibitory antibodies have been described thatrecognize epitopes outside the A2, C2, A3 and apregions. A human monoclonal antibody, LE2E9,is an IgG4k antibody that it is directed against theC1 domain and was derived from a patient withmild hemophilia A who has an Arg 2150 His mutation.47 The antibody recognizes wild-type FVIIIbut not endogenous FVIII, indicating that theantibody recognizes Arg 2150 . The antibody is a type2 inhibitor and blocks FVIII binding to vWf. Similarly,an inhibitor from a patient with mildhemophilia A due to an Arg 593 Cys mutation inhaematologica vol. 88(supplement n. 12):september <strong>2003</strong>
IV International Workshop on Immune Tolerance in Hemophilia 15the A2 domain has been described that recognizeswild-type FVIII but not the endogenous FVIII A2domain. 48An inhibitor with specificity confined to the A1domain was identified in a severe hemophilia Apatient who had a deletion of the A1 domain, butnevertheless produced detectable circulating FVIIIantigen. 49 Three type II inhibitory autoantibodieshave been identified that recognize the A3domain, and not the C2 domain, which typicallyis the target of type II antibodies. 50 These antibodiescirculate as immune complexes with FVIII,which has not been previously described.It is unusual for previously treated patients withhemophilia A to develop an inhibitor if they havenot developed one within the first year of treatment.18,51 Outbreaks of C2-specific inhibitorshave been described in previously treated hemophiliaA patients receiving specific lots of plasmaderivedFVIII. 52,53 The C2-specificity is remarkablebecause it is unusual in the hemophilia A population.The results suggest that exposure to denaturedforms of FVIII or generation of a neoepitopemay be immunogenic.The immunogenicity of FVIII in murinehemophilia AThe analysis of FVIII inhibitors is man is limitedby the inaccessibility of lymphoid tissue andthe difficulty associated with cloning antigen-specificB-cells. The polyclonal nature of the inhibitorantibody population makes functional and structuralanalysis of B-cell epitopes difficult. Only twohuman monoclonal antibodies have been produced(vide supra), in contrast to the potentialease of generation of murine monoclonal antibodies.Thus, the analysis of the immune responseto FVIII in mice holds considerable promise as amodel. There are two strains of FVIII knockoutmice, 54 which were produced by targeted disruptionof the FVIII gene at exons 16 and 17 encodingthe A3 domain. The E16 and E17 mice haveidentical phenotypes in studies to date. The micehave no measurable FVIII and have a bleedingdiathesis. However, they synthesize detectableamounts of FVIII antigen due to the fact that theFVIII gene is disrupted near to the middle of thesequence encoding protein. 55 In contrast, secretionof potentially tolerizing FVIII antigen may bethe exception, rather than the rule, in severehuman hemophilia A. Human hemophilia A isextremely heterogeneous at the genetic level,which probably influences the variability in theimmune response to FVIII. There could be similardifferences in the immune responses in hemophiliaA mice if they are produced by lesions in differentregions of the FVIII gene.When E16 or E17 is infused intravenously withhuman FVIII using a dosage schedule and dosageon a body weight basis that mimics treatment ofsevere hemophilia A in man, almost all develophigh titer inhibitors after two or three doses. 56These mice have a T-cell proliferative response toFVIII, demonstrating that the immune response isT-cell dependent. 56,57 A T-cell proliferativeresponse has also been demonstrated in humanhemophilia A. 58 The murine antibody response isprimarily IgG1-mediated, and to a lesser extentIgG2-mediated. 57 In contrast, when hemophilia Amice are crossed with B7.2 knockout mice, theydo not develop anti-FVIII antibodies or produce aT-cell proliferative response. 59 The fusion proteinCTLA-Ig, which blocks the B7-CD28 interaction,prevents the primary response to FVIII. 59 Theseresults indicate that the B7-CD28 co-stimulatorypathway is necessary for the immune responseto FVIII and suggest a possible therapeuticapproach. Anti-CD40L antibodies also block theprimary response to FVIII in this model. 60,61The murine hemophilia A model has beenadopted widely for development of somatic cellgene therapy of hemophilia A, in which immunogenicityof FVIII 55,62,63 and potential induction oftolerance 64 are major areas of interest.FundingSupported by grants from the National Institutesof Health, R01-HL40921.References1. Laver WG, Air GM, Webster RG, Smith-Gill SJ. Epitopeson protein antigens: misconceptions and realities. Cell1990;61:553-6.2. Jin L, Fendly BM, Wells JA. High resolution functionalanalysis of antibody-antigen interactions. J Mol Biol1992;226:851-65.3. Watts C, Lanzavecchia A. Suppressive effect of antibodyon processing of T cell epitopes. J Exp Med 1993; 178:1459-63.4. Benjamin DC, Berzofsky JA, East IJ, Gurd FR, HannumC, Leach SJ, et al. The antigenic structure of proteins: areappraisal. Ann Rev Immunol 1984;2:67-101.5. Caton AJ, Brownlee GG, Yewdell JW, Gerhard W. Theantigenic structure of the influenza virus A/PR/8/34hemagglutinin (H1 subtype). Cell 1982;31:417-27.6. Diaz M, Klinman NR. Relative roles of somatic and Darwinianevolution in shaping the antibody response.Immunol Res 2000;21:89-102.7. Mamula MJ, Jemmerson R, Hardin JA. The specificity ofhuman anti-cytochrome c autoantibodies that arise inautoimmune disease. J Immunol 1990; 144:1835-40.8. Staudt LM, Gerhard W. Generation of antibody diversityin the immune response of BALB/c mice to influenzavirus hemagglutinin. I. Significant variation in repertoireexpression between individual mice. J Exp Med 1983;157:687-704.9. Davies DR, Padlan EA. Antibody-antigen complexes.Ann Rev Biochem 1990;59:439-73.10. Cunningham BC, Jhurani P, Ng P, Wells JA. Receptorand antibody epitopes in human growth hormone identifiedby homolog-scanning mutagenesis. Science 1989;243:1330-6.11. Slayter H, Loscalzo J, Bockenstedt P, Handin RI. Nativeconformation of human von Willebrand protein. Analysisby electron microscopy and quasi-elastic light scattering.J Biol Chem 1985;260:8559-63.12. Kaufman RJ, Pipe SW, Tagliavacca L, Swaroop M, MoussalliM. Biosynthesis, assembly and secretion of coagulationfactor VIII. Blood Coagul Fibrinol 1997;8 Suppl2:S3-14.13. Lollar P, Parker CG. Subunit structure of thrombin-activatedporcine factor VIII. Biochemistry 1989;28:666-74.14. Vehar GA, Davie EW. Preparation and properties ofbovine factor VIII (antihemophilic factor). Biochemistry1980;19:401-10.15. Parker ET, Pohl J, Blackburn MN, Lollar P. Subunit struc-haematologica vol. 88(supplement n. 12):september <strong>2003</strong>
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