Immunogenicity of biopharmaceuticals in chronic inflammatory diseases

Multiple sclerosis (MS) and rheumatoid arthritis (RA) are chronic inflammatory diseases where genetic and environmental factors influence the pathogenesis. MS is a disease of the central nervous system, while RA primarily affects the joints. Biopharmaceuticals such as interferon beta (IFNβ) and tumor necrosis factor-alpha (TNF-α) inhibitors are widely used treatments to achieve a reduction in disease activity in people with MS and RA respectively. Over time, however, some of the treated patients develop anti-drug antibodies (ADA) or neutralizing ADA (NAb) that can reduce or abrogate the drug efficacy and subsequently lead to loss of clinical response. Five studies are included in this thesis, which assess endogenous immune processes affected and evaluates laboratory methods used for monitoring immunogenicity of IFNβ and TNF-α inhibitors. Collectively, the findings presented in this thesis aim to optimize methods for drug level and ADA screening to allow for easier treatment decisions. Additionally, the thesis highlights the skin site as a potential contributor to ADA development. In study I, we studied the immunomodulatory role of IFNβ and how it was affected by NAb. We found a 3-fold increase of serum IL-7 (genetically associated with MS) in IFNβ treated MS patients and this was related to the lowered IL-7Rα expression on cell surfaces. The presence of high NAb titers to IFNβ resulted in significantly lower serum IL-7 levels compared to NAb negative patients as measured with the myxovirus resistance protein A (MxA) gene expression assay (MGA). Since the MGA method is cumbersome we decided to evaluate a new method, iLite (in study II). We found that the NAb titers had a high degree of correlation between the two assays and that NAb titers of 150 TRU/mL were suggestive of significant neutralization of the drug. However, in the iLite assay (and MGA) NAb titers are calculated using the Kawade principle and this method has statistical limitations. In study III, we therefore continued to validate the iLite assay using a cut-point approach designed to be more sensitive and statistically accurate. By using the cut-point approach, we identified 12% more NAb positive samples compared to using the Kawade method, showing the increased sensitivity achieved with the cut-point design. In study IV, we evaluated different methods for ADA screening of the TNF-α inhibitor infliximab. We showed that ADA could be detected in the majority of samples with low drug levels using ELISA, but that samples with detectable drug levels also tested ADA positive using an acid and dissociation assay (PandA). Thus, the PandA proved useful as a complement to the routinely available ELISA to monitor immunogenicity. Lastly (in study V), to understand the mechanism of ADA induction, we investigated the primary immune response against repetitive injections with biologicals in skin cells. Using a human skin model, we found that the IFNβ injection enhanced dendritic cell maturation and elevated the expression of several inflammatory cytokines, which suggest that the administration triggers an immune response at the injection site. LIST OF PUBLICATIONS I. Wangko Lundström, Christina Hermanrud, Maria Sjöstrand, Susanna Brauner, Marie Wahren Herlenius, Tomas Olsson, Virginija Karrenbauer, Jan Hillert and Anna Fogdell-Hahn. Interferon beta treatment of multiple sclerosis increases serum interleukin7. Multiple Sclerosis Journal, November 2014, 20(13): 1727–1736 II. Christina Hermanrud, Malin Lundkvist Ryner, Elin Engdahl, and Anna FogdellHahn. Anti-Interferon Beta Antibody Titers Strongly Correlate Between Two Bioassays and In Vivo Biomarker Expression, and Indicates That a Titer of 150 TRU/mL Is a Biologically Functional Cut-Point. Journal of Interferon & Cytokine Research, July 2014, 34(7): 498-504 III. Christina Hermanrud, Malin Ryner, Thomas Luft, Poul Erik Jensen, Kathleen Ingenhoven, Dorothea Rat, Florian Deisenhammer, Per Soelberg Sørensen, Marc Pallardy, Dan Sikkema, Elisa Bertotti, Daniel Kramer, Paul Creeke, Anna Fogdell-Hahn. Development and validation of cell-based luciferase reporter gene assays for measuring neutralizing anti-drug antibodies against interferon beta. Journal of Immunological Methods, March 2016, 430: 1–9 IV. Christina Hermanrud, Malin Ryner, Rille Pullerits, Karen Hambardzumyan, Nancy Vivar Pomiano, Per Marits, Inger Gjertsson, Saedis Saevarsdottir, Anna Fogdell-Hahn. Measurement of serum infliximab levels and detection of free and bound anti-infliximab antibodies in patients with rheumatoid arthritis. Manuscript V. Christina Hermanrud, Toni M.M van Capel, Michael Auer, Virginija Karrenbauer, Florian Deisenhammer, Esther C. de Jong and Anna FogdellHahn. Different interferon beta preparations induce the same qualitative immune response in human skin. Manuscript ADDITIONAL PUBLICATIONS I. Clemens Warnke, Christina Hermanrud, Malin Lundkvist, Anna Fogdell-Hahn Anti-drug antibodies Drugs and Therapy Studies 2012; volume 2:e11 II. Jenny Link, Malin Lundkvist Ryner, Katharina Fink, Christina Hermanrud, Izabella Lima, Boel Brynedal, Ingrid Kockum, Jan Hillert, and Anna Fogdell-Hahn. Human leukocyte antigen genes and interferon beta preparations influence risk of developing neutralizing anti-drug antibodies in multiple sclerosis. PLoS One, March 2014, 9(3):e90479 III. Kathleen Ingenhoven, Daniel Kramer, Poul Erik Jensen, Christina Hermanrud, Malin Ryner, Florian Deisenhammer, Marc Pallardy, Til Menge, Hans-Peter Hartung, Bernd C. Kieser, Elisa Bertotti, Paul Creeke, Anna Fogdell-Hahn. Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta. Frontiers in Neurology, July 2017, 8:305