Structural Characterization and Immunogenicity in Wild-Type and Immune Tolerant Mice of Degraded Recombinant Human Interferon Alpha2b

Abstract

Chapter 6 96 AbstractPurpose. To study the influence of protein structure on the immunogenicity in wildtype and immune tolerant mice of well-characterized degradation products of recombinant human interferon alpha2b (rhIFNα2b).Methods. RhIFNα2b was degraded by metal catalyzed oxidation (M), crosslinking 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, reversed-phase HPLC, SDS-PAGE, Western blotting and mass spectrometry. The immunogenicity of the products was evaluated in wildtype mice and in transgenic mice immune tolerant for hIFNα2. Serum antibodies were detected by ELISA or surface plasmon resonance.Results. 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. Native (N) rhIFNα2b was immunogenic in the wildtype mice but not in the transgenic mice, showing that the latter were immune tolerant for rhIFNα2b. The antirhIFNα2b antibody levels in the wildtype 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.Conclusions. 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. Structural characterization and immunogenicity of rhIFNα2b97

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

“…13 Low aggregate content is especially crucial in pharmaceutical formulations as aggregates here may elicit an inflammatory immune response to neutralize the aggregate. 14 This would not only diminish the effect of the drug itself but could also lead to more serious adverse effects related to cross-reactivity with autologous proteins. 15,16 In this study, we used the monoclonal IgG1 antibody Rituximab 17 as a model system to better understand the mechanisms leading to the formation of large aggregates when incubating a multidomain protein under weak thermal stress.…”

Maintenance and break of immune tolerance against human factor VIII in a new transgenic hemophilic mouse model

“…15,16 Expression of human FVIII during this period should therefore lead to the depletion of high-avidity human FVIII-specific T cells and result in specific immune tolerance toward human FVIII. Similar genetically engineered mouse models were described for human insulin analogs, 17 modified human tissue plasminogen activator variants, 18 human IFNs, 19,20 human Fas ligand inhibitory protein (decoy receptor 3, DcR3), 21 and a mutant of human FVIII. 22 Transgenic mouse models for human IFN␣2b (hIFN␣2b) and human IFN␤ (hIFN␤) 19,20 were shown to be predictive of the human immune response, because commercially available hIFN␤ drugs that were found to induce antibodies in a large number of patients were shown to break immune tolerance in these mice.…”

“…Similar genetically engineered mouse models were described for human insulin analogs, 17 modified human tissue plasminogen activator variants, 18 human IFNs, 19,20 human Fas ligand inhibitory protein (decoy receptor 3, DcR3), 21 and a mutant of human FVIII. 22 Transgenic mouse models for human IFN␣2b (hIFN␣2b) and human IFN␤ (hIFN␤) 19,20 were shown to be predictive of the human immune response, because commercially available hIFN␤ drugs that were found to induce antibodies in a large number of patients were shown to break immune tolerance in these mice. 19 In the present study, we describe the selection and characterization of a transgenic mouse line that is immunologically tolerant to native human FVIII but is still able to mount an antibody response to human FVIII when challenged with a modified FVIII protein that possesses increased immunogenicity.…”

“…39 Therefore, the difference in the levels of human F8 cDNA and human F8 mRNA found after successful gene-therapy approaches and after transgenic expression of human F8 cDNA are likely to be substantial. There are several other examples of transgenic mouse models that are immunologically tolerant to specific human proteins such as insulin, 17 IFNs,19,20 or factor IX, 48 but lack detectable human protein in the circulation. Moreover, Connely et al 49 demonstrated that the lack of circulating FVIII protein observed after gene therapy using a vector containing a human F8 cDNA driven by an albumin promoter was not necessarily associated with a lack of the production of functional human FVIII protein.…”

Classification and Characterization of Therapeutic Antibody Aggregates

“…Immunogenicity assays have shown that the nature of the aggregate and the type of protein are critical for eliciting an immune response as seen in several cases, IFN-␣ (9,(15)(16)(17), FVIII (18,19), and recombinant human growth hormone (20). Metal-catalyzed aggregates of IFN-␣ showed the highest level of response and were able to break the tolerance of transgenic mice (9,(15)(16)(17), whereas heat-aggregated FVIII was found to be less immunogenic than the monomeric protein (18,19).…”