Abstract
Therapeutic proteins have become an important class of drugs. One of those proteins currently on the market is recombinant human interferon beta (rhIFN?), used to treat multiple sclerosis. After prolonged treatment, a substantial proportion of patients form antibodies against the protein. The immunological response generally starts with the appearance of
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binding antibodies possibly affecting the clearance of the drug, followed by neutralizing antibodies that block the biological activity of the protein. Such immune response is not expected, since human proteins are in principle tolerated by the human immune system. Several factors contribute to immunogenicity but protein aggregates are believed to play a major role. To study the relation between protein structure and immunogenicity, transgenic animal models immune tolerant for the protein were developed. This thesis aimed at providing mechanistic insight into the relation between rhIFN? aggregation and its immunogenicity. Transgenic C57Bl/6 mice immune tolerant for hIFN? have been applied as tools to study the breaking of immune tolerance by rhIFN?. RhIFN?-1a products formulated with HSA failed to induce antibodies in wildtype C57Bl/6 mice, which led to the conclusion that the transgenic C57Bl/6 mice cannot be used to study the immunogenicity of these products. For this reason, a second mouse model was developed by crossing the original transgenic C57Bl/6 mice with wildtype FVB/N mice. Both wildtype and transgenic C57Bl/6 x FVB/N hybrid offspring were used to evaluate the immunogenicity of three marketed products, Rebif (rhIFN?-1a), Avonex (rhIFN?-1a) and Betaferon (rhIFN?-1b). All products were immunogenic in wildtype hybrid mice, while the immunogenicity in transgenic hybrid mice was product dependent and in line with clinical observations. In conclusion, the hybrid mouse model offered unique possibilities to analyze the immunogenicity of a wide range of rhIFN? preparations, study factors that are of influence and look into the mechanism that is responsible for overcoming immune tolerance. The research described in this thesis led to greater insight in the mechanism by which rhIFN? aggregates overcome immune tolerance for the human protein. The transgenic immune tolerant mouse model that was developed acted as useful instrument for investigating the immunogenicity of a wide range of aggregated products. The transgenic mice enabled us to look into the features of the immune response induced by rhIFN? aggregates, the results of which indicate that CD4+ T cells as well as MZ B cells are involved but NABs and immunological memory are lacking within the time frame of the experiments. Not all rhIFN? aggregates were equally immunogenic, but removing aggregates eliminated the immune response against rhIFN? in transgenic mice. Aggregated rhIFN? preparations that were immunogenic in transgenic mice appeared to contain native-like protein. Moreover, it was shown that different pathways of oxidizing rhIFN? may lead to the formation of aggregates with greatly enhanced immunogenicity. Finally, it was demonstrated that rhIFN? adsorbs readily to common surface materials such as glass, metal and polystyrene. In particular complexes of rhIFN? with metal microparticles induced high levels of anti-rhIFN? IgG in transgenic mice, highlighting the importance of measuring and characterizing (sub)visible particulates in protein formulations
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