Structural Characterization and Immunogenicity in Wild-Type and Immune Tolerant Mice of Degraded Recombinant Human Interferon Alpha2b
- 490 Downloads
This study was conducted to study the influence of protein structure on the immunogenicity in wild-type and immune tolerant mice of well-characterized degradation products of recombinant human interferon alpha2b (rhIFNα2b).
RhIFNα2b was degraded by metal-catalyzed oxidation (M), cross-linking with glutaraldehyde (G), oxidation with hydrogen peroxide (H), and incubation in a boiling water bath (B). The products were characterized with UV absorption, circular dichroism and fluorescence spectroscopy, gel permeation chromatography, reverse-phase high-pressure liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and mass spectrometry. The immunogenicity of the products was evaluated in wild-type mice and in transgenic mice immune tolerant for hIFNα2. Serum antibodies were detected by enzyme-linked immunosorbent assay or surface plasmon resonance.
M-rhIFNα2b contained covalently aggregated rhIFNα2b with three methionines partly oxidized to methionine sulfoxides. G-rhIFNα2b contained covalent aggregates and did not show changes in secondary structure. H-rhIFNα2b was only chemically changed with four partly oxidized methionines. B-rhIFNα2b was largely unfolded and heavily aggregated. Nontreated (N) rhIFNα2b was immunogenic in the wild-type mice but not in the transgenic mice, showing that the latter were immune tolerant for rhIFNα2b. The anti-rhIFNα2b antibody levels in the wild-type mice depended on the degradation product: M-rhIFNα2b > H-rhIFNα2b ∼ N-rhIFNα2b ≫ B-rhIFNα2b; G-rhIFNα2b did not induce anti-rhIFNα2b antibodies. In the transgenic mice, only M-rhIFNα2b could break the immune tolerance.
RhIFNα2b immunogenicity is related to its structural integrity. Moreover, the immunogenicity of aggregated rhIFNα2b depends on the structure and orientation of the constituent protein molecules and/or on the aggregate size.
Key Wordsaggregates immune tolerance immunogenicity interferon alpha2 protein structure transgenic mice
bovine serum albumin
dynamic light scattering
enzyme-linked immunosorbent assay
electrospray ionization-time of flight
gel permeation chromatography
human interferon alpha2
matrix-assisted laser desorption ionization time of flight/time of flight
polyacrylamide gel electrophoresis
sodium phosphate buffer, pH 7.2
recombinant human interferon alpha2b
reverse-phase high-pressure liquid chromatography
sodium dodecyl sulfate
surface plasmon resonance
The authors wish to thank Dr. Viscomi and Lucia Scapol for supplying the rhIFNα2b solutions, the standard anti-rhIFNα2b serum, and for valuable discussions. Dr. Pestka is kindly thanked for supplying the transgenic immune tolerant mice. Janny Westdijk is kindly acknowledged for her help with the Biacore experiments and valuable discussions. We thank Ronald van Ooijen and Georgina Gal for performing the mass spectrometric analyses. This work was financially supported by the European Union through the 5th Framework Program “Competitive and Sustainable Growth,” LYOPRO project (Contract no. G1RD-CT2002-00736).
- 13.Pfeffer, L. M., Dinarello, C. A., Herberman, R. B., Williams, B. R., Borden, E. C., Bordens, R., Walter, M. R., Nagabhushan, T. L., Trotta, P. P., Pestka, S. 1998Biological properties of recombinant alpha-interferons: 40th anniversary of the discovery of interferonsCancer Res.5824892499PubMedGoogle Scholar
- 17.Interferon alpha2b. European Pharmacopeia 5.0, Vol. 2, 2004, pp. 1812–1815.Google Scholar
- 20.ExPASy. Peptide Mass.http://ca.expasy.org/tools/peptide-mass. html (accessed 12 December 2004).
- 21.Kueltzo, L. A., Midddaugh, C. R. 2005
Ultraviolet absorption spectroscopyJiskoot, W.Crommelin, D. J. A. eds. Methods for Structural Analysis of Protein Pharmaceuticals AAPS PressArlington125Google Scholar
- 22.Bremer, E. T. J., Heck, A. J. R. 2005
Mass spectrometry: protein conformational analysis and molecular recognitionJiskoot, W.Crommelin, D. J. A. eds. Methods for Structural Analysis of Protein PharmaceuticalsAAPS PressArlington435464Google Scholar
- 23.ExPASy. ExPASy Proteomics Server.http://ca.expasy.org/uniprot/Q86UP4 (accessed 1 June 2005).