Analysis of Samples from Patients Treated with ReFacto® for the Presence of Anti-SQ-Peptide Specific Antibodies

ABSTRACT

ReFacto® lacks the major part of the B-domain, except for 14 amino acid residues from the N-terminal and C-terminal ends, which are linked into a 14 amino acid SQ-peptide tail in the C-terminal end of the 90 kD heavy chain, thereby introducing a new peptide sequence in the factor VIII (FVIII) molecule. Therefore, samples from some of the patients enrolled in the ReFacto clinical trials were analyzed for the emergence of anti-SQ-peptide antibodies in addition to determinations for inhibitors by Bethesda assay and for anti-FVIII antibodies by enzyme-linked immunosorbent assay (ELISA). An anti-peptide ELISA was developed using six partially overlapping 15-16 mer peptides covering the SQ-linker region. Results from ReFacto-treated anti-FVIII antibody-positive or -negative patients were compared with those for normal healthy adults and patients treated with other FVIII products. The pattern was compared with that for SQ-peptide specific monoclonal antibodies, each recognizing three or four overlapping SQ-peptides. Antibodies recognizing mainly one of the SQ-peptides were found in samples in some individuals from all investigated groups. This means that the anti-SQ reactivity found in patients treated with ReFacto is not induced by the SQ-sequence of the molecule but rather by some other agent, giving rise to antibodies cross-reactive with SQ-peptides.

KEYWORDS

ReFactor® - SQ-peptide - continuous epitope - immunogenicity - human antibodies

REFERENCES

• 1 Lind P, Larsson K, Spira J. Novel forms of B-domain deleted recombinant factor VIII molecules. Construction and biochemical characterization.  Eur J Biochem . 1995;  232 19-27
• 2 Sandberg H, Almstedt A, Brandt J. Structure and functional characteristics of the B-domain-deleted recombinant factor VIII protein, r-VIII SQ.  Thromb Haemost . 2001;  85 93-100
• 3 Bjorkman P J. MHC restriction in three dimensions: a view of T cell receptor/ligand interactions.  Cell . 1997;  89 167-170
• 4 York I A, Goldberg A L, Mo X Y, Rock K L. Proteolysis and class I major histocompatibility complex antigen presentation.  Immunol Rev . 1999;  172 49-66
• 5 Kropshofer H, Hämmerling G J, Vogt A B. The impact of the non-classical MHC proteins HLA-DM and HLA-DO on loading of MHC class II molecules.  Immunol Rev . 1999;  172 267-278
• 6 Pinilla C, Martin R, Gran B. Exploring immunological specificity using synthetic peptide combinatorial libraries.  Curr Opin Immunol . 1999;  11 193-202
• 7 Kabat E A. Heterogeneity and structure of antibody-combining sites.  Ann N Y Acad Sci . 1970;  169 43-54
• 8 Berzofsky J A. Intrinsic and extrinsic factors in protein antigenic structure.  Science . 1985;  229 932-940
• 9 Graeme Laver W, Air G M, Webster R G, Smith-Gill S J. Epitopes on protein antigens: misconceptions and realities.  Cell . 1990;  61 553-556
• 10 Zeder-Lutz G, Altschuh D, Geysen H M. Monoclonal antipeptide antibodies: affinity and kinetic rate constants measured for the peptide and the cognate protein using a biosensor technology.  Mol Immunol . 1993;  30 145-155
• 11 Pinilla C, Appel J R, Houghten R A. Functional importance of amino acid residues making up peptide antigenic determinants.  Mol Immunol . 1993;  30 577-585
• 12 Enomoto A, Shon D-H, Aoki Y, Yamauchi K, Kaminogawa S. Antibodies raised against peptide fragments of bovine ;ia s1-casein cross-react with the intact protein only when the peptides contain both B and T cell determinants.  Mol Immunol . 1990;  27 581-586
• 13 Trifilieff E, Dubs M C, Van Regenmortel H V M. Antigenic cross-reactivity potential of synthetic peptides immobilized on polyethylene rods.  Mol Immunol . 1991;  28 889-896
• 14 Devaux C, Juin M, Mansuelle P, Granier C. Fine molecular analysis of the antigenicity of the Androctonus australis hector scorpion neurotoxin II: a new antigenic epitope disclosed by the pepscan method.  Mol Immunol . 1993;  30 1061-1068
• 15 Stemmer C, Briand J P, Muller S. Mapping of linear epitopes of human histone H1 recognized by rabbit anti-H1/H5 antisera and antibodies from autoimmune patients.  Mol Immunol . 1994;  31 1037-1046
• 16 Williams R C, Staud R, Malone C C. Epitopes on proteinase-3 recognized by antibodies from patients with Wegener's granulomatosis.  J Immunol . 1994;  152 4722-4737
• 17 Atassi M Z, Dolimbek B Z, Manshouri T. Antibody and T-cell recognition of ;ia-bungarotoxin and its synthetic loop-peptides.  Mol Immunol . 1995;  32 919-929
• 18 Apostolidis V A, Browne K A, Smyth M J, Trapani J A. The peptide loop consisting of amino acids 139-157 of human granzyme B (fragmentin 2) contains an immunodominant epitope recognized by the mouse.  Mol Immunol . 1995;  32 909-917
• 19 Warren K G, Catz I, Steinman L. Fine specificity of the antibody response to myelin basic protein in the central nervous system in multiple sclerosis: the minimal B-cell epitope and a model of its features.  Proc Natl Acad Sci USA . 1995;  92 11061-11065
• 20 Hellmark T, Brunmark C, Trojnar J, Wieslander J. Epitope mapping of anti-glomerular basement membrane (GBM) antibodies with synthetic peptides.  Clin Exp Immunol . 1996;  105 504-510
• 21 Cane P A. Analysis of linear epitopes recognised by the primary human antibody response to a variable region of the attachment (G) protein of respiratory syncytial virus.  J Med Virol . 1997;  51 297-304
• 22 Delvig A A, Michaelsen T E, Aase A, Hoiby E A, Rosenqvist E. Vaccine-induced IgG antibodies to the linear epitope on the PorB outer membrane protein promote opsonophagocytosis of Neisseria meningitidis by human neutrophils.  Clin Immunol Immunopathol . 1997;  84 27-35
• 23 Rharbaoui F, Granier C, Kellou M. Peptide specificity of high-titer anti-glutamic acid decarboxylase (GAD) 65 autoantibodies.  Immunol Lett . 1998;  62 123-130
• 24 Motrán C C, Cerbán F M, Rivarola W, Iosa D, de Cima V E. Trypanosoma cruzi: immune response and functional heart damage induced in mice by the main linear B-cell epitope of parasite ribosomal P proteins.  Exp Parasitol . 1998;  88 223-230
• 25 Banerjee B, Greenberger P A, Fink J N, Kurup V P. Conformational and linear B-cell epitopes of Asp f 2, a major allergen of Aspergillus fumigatus, bind differently to immunoglobulin E antibody in the sera of allergic bronchopulmonary aspergillosis patients.  Infect Immun . 1999;  67 2284-2291
• 26 Plotnicky-Gilquin H, Goetsch L, Huss T. Identification of multiple protective epitopes (protectopes) in the central conserved domain of a prototype human respiratory syncytial virus G protein.  J Virol . 1999;  73 5637-5645
• 27 Wikén M, Novak V, Sjöström M. Immunogenicity in cynomolgus monkeys of r-VIII SQ, a recombinant truncated form of factor VIII (Abst 374).  XX International Congress of the World Federation of Hemophilia 1992.
• 28 Wikén M. Evaluation of the role of the SQ-peptide of r-VIII SQ in the activation of T cells in mice (Abst 307).  Haemophilia . 1998;  4 232
• 29 Gruppo R, Chen H, Schroth P, Bray G L. Safety and immunogenicity of recombinant factor VIII (Recombinate™) in previously untreated patients (PUPs): a 7.3 year update (Abst 291).  Haemophilia . 1998;  4 228
• 30 Lusher J, Arkin S, Abildgaard C F, Hurst D. Recombinant FVIII (Kogenate) treatment of previously untreated patients (PUPs) with hemophilia A: update of safety, efficacy and inhibitor development after seven study years (Abst PD-664).  Thromb Haemost . 1997;  78 (Suppl)
• 31 Schulman S. B-domain deleted human recombinant factor VIII.  International Monitor on Hemophilia . 1999;  2 5-9
• 32 Geysen H M, Rodda S J, Mason T J, Tribbick G, Schoofs P G. Strategies for epitope analysis using peptide synthesis.  J Immunol Methods . 1987;  102 259-274
• 33 Geysen H M, Rodda S J, Mason T J. A priori delineation of a peptide which mimics a discontinuous antigenic determinant.  Mol Immunol . 1986;  23 709-715
• 34 Balass M, Heldman Y, Cabilly S. Identification of a hexapeptide that mimics a conformation-dependent binding site of acetylcholine receptor by use of a phage-epitope library.  Proc Natl Acad Sci USA . 1993;  90 10638-10642
• 35 Folgori A, Tafi R, Meola A. A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera.  EMBO J . 1994;  13 2236-2243
• 36 Williams R C, Malone C C, Kolaskar A S, Kulkarni-Kale U. Antigenic determinants reacting with rheumatoid factor: epitopes with different primary sequences share similar conformation.  Mol Immunol . 1997;  34 543-556