Investigation of the role of antagonism of the Interleukin-7 receptor in the treatment of multiple sclerosis in humans and in vitro differences between genetically stratified subjects based on Interleukin-7 receptor genotype Dr Onajite Kousin-Ezewu Department of Clinical Neurosciences University of Cambridge Homerton College July 2019 This dissertation is submitted for the degree of Doctor of Philosophy DECLARATION This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except as declared in the Preface and specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University of similar institution except as declared in the Preface and specified in the text. It does not exceed the prescribed word limit for the relevant degree committee. 2 ABSTRACT Dr Onajite Kousin-Ezewu Investigation of the role of antagonism of the Interleukin-7 receptor in the treatment of multiple sclerosis in humans and in vitro differences between genetically stratified subjects based on Interleukin-7 receptor genotype Multiple Sclerosis is an autoimmune disease mediated by activated lymphocytes entering the central nervous system. Treatments with the greatest efficacy either prevent the entry of activated lymphocytes, or deplete the lymphocyte population, before allowing lymphocyte reconstitution. IL-7Rα was identified by genetic studies in MS pathogenesis and is involved in the homeostasis and proliferation of lymphocytes. This thesis investigates the role of antagonism of IL-7Rα in the treatment of MS in humans and in vitro differences between genetically stratified subjects based on IL- 7Rα genotype. It also explores the role for biomarkers during reconstitution of lymphocytes after Alemtuzumab treatment in MS, in which IL-7Rα plays a major role. Chapter 3 describes the prematurely aborted clinical trial of subjects with an IL-7Rα antagonist. This first-time-in-human trial demonstrated the drug was safe and well tolerated in this limited cohort of subjects. Chapter 4 investigated the differences between individuals based on IL-7Rα genotype with in vitro IL-7Rα antagonism and stimulation. It demonstrated greater activation through IL-7Rα in individuals with the protective genotype. Differences in negative feedback mechanisms of IL-7Rα were explored. Chapter 5 investigated the tolerability of palifermin, a keratinocyte growth factor, with alemtuzumab, which was well tolerated as part of a dose escalation sub-study of the CAMTHY trial. The main CAMTHY trial investigated if palifermin could cause increased thymic lymphopoiesis, offsetting the IL-7 driven homeostatic proliferation of lymphocytes and secondary autoimmunity associated with alemtuzumab. 3 Chapter 6 investigated the use of CD4+ lymphocytes as a biomarker for relapses after Alemtuzumab treatment. This contradicted the findings of a previously published paper, using a much larger cohort of patients in Cambridge. This work underlines the importance of IL-7 in the pathogenesis and treatment of MS. It points towards the IL-7Rα pathway as a future avenue for biomarkers and novel treatments for MS. 4 PREFACE The overall aim at the start of my PhD was to train in translational medicine in order to become a clinical trials specialist. Therefore the funding from the Wellcome Trust for the translational medicine and therapeutics PhD incorporated funding from the Wellcome Trust and GlaxoSmithKline (GSK). The aim was to take a translational project at GSK. This was a first time in human trial of an IL-7Rα antagonist. During the first two parts of the trial I was due to work as a sub-investigator on the trial, learning about clinical trials in healthy volunteers. The final part of the trial was a novel approach to Phase 1 trials with the introduction of Multiple Sclerosis (MS) patients early in the clinical trial process. It was intended that I would be the chief investigator leading this part of the trial. Unfortunately after dosing 16 healthy volunteers my PhD had to change course due to the trial being prematurely terminated by GSK due to data fraud in the pre-clinical scientific work. My experiences of this is described in more detail in chapter 3, where I also describe the observations of the healthy volunteers that were dosed prior to termination of the trial. After termination of the IL-7Rα trial, GSK stopped all work on the IL-7Rα pathway within the organization. Therefore I had to change course during my research period. However once work could re-start on the IL-7Rα pathway after an investigation into what had led up to the data fraud in China, I moved my focus away from the clinical aspect of IL-7Rα and focused on signaling through IL-7Rα. This was due to the fact that there was continuing uncertainty about how blocking IL-7Rα would affect patients. This led to a genetic study, with the genetic stratification by IL-7Rα genotype drawn up by geneticists at GSK before I began my period of research. This investigated if IL-7Rα antagonism affected subjects differently according to IL-7Rα genotype. The data from this first project led to further questions about if the differences seen between the genetic groups in the first project was due to negative feedback mechanisms such as downregulation of IL-7Rα after stimulation of the receptor. Therefore the second project in chapter 4 investigated this further with an IL-7 stimulation in vitro of blood from subjects stratified according to IL-7Rα genotype. 5 During the period when work on the IL-7Rα pathway had been stopped by GSK, I wanted to continue my interest in translational medicine, the original purpose of my PhD. Therefore I became a sub-investigator on the CAMTHY trial, a study investigating lymphocyte reconstitution post alemtuzumab (in which IL-7 plays a major role), a drug known to deplete lymphocytes, which is a highly effective treatment for MS. The aim of the study was to use palifermin, a keratinocyte growth factor, in order to drive thymic reconstitution of lymphocytes and increase the diversity of the lymphocyte population, rather than peripheral reconstitution of lymphocytes that were not depleted after alemtuzumab. Prior to this trial commencing a safety sub-study was performed to investigate if palifermin was tolerable as this was a dose that had never before been used in humans. I was heavily involved in the practical administration of this study whilst also investigating and collecting the clinical data on the patients that were dosed. I have described my experiences of this study in chapter 5. I have also included in the appendix a paper, which has been published, of the main CAMTHY trial, which followed this sub-study (Coles et al., 2019). During the period when I was unable to work on the IL-7Rα pathway at GSK I also did further work investigating the reconstitution of lymphocytes post alemtuzumab. I investigated if there was a relationship between the level of reconstitution of lymphocytes and the clinical outcome in MS patients such as disability, relapses and MRI imaging. This work followed a controversial paper, which claimed increased CD4+ counts post alemtuzumab was associated with increased MS disease activity. I have summarized this work in chapter 6, which led to a publication in the journal Neurology (Kousin-Ezewu et al., 2014). In summary this thesis explores the role of the IL-7Rα pathway in MS, first through antagonism of IL-7Rα in vitro, but also in a first time in human trial. It also explores the effects in vitro of stimulation of the IL-7Rα pathway and how this differs between genetic groups stratified according to IL-7Rα genotype. Whilst working with alemtuzumab, a potent lymphocyte depleting monoclonal antibody, I was able to explore reconstitution of lymphocytes, a process in which IL-7 and its receptor play a very important role. I also explored if this reconstitution could be altered in humans by attempting to stimulate increased thymic reconstitution, ultimately attempting to reduce the autoimmune clinical side effects of alemtuzumab, which could improve the suitability of this highly effective drug for a wider range of MS patients. 6 ACKNOWLEDGEMENTS I would like to thank my mother and brothers for continuing to encourage my study through the years and for helping to keep me grounded. I would like to thank the Therapeutic Immunology Group who have guided me through the PhD process. Finally I would like to give my love and thanks to Louisa and Dorothy. Louisa has tirelessly listened when things have not gone according to plan and has always suggested sensible ways forward. She uniquely understands the difficulty of doing a PhD in our family and I will forever be in her debt. I look forward to enjoying family life with her, Dorothy and Urias. 7 PUBLICATIONS AND PRESENTATIONS ARISING FROM THIS WORK Kousin-Ezewu, O. & Coles, A. 2013. Alemtuzumab in multiple sclerosis: latest evidence and clinical prospects. Ther Adv Chronic Dis 4(3):97-103. This review was published in the journal Therapeutic Advances in Chronic Disease. Kousin-Ezewu, O., Azzopardi, L., Parker, R. A., Tuohy, O., Compston, A., Coles, A. & Jones, J. 2014. Accelerated lymphocyte recovery after alemtuzumab does not predict multiple sclerosis activity. Neurology, 82,2158-64. This paper was published in the Neurology Journal. Joanne Ellis, Andre van Maurik, Lea Fortunato, Sophie Gisbert, Keguan Chen, Ann Schwartz, Simon McHugh, Andrew Want, Sara Santos Franco, Joao-Joaquim Oliveira, Jeffrey Price, Kim Brown, Donna Su, Jenny Craigen, Jiansong Yang, Sara Brett, Bill Davis, Joseph Cheriyan, Onajite Kousin-Ezewu, Frank Gray, Paul Thompson, Alasdair Coles and Disala Fernando.