title: Immunopharmacological consequences of immune responses to therapeutic interferon beta
Creators: name: Govindappa, Karthik
Publisher: name: University of Liverpool Repository
Year: 2014

description: Protein therapeutics or biologics represent 30 % of current licensed pharmaceutical products. In general, biologics offer superior safety profiles compared to small molecules. However, significant clinical concerns have emerged in terms of development of anti-drug antibodies (ADAs), a phenomenon that is covered under the term, immunogenicity. Anti-drug antibodies can alter pharmacokinetics, reduce efficacy of the therapeutic and also can in some cases induce allergic reactions. Human recombinant interferon beta (IFN-β) is a biologic used for the treatment of multiple sclerosis (MS) – a chronic, inflammatory and demyelinating disease of the central nervous system. Long-term treatment with IFN-β has been shown to lead to the development of anti-IFN-β antibodies that can cause total loss or reduced efficacy. Anti-drug antibodies can be non-neutralising (N-NAb) and neutralising (NAb) depending on the site to which they bind. This study aimed to conduct a systematic review to determine factors affecting the formation of neutralising antibodies against three different formulations of IFN-β Avonex™, Rebif™ and Betaseron/Betaferon™. Findings from the systematic review highlight the relative differences in immunogenicity risk of different IFN-β formulations Avonex™, Rebif ™ and Betaseron/Betaferon™ with Avonex™ having the lowest risk, Rebif™ has moderate risk and Betaseron/Betaferon™ has high risk. Characterising the immunoglobulin profile of the IFN-β ADAs from the plasma of ADA positive MS patients revealed that IFN-β ADAs are predominantly of the IgG1 and IgG4 subclass. We also characterised the neutralising potential of the major ADA IgG4 subclass using a IFN-β bioactivity assay and show that IgG4 antibodies may likely contribute to the neutralisation activity. The potential of the neutralising ADAs to cross-react with endogenous IFN-β was investigated using an in vitro bioactivity assay. Findings from this set of experiments revealed varying degrees of neutralisation of endogenous IFN-β. We next explored the potential immunological consequence of ADA with regards to formation of immune complexes and activation of complement. The interaction of ADAs with the biologic can result in formation of immune complex. Immune complexes can activate the complement system. The data revealed IFN-β ADAs can form immune complexes with IFN-β and therefore activate complement. We also attempted to identify IFN-β linear epitopes that the ADAs had the ability to bind. However, a combination of multipin peptide technology and in vitro peptide competitive binding assay failed to reveal a definitive linear epitope although there was some evidence for the existence of potential linear epitopes. We also examined the involvement of T helper cells and T regulatory cells (Tregs) in ADA development. The data revealed no significant differences in the frequency of Tregs among IFN-β ADAs positive, negative and healthy donors. Attempts were also made to identify T helper epitopes within IFN-β that could potentially drive immunogenicity. Using T-cell epitope prediction tools (IEDB-AR and ProPred) and T-cell functional assays we identified an immunogenic sequence of 36 amino acids within IFN-β (position 130-166). Our data revealed that one IFN-β peptide within this sequence is a potential T-cell immunogenic epitope. In addition we identified a possible association of one IFN-β derived peptide with the DRB1*1501HLA haplotype. In summary, the results presented in this thesis have provided essential information on subclass profile of IFN-β ADAs, the possible involvement of T helper cells and potential antibody epitopes within IFN-β. Future studies should be aimed at providing greater detail on the evolution of the ADA response and test strategies to remove immunogenic determinants from IFN-β.