Structural aspects of the immunogenicity of therapeutic proteins : transgenic animals as predictors for breaking immune tolerance

Animal antisera were the first proteins introduced in medicine more than a century ago, followed in the 1920s by insulin from porcine and bovine origin. This first generation of therapeutic proteins proved to be immunogenic which is expected from foreign proteins. Today, most proteins used in medicine are made by recombinant DNA technologies. Many of these proteins are copies of human proteins but the majority of these proteins still induces antibodies.\n \nMany factors are known to influence the immunogenicity, but their interplay is highly complex and it is still difficult to predict the occurrence of immune reactions in patient populations and, even more so, in individual patients. \n \nTherefore, it was the aim of this thesis to contribute to the development of a generic strategy to predict the immunogenicity of therapeutic proteins in clinical settings. Different conformations of proteins were created and physicochemically characterized. Their immunogenicity was assessed in wildtype and immune tolerant transgenic mice. Also, the effect of formulation excipients on protein structure was studied and attempts were made to correlate structural properties of the protein with immunogenicity. Finally, a start was made to evaluate the value of the animal models, wildtype and transgenic immune tolerant mice, in predicting the immune response in patients. \n \nTransgenic immune tolerant mice carry the gene for the human protein in their chromosomal DNA and therefore share immune tolerance for this protein with humans. The administered protein will be recognized as a self-protein by the immune system and no immune response should be elicited. \nThe presence of aggregates in a protein formulation is known to induce an immune response. The transgenic animal model was used to evaluate the effect of aggregate content, aggregate type and route of administration of protein formulations on the immunogenicity. It was shown that not all aggregates were able to break the tolerance of the transgenic mice. Only aggregates with a native-like structure were able to break the tolerance of the transgenic mice. A high content of aggregates was needed to increase the immune response in the wildtype mice, while a small amount of aggregates in the protein formulation was already able to break the tolerance of the transgenic mice. The transgenic mouse model was shown to be able to simulate clinical data: an interferon beta formulation known to induce an immune response in 90 % of the patients was able to break the tolerance of the transgenic mice. \nImmunogenicity testing in transgenic mice and wildtype mice, will be a good method to compare the immunogenicity of protein formulations before the start of large clinical trials.