Influence of aggregation on immunogenicity of recombinant human Factor VIII in hemophilia A mice.

Recombinant human factor VIII (rFVIII), a multidomain glycoprotein is used in replacement therapy for treatment of hemophilia A. Unfortunately, 15%-30% of the treated patients develop inhibitory antibodies. The pathogenesis of antibody development is not completely understood. The presence of aggregated protein in formulations is generally believed to enhance the immune response. rFVIII has a tendency to aggregate but the effect of such aggregation on the immunogenicity of rFVIII is not known. We have, therefore, characterized aggregated rFVIII produced by thermal stress and evaluated its effect on the immunogenicity of rFVIII in hemophilia A mice. Aggregated rFVIII alone and mixtures of rFVIII with aggregated rFVIII were less immunogenic than native rFVIII. In vitro Th-cell proliferation studies and cytokine analyses conducted on splenocytes obtained from immunized animals suggest that aggregated rFVIII behaves as a unique antigen compared to native monomeric rFVIII. The antigenic properties of the aggregated and native rFVIII were compared using ELISAs (epitope availability) and cathepsin-B (an antigen processing enzyme) digestion. The data suggest significant differences in the antigenic properties of rFVIII and aggregated rFVIII. Overall it appears that aggregated rFVIII does not enhance the immunogenicity (inhibitor development) of rFVIII in hemophilia A mice but rather acts as a distinct antigen.

[1]  S. Balasubramanian,et al.  Lipid binding region (2303-2332) is involved in aggregation of recombinant human FVIII (rFVIII). , 2005, Journal of pharmaceutical sciences.

[2]  A. Schoppmann,et al.  A caution on the use of murine hemophilia models for comparative immunogenicity studies of FVIII products with different protein compositions , 2003, Thrombosis and Haemostasis.

[3]  B. Keyt,et al.  Structure of human factor VIII , 1984, Nature.

[4]  Huub Schellekens,et al.  Immunogenicity of rDNA-derived pharmaceuticals. , 2002, Trends in pharmacological sciences.

[5]  H. Kazazian,et al.  Lower Inhibitor Development in Hemophilia A Mice following Administration of Recombinant Factor VIII-O-Phospho-L-serine Complex* , 2005, Journal of Biological Chemistry.

[6]  L. Brown,et al.  Immunological parameters associated with antigenic competition in a multivalent footrot vaccine. , 1995, Vaccine.

[7]  H. Schwarz,et al.  Characterization of Antibodies Induced by Human Factor VIII in a Murine Knockout Model of Hemophilia A , 2000, Thrombosis and Haemostasis.

[8]  H. Schellekens,et al.  The immunogenicity of biopharmaceuticals. Lessons learned and consequences for protein drug development. , 2003, Developments in biologicals.

[9]  P. Fay Reconstitution of human factor VIII from isolated subunits. , 1988, Archives of biochemistry and biophysics.

[10]  C. R. Middaugh,et al.  Aggregation kinetics of recombinant human FVIII (rFVIII). , 2005, Journal of pharmaceutical sciences.

[11]  B. Ferraiolo,et al.  The Role of Pharmacokinetics in the Development of Biotechnologically Derived Agents , 1992, Clinical pharmacokinetics.

[12]  G. Knutson,et al.  Molecular cloning of a cDNA encoding human antihaemophilic factor , 1984, Nature.

[13]  A. Dorner,et al.  Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells. , 1988, The Journal of biological chemistry.

[14]  S. Constant,et al.  Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. , 1997, Annual review of immunology.

[15]  W V Moore,et al.  Role of aggregated human growth hormone (hGH) in development of antibodies to hGH. , 1980, The Journal of clinical endocrinology and metabolism.

[16]  A. Ménez,et al.  Antigen Stability Controls Antigen Presentation* , 2004, Journal of Biological Chemistry.

[17]  D. Scott,et al.  Immunodominant T-cell epitopes in the factor VIII C2 domain are located within an inhibitory antibody binding site , 2004, Thrombosis and Haemostasis.

[18]  H. Kazazian,et al.  Inhibitor Antibody Development and T Cell Response to Human Factor VIII in Murine Hemophilia A , 1999, Thrombosis and Haemostasis.

[19]  Huub Schellekens,et al.  Immunogenicity of therapeutic proteins: clinical implications and future prospects. , 2002, Clinical therapeutics.

[20]  M. van den Berg,et al.  The Nijmegen Modification of the Bethesda Assay for Factor VIII:C Inhibitors: Improved Specificity and Reliability , 1995, Thrombosis and Haemostasis.

[21]  N. Katunuma,et al.  Insights into the Roles of Cathepsins in Antigen Processing and Presentation Revealed by Specific Inhibitors , 2003, Biological chemistry.

[22]  J. Collinge,et al.  Protein Conformation Significantly Influences Immune Responses to Prion Protein1 , 2005, The Journal of Immunology.

[23]  R. Brunham,et al.  Aggregate content influences the Th1/Th2 immune response to influenza vaccine: Evidence from a mouse model , 2004, Journal of medical virology.

[24]  M. Reding,et al.  Distribution of Th1- and Th2-induced Anti-factor VIII IgG Subclasses in Congenital and Acquired Hemophilia Patients , 2002, Thrombosis and Haemostasis.

[25]  C. Watts,et al.  Capture and processing of exogenous antigens for presentation on MHC molecules. , 1997, Annual review of immunology.

[26]  Huub Schellekens,et al.  Bioequivalence and the immunogenicity of biopharmaceuticals , 2002, Nature Reviews Drug Discovery.

[27]  J. Oldenburg,et al.  Environmental and genetic factors influencing inhibitor development. , 2004, Seminars in hematology.

[28]  P. Foster,et al.  Factor VIII structure and function. , 1989, Blood reviews.

[29]  J. Saint-Remy,et al.  Von Willebrand Factor Modulates Factor VIII Immunogenicity: Comparative Study of Different Factor VIII Concentrates in a Haemophilia A Mouse Model , 2002, Thrombosis and Haemostasis.

[30]  J. Lakowicz Principles of fluorescence spectroscopy , 1983 .

[31]  D. Parker T cell-dependent B cell activation. , 1993, Annual review of immunology.

[32]  G. Houen,et al.  A comparison of the immunogenicity of the native and denatured forms of a protein , 1996, APMIS : acta pathologica, microbiologica, et immunologica Scandinavica.

[33]  C. Wong,et al.  Characterization of aggregates of recombinant human factor VIII by size‐exclusion chromatography and immunoassay , 1996, Biotechnology and applied biochemistry.

[34]  S. Antonarakis,et al.  Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A , 1995, Nature Genetics.

[35]  H. Rammensee,et al.  Priming of cytotoxic T lymphocytes by five heat‐aggregated antigens in vivo: Conditions, efficiency, and relation to antibody responses , 1997, European journal of immunology.

[36]  J. Sodroski,et al.  Factors limiting the immunogenicity of HIV-1 gp120 envelope glycoproteins. , 2004, Virology.

[37]  C. Kessler,et al.  Factor VIII immunogenicity , 1998, Haemophilia : the official journal of the World Federation of Hemophilia.

[38]  S. Lacroix-Desmazes,et al.  Inhibitors in haemophilia: pathophysiology , 2004, Haemophilia : the official journal of the World Federation of Hemophilia.

[39]  D. Kelner,et al.  Correlation of rFVIII Inactivation with Aggregation in Solution , 2003, Pharmaceutical Research.

[40]  C. Russell Middaugh,et al.  Formulation Design of Acidic Fibroblast Growth Factor , 1993, Pharmaceutical Research.

[41]  M. Vandenbranden,et al.  Heat denaturation affects the ProDer p 1 IgE reactivity and downregulates the development of the specific allergic response. , 2004, The Journal of allergy and clinical immunology.

[42]  W. Vanaken The potential impact of recombinant factor VIII on hemophilia care and the demand for blood and blood products. , 1997, Transfusion medicine reviews.

[43]  J. Healey,et al.  Factor VIII inhibitors. , 2001, Advances in experimental medicine and biology.

[44]  H. Wu,et al.  Mechanism of the Immune Response to Human Factor VIII in Murine Hemophilia A , 2001, Thrombosis and Haemostasis.

[45]  A. Grillo,et al.  Conformational origin of the aggregation of recombinant human factor VIII. , 2001, Biochemistry.

[46]  H. Schwarz,et al.  Single Cell Analysis of Factor VIII-specific T Cells in Hemophilic Mice after Treatment with Human Factor VIII , 2002, Thrombosis and Haemostasis.

[47]  Jochem Alsenz,et al.  Protein Aggregates Seem to Play a Key Role Among the Parameters Influencing the Antigenicity of Interferon Alpha (IFN-α) in Normal and Transgenic Mice , 1997, Pharmaceutical Research.