IL-3Rα as a novel therapeutic target in Systemic Lupus Erythematosus

A thesis submitted in total fulfillment of the requirements of the degree of

Doctor of Philosophy

by

Shereen Pek Chuen Oon

The Walter and Eliza Hall Institute of Medical Research

Department of Medical Biology

The University of Melbourne

June 2017

ORCID ID: orcid.org/0000-0002-6822-5711

Abstract

Systemic lupus erythematosus (SLE) is a prototypic human autoimmune disease characterized by impaired immune tolerance, resulting in the generation of pathogenic autoantibodies and immune complexes. Despite currently available therapies, there is still a significant morbidity, and unacceptable mortality, associated with the disease, and there is therefore great interest in the development of more effective therapies for SLE. Although autoreactive B lymphocytes have been the major targets for biologic therapies in SLE to date, the importance of the innate immune system, and in particular, the plasmacytoid dendritic cell (pDC)-interferon (IFN) pathway, has emerged, with monoclonal antibodies (mAbs) which block IFNα and the IFN receptor entering clinical trials.

The work of this thesis, conducted ex vivo in a heterogeneous population of SLE patients, and in healthy and autoimmune donors, in addition to in vivo in cynomolgus macaques, has revealed the IL-3Rα/CD123 as a potential therapeutic target in SLE. CSL362 is a fully humanized monoclonal antibody which targets the IL-3 receptor alpha chain (CD123) and has two mechanisms of action - neutralizing IL-3 signaling, and effecting antibody-dependent cell-mediated cytotoxicity (ADCC) against CD123 expressing cells.

CSL362 was found to alter a variety of key pathogenic cell types and cytokines in SLE. CSL362 potently depleted pDCs, which highly express CD123. Through pDC depletion, type I IFN production in response to TLR7 and TLR9 agonists, and to SLE serum, was markedly reduced, as was the expression of IFN-inducible genes, and the expansion of plasmablasts. These effects were more pronounced than with type I IFN blockade alone, possibly because pDC depletion reduced the production of other IFN types (type III IFN) and other cytokines produced by activated pDCs (such as IL-6, which was found to contribute to plasmablast expansion together with IFNα).

The anti-CD123 mAb was also found to deplete CD123hi basophils, and this effect, together with its ability to neutralize IL-3, potentially sets this therapeutic strategy apart

ii from other agents that target pDCs. Relatively little is known about the role of IL-3 in SLE specifically, although murine studies have indicated it may play a direct role in the progression of lupus nephritis. In this study, a novel association between IL-3 and IFN (type I and type III) was found in the serum of SLE patients and healthy donors, which was supported by findings from transcriptional profiling of whole blood, with an ‘IL-3 gene signature’ in SLE patients found to correlate with an ‘IFN gene signature’. This raises the possibility of utilizing a dual IL-3/IFN gene signature to predict potential benefit from the use of CSL362 in patients.

This study also confirmed in vivo efficacy with subcutaneous administration of CSL362 in depleting pDCs and basophils, and decreasing an IFN gene signature, in cynomolgus macaques. The subcutaneous route is typically preferred in chronic diseases such as SLE and can allow self administration. Importantly, CSL362 was well tolerated in this non-human primate study, in particular, there were no cytopenias or serious infections.

These data have revealed a unique spectrum of effects from an anti-CD123 mAb which may be of therapeutic benefit in SLE, and related diseases, providing a strong pre- clinical rationale for the therapeutic evaluation of CSL362 in SLE.

iii Declaration

This is to certify that:

1. This thesis comprises my original work;

2. Due acknowledgement has been made in the text to all other material used; and

3. This thesis is less than 100,000 words in length, exclusive of tables, figures, references and appendices.

Shereen Oon

Reid Rheumatology Laboratory Inflammation Division The Walter and Eliza Hall Institute of Medical Research

iv

Preface

The work presented in this thesis was performed at CSL Limited and The Walter and Eliza Hall Institute of Medical Research. Shereen Oon was supported by an NHMRC Postgraduate Scholarship (#1039026), an Edith Moffat Postgraduate Scholarship from The Walter and Eliza Hall Institute of Medical Research, and a stipend from CSL Limited.

I assess my contribution to each chapter to be:

Chapter 1: 100% Chapter 2: 100% Chapter 3: 90% Huy Huynh (CSL) performed the experiments for Figures 3-6A, 3-11 and 3-26A and E; Katherine Monaghan (CSL) performed the experiment for Figure 3-12; Nick Wilson (CSL) performed the experiments for Figures 3-14, 3-18 and 3-23; Tsin Yee Tai (CSL) performed the experiments for Figures 3-19, 3-20 and 3-21; Mark Biondo (CSL) helped analyse the experiments for Figures 3-26A and B. Heat maps were created by Milica Ng (CSL). Chapter 4: 95% Katherine Monaghan performed the experiments which formed Figure 4-12. Heat maps were created, and bioinformatic analysis performed, by Milica Ng. Chapter 5: 100%

My overall contribution to the work presented in this thesis is therefore >95%.

v Publications arising from this thesis

Parts of this thesis have been published, or manuscripts are in preparation, as follows:

Published

Oon S, Huynh H, Tai TY, Ng M, Monaghan K, Biondo M, Vairo G, Maraskovsky E, Nash AD, Wicks IP†, Wilson NJ†. A cytotoxic anti-IL-3Rα antibody targets key cells and cytokines implicated in systemic lupus erythematosus. JCI Insight. 2016;1:e86131. This publication contains data found in Chapter 3.

Oon S, Wilson NJ†, Wicks IP†. Targeted therapeutics in SLE – emerging strategies to modulate the interferon pathway. Clin Trans Immunol. 2016;5:e81.

Manuscripts in preparation

Oon S, Monaghan K, Ng M, Keeble J, Speed T, Hoi A, Morand E, Vairo G, Maraskovsky E, Nash AD, Wilson NJ†, Wicks IP†. Serum cytokine analysis and whole blood transcriptional profiling reveal an association between IL-3 and IFN in human SLE.

† These authors contributed equally

vi

Conference abstract presentations

Oral presentations

Oon S, Huynh H, Tai TY, Ng M, Monaghan K, Vairo G, Maraskovsky E, Nash AD, Wicks IP†, Wilson NJ†. IL-3Rα as a novel therapeutic target in systemic lupus erythematosus. Australian Rheumatology Association Annual Scientific Meeting, Darwin 2016.

Oon S, Wilson N†, Wicks I†. Targeted plasmacytoid dendritic cell (pDC) depletion with an anti-CD123 mAb – a potential novel treatment for systemic lupus erythematosus. Australian Rheumatology Association Annual Scientific Meeting, Adelaide 2015.

Oon S, Wilson N†, Wicks I†. Targeted plasmacytoid dendritic cell depletion with an anti-CD123 mAb – a potential novel treatment for systemic lupus erythematosus. Melbourne Health Research Week, Melbourne 2015.

Oon S, Wilson N†, Wicks I†. An anti CD123 monoclonal antibody (CSL362) depletes plasmacytoid dendritic cells and inhibits CpG upregulated IFNα production and IFNα- inducible gene expression in peripheral blood mononuclear cells from patients with systemic lupus erythematosus. American College of Rheumatology Annual Scientific Meeting, Boston 2014.

vii Poster presentations

Oon S, Monaghan K, Ng M, Hoi A, Morand E, Wicks IP†, Wilson NJ†. Serum cytokine analysis and transcriptional blood profiling reveal an association between IL-3 and IFN in human SLE. Australian Rheumatology Association and New Zealand Rheumatology Association Annual Scientific Meeting, Auckland 2017.

Oon S, Monaghan K, Ng M, Hoi A, Morand E, Wicks IP†, Wilson NJ†. Serum cytokine analysis and transcriptional blood profiling reveal an association between IL-3 and IFN in human SLE. 12th International Congress on Systemic Lupus Erythematosus, Melbourne 2017.

Oon S, Huynh H, Tai TY, Ng M, Monaghan K, Vairo G, Maraskovsky E, Nash AD, Wicks IP†, Wilson NJ†. IL-3Rα as a novel therapeutic target in systemic lupus erythematosus. International Congress of Immunology, Melbourne 2016.

Oon S, Huynh H, Tai TY, Ng M, Monaghan K, Vairo G, Maraskovsky E, Nash AD, Wicks IP†, Wilson NJ†. IL-3Rα as a novel therapeutic target in systemic lupus erythematosus. European League Against Rheumatism (EULAR) Annual Congress, London 2016.

Oon S, Wilson N†, Wicks I†. Targeted plasmacytoid dendritic cell (pDC) depletion with an anti-CD123 mAb – a potential novel treatment for systemic lupus erythematosus. 11th International Congress on Systemic Lupus Erythematosus, Vienna 2015.

† equal contributors

viii Acknowledgements

It has been a privilege to have been given the opportunity to undertake this work at both The Walter and Eliza Hall Institute of Medical Research, and CSL Limited. I would like to thank CSL for facilitating the collaborations on this project.

I am grateful for the contributions of the many people who have assisted me during my PhD journey. I would particularly like to thank Professor Ian Wicks and Dr Nicholas Wilson, for their supportive mentorship, and wise and patient guidance as my co- primary supervisors. I am grateful also for the warm advice and words of encouragement offered by my committee members, Dr Sandra Nicholson and Professor Lynn Corcoran.

I would also like to thank the following people (in no particular order) for their technical advice, practical assistance, or moral support, and for providing a friendly working environment at:

The Walter and Eliza Hall Institute of Medical Research

• Wicks laboratory: Gabrielle Goldberg, Emma Stuart, Rhiannon Jones, Tsin Yee Tai, Jo Keeble, Simon Chatfield, Jacinta Hansen, Willy-John Martin, Damian D’Silva, Jane Murphy, Ee Shan Pang, Tommy Liu, Devi Ngo, Andrew Foers, Marilou Barrios, Tan Nguyen, Ken Pang, Blake Smith, Man Lyang Kim, Angus Stock, Rebecca Stewart, Cynthia Louis, Rowena Lewis, Kathrin Grebe, Bartjin Pieters, Annemarie van Nieuwenhuijze. • Inflammation division: Paul Baker, Edmond Linossi, Nga Lam, Kate Lawlor, Dom De Nardo, Fiona Moghaddas, Paul Nguyen, Akshay D’Cruz, Maryam Rashidi, Suad Abdirahman, Swarna Vijayaraj, Ashleigh Poh, Hazel Tye, Thomas Hayman, Tatiana Kolesnik, Lukasz Kedzierski, Tracy Putoczki, Seth Masters, James Vince. • Clinical translation centre: Jenni Harris, Lina Laskos, Cathy Quillici, Naomi Sprigg, Kimvan Le (for assistance with healthy donor recruitment). • Statistical analysis: Terry Speed.

ix • Other: Graphics and media (Rachel Bucknall, Peter Maltezos), grants (Julie Mercer, Annette Wilson, Lynne Hartley), IT (Chris Fitzgerald, Andrew McInneny, Edy Huynh, Chris Ham, Jason Cutler), education (Sue Hardy, Keely Bumsted-O’Brien) purchasing (Dorothy Pilarinos, Claudia Kerstovitch, Boris Trajcevski), stores (Luke Baltrunas, Richard Reeve), administration (Mabel Kiang).

CSL Limited

• Pre-clinical biology: Huy Huynh, Katherine Monaghan, Mark Biondo, Alexander Karnowski, Laura McMillan, Vicki Sorto, Karen Scalzo-Iguanti, Anne McDonald, Mae Wong, Samantha Busfield. • Translational research: Catherine Tarlinton (cell sorting), Sandra Koernig, Yun Dai, David Leong, Karoline Krstevska, Ingela Vickstrom, Peter Schmidt, Larissa Provan, Snezana (Nina) Koso, Pino Maccarone, Adele Mount, Soosan Wan, Jessica Petracca, Jenny Chia, Anabel Silva, Therese Lynch, Ian Campbell, Anne Walter, Isabelle Glauser, Esin Donaldson, David Pejoski, Dorit Becher, Hua Feng, Helen Cao, Sandro Prato, Adrian Zuercher. • Bioinformatic analysis: Milica Ng, Monther Alhamdoosh. • Research management: Gino Vairo, Eugene Maraskovksy, Andrew Nash, Zdenka Bolcevic.

The Royal Melbourne Hospital

For assistance with donor recruitment: • Rheumatology department: Susan Tadros, Clare O’Neill, Malisha Hettiararchy, Gene-Siew Ngian, Sabina Ciciriello, John Moi, Simon Chatfield, David Liew, Melissa Northcott, Katie Morrisroe, Andrew Teichtahl, Jessica Day, Louisa Chou, Laura Beaton, Lucy Croyle, Kate Franklyn, Emma Mitchell, Leanne Albas. • Nephrology department: Kathy Nicholls, Peter Hughes, Scott Wilson. • Dermatology department: George Varigos, Amanda Saracino.

x

Monash Medical Centre

For assistance with donor recruitment and collection of clinical information, I thank Eric Morand, Alberta Hoi and Sue Morton.

I would additionally like to thank the patients and healthy volunteers who have generously contributed samples to these studies.

Funding towards this project was gratefully received from CSL Limited, Arthritis Australia (South Australia Lupus, Scleroderma and Sjögren’s Support Group Grant), and The Reid Charitable Trusts, which supports the Wicks Laboratory.

Lastly, but by no means least, I would like to thank my family and friends, in particular my parents Alan and Quee Sin Oon, my sister Elaine Oon, and my aunt, uncle and cousin, Quee Teen Boon, Kok Cheng Boon and Michael Boon, for their steadfast encouragement and support during this time.

xi Table of contents

Abstract ...... ii

Declaration ...... iv

Preface ...... v

Publications arising from this thesis ...... vi Conference abstract presentations ...... vii

Acknowledgements ...... ix

List of Tables and Figures ...... xviii

Abbreviations ...... xxiii

Chapter 1: Introduction ...... 1

1.1 Systemic lupus erythematosus - a complex chronic autoimmune disease for which novel therapies are required ...... 1 1.2. Overview of SLE immunopathogenesis ...... 2 1.2.1 The immunopathogenesis of SLE ...... 2 1.2.2 Genetic, environmental and hormonal abnormalities in SLE ...... 5 1.3. Current therapeutics in SLE ...... 6 1.3.1 B cells are a major therapeutic target in SLE ...... 7 1.4. Type I interferon - a promising therapeutic target in SLE ...... 11 1.4.1 Other interferon types contribute to SLE pathogenesis ...... 15 1.4.2 Emerging strategies to modulate the type I IFN pathway ...... 17 1.4.3 TLRs as therapeutic targets ...... 20 1.4.4 Host nucleic acids as therapeutic targets ...... 22 1.4.5 JAK/STAT inhibition as a therapeutic strategy ...... 23 1.4.6 pDCs as a therapeutic target ...... 25 1.5 Other therapeutic targets in SLE ...... 27 1.5.1 Anti–cytokine therapies in SLE ...... 27 1.5.2 Targeting T cells in SLE ...... 34 1.6 Anti-IL-3Rα mAb - a novel way of targeting pDCs in SLE? ...... 39

xii 1.6.1 Basophils express CD123 and may play a role in SLE pathogenesis ...... 40 1.6.2 Therapeutic targeting of IL-3 may be beneficial in SLE ...... 41 1.7 Hypothesis and aims ...... 42

Chapter 2: Subjects, clinical characterisation and sample collection ...... 44

2.1 Subjects ...... 44 2.1.1 Subject recruitment ...... 44 2.1.2 Study approval ...... 44 2.1.3 Inclusion and exclusion criteria ...... 45 2.2 Clinical characterisation of donors ...... 45 2.2.1 SLE donor characteristics ...... 45 2.2.2 Autoimmune disease donor characteristics ...... 51 2.2.3 Healthy donor characteristics ...... 52 2.3 Sample collection and processing ...... 53 2.3.1 Blood sample collection and storage ...... 53 2.3.2 Standard tissue culture ...... 53 2.4 Discussion ...... 54

Chapter 3: Anti-CD123 monoclonal antibody, CSL362, alters key cells and cytokines implicated in systemic lupus erythematosus ...... 55

3.1 Introduction ...... 55 3.2 Materials and methods ...... 56 3.2.1 Human subjects, sample collection and standard tissue culture ...... 56 3.2.2 Study approval – human and cyno ...... 56 3.2.3 Flow cytometry, antibodies and reagents ...... 56 3.2.4 Measurement of cell surface CD123 expression ...... 58 3.2.5 Evaluating the effect of CSL362 on pDCs, basophils, myeloid dendritic cells, and NK, B and T cell subsets ex vivo ...... 59 3.2.6 Evaluating the effect of CSL362 on NK cell activation ex vivo ...... 60

xiii 3.2.7 Evaluating the effect of CSL362, and two anti-IFNα mAbs, on TLR1-9 agonist, or SLE serum-stimulated IFNα production and IFN-upregulated gene expression ex vivo ...... 60 3.2.8 Evaluating the effect of depleting pDCs and basophils on IFNα production ex vivo ...... 61 3.2.9 Extraction of RNA from PBMCs and whole blood for assessment of IFN- inducible gene expression ...... 62 3.2.10 RNA Sequencing of isolated pDCs ...... 62 3.2.11 Evaluating the effect of CSL362, anti-IFNα mAbs and an anti-IFNAR mAb on TLR7 and TLR9 agonist-, and CD40L-induced, plasmablast expansion and proliferation ex vivo ...... 63 3.2.12 Evaluating the effect of reconstituting CSL362-treated PBMCs with pDCs or basophils, or pDC and basophil conditioned medium, on TLR7- and TLR9- induced plasmablast expansion ...... 63 3.2.13 Enzyme-linked immunosorbent assays ...... 64 3.2.14 Luminex and Bioplex assays ...... 65 3.2.15 Evaluating the effect of blockade of IFNα, IL-6 and TNF-α by neutralizing mAbs on plasmablast expansion restored by conditioned medium produced by stimulating pDCs with CpG C ...... 65 3.2.16 Evaluating the effect of subcutaneously administered CSL362 on pDCs, basophils, and IFNα-inducible gene expression in cynomolgus macaques ...... 66 3.2.17 Statistical analyses ...... 66 3.3 Results ...... 67 3.3.1 pDCs and basophils have high CD123 expression and are selectively depleted by CSL362 ...... 67 3.3.2 CSL362 activates NK cells, via its modified IgG1-Fc ...... 74 3.3.3 Depletion of pDCs by CSL362 inhibits TLR7-, TLR9- and SLE serum- stimulated IFNα-production and IFNα-inducible gene expression ...... 76 3.3.4 CSL362 inhibits IFN-inducible gene expression more effectively than IFNα blockade alone, and inhibits type III IFN production ...... 83 3.3.5 CSL362 inhibits TLR7- and TLR9-induced plasmablast expansion and proliferation ...... 87

xiv 3.3.6 pDCs activated by TLR7 and TLR9 stimulation produce IFNα and IL-6, which promote plasmablast expansion ...... 93 3.3.7 CSL362 inhibits TLR9-induced plasmablast expansion more effectively than IFNα or IFNAR blockade alone ...... 98 3.3.8 Subcutaneous administration of CSL362 to cynomolgus macaques depletes pDCs and basophils in vivo and inhibits TLR9-induced IFNα-inducible gene expression ...... 102 3.4 Discussion ...... 105

Chapter 4: Investigating a potential role for IL-3 in SLE through serum cytokine analysis and transcriptional profiling ...... 110

4.1 Introduction ...... 110 4.1.1 Potential role for IL-3 in SLE ...... 110 4.2 Material and methods ...... 112 4.2.1 Human subjects and collection of biological samples and clinical information ...... 112 4.2.2 Serum cytokine analysis ...... 112 4.2.3 Measurement of peripheral blood cell types (pDCs, basophils, B cells and T cells) ...... 114 4.2.4 Antibodies for flow cytometry ...... 118 4.2.5 Assessing alterations in gene expression in response to IL-3 stimulation in healthy donor whole blood ...... 119 4.2.6 Analysis of whole blood gene expression in SLE and healthy donors ...... 120 4.2.7 Statistical analyses ...... 120 4.2.8 RNASeq bioinformatics ...... 120 4.3 Results ...... 121 4.3.1 Serum IL-3 levels in SLE patients and healthy controls ...... 121 4.3.2 Correlations between serum IL-3 levels and measures of disease activity in SLE ...... 122 4.3.3 Correlations between serum IL-3 levels and disease manifestations and medication use in SLE ...... 124

xv 4.3.4 Correlations between serum IL-3 levels and peripheral blood pDCs, basophils, B cells and T cells ...... 128 4.3.5 Serum levels of IL-3 correlate with IFNα and type III IFN in SLE and healthy donors ...... 131 4.3.6 An ‘IL-3 gene signature’ differentiates SLE and healthy donors ...... 140 4.3.7 Presence of an ‘IL-3 gene signature’ correlates with an ‘IFN gene signature’ in SLE ...... 147 4.4 Discussion ...... 150

Chapter 5: Overall discussion, conclusions and future directions ...... 153

References ...... 161

Appendix 1 ...... 196

SELENA-SLEDAI score ...... 196

Appendix 2 ...... 198

SDI Damage Index ...... 198

Appendix 3 ...... 200

Gating strategies for assessment of CD123 cell surface expression on peripheral blood cell types ...... 200

Appendix 4 ...... 203

Gating strategies for assessment of the effect of CSL362 on peripheral blood cell types ...... 203

Appendix 5 ...... 206

Gating strategies for assessment of peripheral blood cell counts ...... 206

Appendix 6 ...... 208

Differentially expressed genes between IL-3 stimulated, and unstimulated lysed whole blood from n = 7 healthy donors, at both 6 and 24 hours...... 208

xvi Appendix 7 ...... 213

Differentially expressed genes between IL-3 stimulated, and unstimulated lysed whole blood from n = 7 healthy donors, at 24 hours ...... 213

Appendix 8 ...... 230

Differentially expressed genes between IL-3 stimulated, and unstimulated lysed whole blood from n = 7 healthy donors, at 6 hours ...... 230

xvii List of Tables and Figures

List of Tables

Table 1-1: B cell targeted therapies in SLE ...... 8

Table 1-2: Emerging strategies to target the type I IFN pathway ...... 19

Table 1-3: Anti-cytokine therapies in SLE ...... 27

Table 1-4: T cell targeted therapies in SLE ...... 34

Table 2-1: SLE donor characteristics (n = 42) ...... 47

Table 2-2: Autoimmune disease donor characteristics ...... 51

Table 2-3: Age, sex and ethnicity of healthy, SLE and autoimmune disease donors .... 52

Table 3-1: Antibodies and reagents ...... 57

Table 3-2: Defining cell surface markers for assessment of CD123 cell surface expression on peripheral blood cell types ...... 58

Table 3-3: Defining cell surface markers for assessment of the effect of CSL362 on different peripheral blood cell types ...... 59

Table 3-4: CD123 expression on SLE, healthy and autoimmune donor cell types ...... 69

Table 4-1: Studies evaluating peripheral blood pDC numbers and frequency in SLE 114

Table 4-2: Studies evaluating peripheral blood basophil numbers and frequency in SLE ...... 115

Table 4-3: Studies evaluating peripheral blood B cell numbers and frequency in SLE ...... 116

Table 4-4: Defining cell surface markers for peripheral blood pDCs, basophils, B cells and T cells ...... 118

xviii Table 4-5: Antibodies for flow cytometry ...... 119

Table 4-6: Mean IL-3 levels compared by disease activity in SLE patients, and healthy controls ...... 123

Table 4-7: Correlation between serum IL-3 levels and disease activity measures in SLE patients ...... 124

Table 4-8: Mean serum IL-3 levels in SLE patients by disease manifestation ...... 125

Table 4-9: Mean serum IL-3 levels in SLE patients by medication use ...... 127

Table 4-10: Cytokine levels in SLE compared to healthy donors ...... 133

Table 4-11: Correlations between serum IL-3 levels and cytokines in SLE and healthy donors ...... 137

Table 4-12: SLE donor characteristics (n = 31) ...... 142

Table 4-13: Differentially expressed genes in response to IL-3 stimulation, and between SLE and healthy donor whole blood ...... 144

List of Figures

Figure 1-1: Overview of SLE pathogenesis...... 3

Figure 1-2: Effects of type I interferon which promote autoimmunity...... 12

Figure 1-3: Overlapping type I, II and III IFN signaling pathways...... 16

Figure 1-4: Emerging strategies to target the type I IFN pathway...... 18

Figure 1-5: Potential role for basophils in SLE pathogenesis...... 40

Figure 1-6: Proposed mechanism by which CSL362 may have therapeutic efficacy in SLE...... 43

Figure 2-1: SLE donor disease activity...... 48

xix Figure 2-2: Positive SLEDAI components...... 49

Figure 2-3: Organ damage as assessed by SDI...... 50

Figure 3-1: CD123 expression is highest on pDCs and basophils in SLE, autoimmune and healthy donors...... 68

Figure 3-2: pDCs and basophils are depleted by CSL362...... 71

Figure 3-3: IL-3 blockade with Fab’CSL362 depletes pDCs, but not basophils, at higher doses...... 72

Figure 3-4: No depletion of CD123lo cells with CSL362 treatment...... 73

Figure 3-5: CSL362 activates NK cells, via its modified IgG1-Fc...... 75

Figure 3-6: CSL362 potently and specifically inhibits TLR7- and TLR9-induced IFNα production and IFNα-inducible gene expression...... 76

Figure 3-7: Depletion of pDCs, not basophils, by CSL362 inhibits TLR7- and TLR9- induced IFNα production...... 77

Figure 3-8: IFN-inducible gene expression is higher in SLE donors compared to healthy controls...... 78

Figure 3-9: CSL362 potently inhibits TLR7- and TLR9- induced IFNα production and IFN-inducible gene expression in SLE, autoimmune disease, and healthy donors...... 80

Figure 3-10: CSL362 inhibits SLE serum-induced IFNα production and IFN-inducible gene expression from healthy donor PBMCs...... 82

Figure 3-11: CSL362 more effectively inhibits TLR7- and TLR9-stimulated IFNα production and IFN-upregulated gene expression compared to type I IFN blockade. ... 84

Figure 3-12: Activation of pDCs with TLR7 and TLR9 agonist stimulation upregulates type I and type III IFN gene expression...... 85

Figure 3-13: CSL362 inhibits TLR7- and TLR9- stimulated type III IFN (IFNλ), but not type II IFN (IFNγ) production...... 86

xx Figure 3-14: Expansion of plasmablasts is maximal after six days of stimulation with TLR7 and TLR9 agonists...... 88

Figure 3-15: CSL362 inhibits TLR7- and TLR9-induced plasmablast expansion...... 89

Figure 3-16: Inhibition of plasmablast expansion by CSL362 is more effective in healthy donors compared to SLE donors...... 90

Figure 3-17: TLR9 induces greater plasmablast expansion than TLR7...... 91

Figure 3-18: CSL362 inhibits proliferation of memory B cells and plasmablasts, but not naïve B cells...... 92

Figure 3-19: Reconstitution of pDCs, but not basophils, into CSL362 pre-treated cultures, restores plasmablast expansion...... 94

Figure 3-20: Conditioned medium from activated pDCs restores plasmablast expansion that is inhibited by CSL362...... 95

Figure 3-21: IFNα, IL-6 and TNF-α are elevated in conditioned medium produced by stimulating pDCs with TLR9 agonist CpG C...... 96

Figure 3-22: Inhibition of IFNα and IL-6, but not TNF-α, by neutralizing mAbs, prevents restoration of plasmablast expansion with TLR9-stimulated pDC CM, in CSL362 pre-treated cultures...... 97

Figure 3-23: CSL362 more effectively inhibits TLR7- and TLR9-, but not CD40L- induced plasmablast expansion than IFNα blockade alone...... 99

Figure 3-24: CSL362 more effectively inhibits TLR9-induced plasmablast expansion compared with IFNAR blockade...... 100

Figure 3-25: No difference in immunoglobulin levels after CSL362 treatment...... 101

Figure 3-26: Subcutaneous administration of CSL362 to cynomolgus macaques depletes pDCs and basophils, and inhibits TLR9-induced IFN-upregulated gene expression...... 104

Figure 4-1: Serum IL-3 levels in SLE patients and healthy controls...... 121

xxi Figure 4-2: Decreased pDCs, basophils, naïve and memory B cells, CD4+ and CD8+ T cells in SLE compared to healthy donors...... 129

Figure 4-3: Correlation between serum IL-3 levels and pDCs and basophils in SLE patients...... 130

Figure 4-4: Altered serum levels of BAFF, GM-CSF, IL-2, IP-10 and IL-13 in SLE patients compared to healthy donors...... 132

Figure 4-5: Serum IL-3 levels correlate with IFNα levels in SLE and healthy donors...... 134

Figure 4-6: Serum IL-3 levels correlate with type III IFN levels in SLE patients and healthy donors...... 135

Figure 4-7: Serum IL-3 levels correlate with IL-4 and IL-5 levels in SLE and healthy donors...... 136

Figure 4-8: Longitudinal serum IL-3, IFNα, IFNλ and BAFF levels in SLE patients...... 139

Figure 4-9: Serial serum IL-3 levels correlate with IFNα and type III IFN in SLE patients over time...... 140

Figure 4-10: Numbers of differentially expressed genes between IL-3 stimulated, or non-stimulated lysed whole blood from healthy donors...... 141

Figure 4-11: Expression of IL-3 regulated genes in SLE and healthy donors ...... 146

Figure 4-12: Expression of IFN regulated genes in SLE and healthy donors ...... 148

Figure 4-13: Correlation between IL-3 and IFN gene signature scores in SLE and healthy donors...... 149

Figure 5-1: Proposed mechanism by which anti-IL-3Rα mAb CSL362 may be therapeutically useful in SLE ...... 160

xxii Abbreviations

Ab antibody ADCC antibody-dependent cell-mediated cytotoxicity AML acute myeloid leukemia APC antigen presenting cell APRIL a proliferation-inducing ligand BAFF/BLyS B-cell activating factor/B lymphocyte stimulator BCR B cell receptor CI confidence interval CNS central nervous system CRP C-reactive protein CTLA cytotoxic T-lymphocyte antigen DC dendritic cell DRESS drug reaction with eosinophilia and systemic symptoms ELISA enzyme-linked immunosorbent assay ER endoplasmic reticulum ESR erythrocyte sedimentation rate GM-CSF granulocyte-macrophage colony-stimulating factor ICOS inducible T-cell costimulator IFN interferon IFNAR interferon α/β receptor/type I interferon receptor IFNGR IFNγ receptor/type II interferon receptor Ig immunoglobulin IL interleukin IRAF interleukin receptor-associated kinase IRF interferon regulatory factor JAK Janus kinase mAb monoclonal antibody M-CSF macrophage colony-stimulating factor mDC myeloid derived dendritic cells MHC major histocompatibility complex

xxiii MIF macrophage migration inhibitor factor MMP matrix metalloproteinase NETs neutrophil extracellular traps NK natural killer PBMC peripheral blood mononuclear cell pDC plasmacytoid dendritic cell PRR pattern recognition receptor RIG-I retinoic-acid-inducible gene I RLRs RIG-I-like receptors SDI Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index SLE Systemic lupus erythematosus SLEDAI SLE Disease Activity Index STAT signal transducer and activator of transcription STING stimulator of interferon genes SYK spleen tyrosine kinase TACI transmembrane activator and calcium-modulating ligand interactor TBK1 TANK-binding kinase 1 TCR T cell receptor Tfh T follicular helper Tg transgenic Th T helper TLR toll-like receptor TNF tumour necrosis factor Treg T regulatory cell TWEAK TNF-like weak inducer of apoptosis TYK tyrosine kinase WBC white blood cell

xxiv

Chapter 1: Introduction

1.1 Systemic lupus erythematosus - a complex chronic autoimmune disease for which novel therapies are required

Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune disease that predominantly affects women of childbearing age. Its prevalence is reported to range between 20-150/100,000 and its incidence from 1-10/100,000;1 the latter has risen in recent years, most likely due to better detection of milder disease. The incidence of SLE is higher in certain ethnic groups, such as Asians, Hispanics, African Americans, and Australian Aborigines. The disease usually follows a relapsing-remitting course. Regarded as a prototypic autoimmune disease, the pathogenesis of SLE is complex, and still being elucidated, however, is thought to result from the interplay of a variety of immunological, genetic and environmental factors.

A fundamental derangement of the immune system in SLE is loss of self tolerance, with autoreactive B cells producing autoantibodies which form immune complexes with self-antigens. A characteristic feature of SLE immune complexes is that they contain autoantibodies directed against self nucleic acids. These immune complexes deposit in various organs, causing inflammation and tissue damage. Almost any organ system can be affected, with disease manifestations and severity displaying heterogeneity within and between patients, ranging from the more common involvement of skin and joints, to life threatening renal or central nervous system (CNS) lupus. This heterogeneity presents challenges in the diagnosis and management of SLE, and also for the design of SLE clinical trials. SLE causes significant morbidity and an increased risk of mortality,2 despite currently available treatments. It is also responsible for a large economic burden, with estimated direct and indirect (for example, due to loss of work capacity) costs per patient of up to $28,000 US dollars per year.3

1 SLE has traditionally been treated with non-specific immunosuppression, including corticosteroids and cyclophosphamide. To date, B cells have been the major targets of specific biologic therapies in SLE, given the importance of autoantibodies in driving the pathogenesis. However, other promising therapeutic targets have emerged, many of which are involved in a plasmacytoid dendritic cell (pDC)-type I interferon (IFN) pathway. The most advanced in development of these therapeutics targeting the IFN pathway are monoclonal antibodies (mAbs) that block type I interferon (IFNα) or its receptor (IFNAR); the latter has commenced a phase 3 clinical trial (NCT02446899). However, recent data suggests that alternate ways of modulating the interferon pathway, such as by targeting the primary IFN-producing cell, the pDC, may also be feasible.

This thesis develops the pre-clinical rationale for a novel mAb (known as CSL362) that targets the interleukin (IL)-3Rα (CD123). The IL-3Rα is strongly expressed on cell types relevant to SLE, including pDCs and basophils. Modulation of these pathogenic cell types with CSL362 may provide a new therapeutic approach in SLE.

1.2. Overview of SLE immunopathogenesis

1.2.1 The immunopathogenesis of SLE

The pathogenesis of SLE is complex (Figure 1-1), and as yet not fully elucidated; however, abnormalities in almost every aspect of the immune system have been documented. SLE has traditionally been considered to be caused by cells of the adaptive immune system.4 However, it has become evident that aberrations in the innate immune system, including in dendritic cells (DCs) and phagocytes, are also important, as these cells contribute to the production, processing and presentation of autoantigens that might initiate or perpetuate disease.

2

Macrophage

TLR7 TLR9 Impaired phagocytosis Type I IFN

Monocyte

pDC

mDC Apoptotic cells Neutrophil NETs

Autoantigens - self nucleic acids T cell BAFF APRIL B cell Immune B and T cell autoreactivity complexes Autoantibodies

Tissue Immune complex deposition, complement activation, inflammation and organ damage

Figure 1-1: Overview of SLE pathogenesis.

Impaired phagocytosis and/or increased NET (neutrophil extracellular trap) formation results in a higher burden of apoptotic material in SLE, increasing exposure of potential autoantigens to the immune system. A pathological cascade is triggered, with interaction between autoreactive T and B cells leading to the production of autoantibodies. These form immune complexes with self-antigens, depositing in tissues and causing inflammation and organ damage. Type I interferon is produced by plasmacytoid dendritic cells (pDCs) activated by self nucleic acids contained in immune complexes, or released by dying neutrophils. Figure from Oon et al, Clin Trans Immunol 2016.5

3 During apoptosis, there is transient expression of autoantigens on apoptotic cell membranes and the generation of apoptotic cell debris. In SLE, there is an increased burden of apoptotic material, with elevated levels of circulating DNA, RNA and nuclear proteins.6 Why this occurs is incompletely understood, but these host-derived molecules can be recognized as antigenic by the immune system, triggering an inflammatory cascade. In humans, the increased apoptotic burden might result from impaired phagocytosis,7 with decreased phagocytosis observed in SLE monocyte- derived macrophages in vitro;8 increased apoptosis of lymphocytes, neutrophils and macrophages has also been observed.9-11

Autoantigenic material is opsonized by autoantibodies and internalized as immune complexes by dendritic cells after binding cell surface Fc receptors. Binding of immune complexes to Fc receptors can stimulate the immune system through receptor cross-linking. Pattern recognition receptors (PRRs) such as the toll-like receptors (TLRs) are also activated by self nucleic acids contained within immune complexes. Of particular relevance in SLE are TLR7 and TLR9, which are intracellular TLRs that recognize RNA and DNA respectively. Both TLR7 and TLR9 are expressed by pDCs and activation initiates the release of type I IFN. A specific form of cell death in neutrophils, called NETosis, results in the release of NETs (neutrophil extracellular traps), that are meshwork-like structures containing chromatin and neutrophil-derived proteins with anti-microbial activity. Some SLE patients display abnormal NET accumulation, possibly due to low DNase (deoxyribonuclease) I activity, which is the main enzyme responsible for NET clearance in humans.12 NETs have also been shown to trigger TLR7 and TLR9. Other sources of DNA and RNA have been implicated in lupus pathogenesis, including chromatin in microparticles of apoptotic cells,13 and oxidized mitochondrial DNA released by activated neutrophils.14, 15

Normally, immature DCs present self-antigens in the absence of costimulatory signals, inducing a tolerogenic effect on autoreactive lymphocytes. However, in SLE, self-antigen presentation can occur in the presence of costimulatory signals.16 T cell hyperreactivity has also been reported, due to exaggerated intracellular calcium influx, resulting from abnormalities in the T cell receptor (TCR) signaling pathway. These include accelerated tyrosine phosphorylation of signaling intermediates and decreased expression of the TCR CD3ζ chain, with increased expression of the Fc

4 receptor gamma chain. The latter recruits spleen tyrosine kinase (SYK) in preference to the normally recruited ZAP70.17 Exaggerated T helper (Th)1, Th2 and Th17 responses can occur, as can a reduced ability to suppress autoreactive T cells due to a decrease in, or defective function of, T regulatory cells (Tregs).17

B lymphocytes play a central role in SLE, due to the production of pathogenic autoantibodies against soluble and cellular components, which form immune complexes that subsequently deposit in various organs and cause tissue damage. However, B cells can also act as antigen presenting cells (APCs), presenting autoantigens to activate T cells.18, 19 Similar to T cells, B cells have been reported to exhibit hyperactivation in SLE, with augmented calcium influx following cross- linking of the B cell receptor (BCR), and increased antigen-receptor mediated phosphorylation of downstream protein tyrosine residues observed.20, 21 Elevated levels of cytokines that influence B cell activation, proliferation and survival have been documented in SLE, such as BAFF/BLyS (B-cell activating factor/B lymphocyte stimulator) and APRIL (a proliferation-inducing ligand).22, 23 Additionally, TLR activation in B cells by RNA and DNA-associated antigens may also promote B cell activation and differentiation.24

1.2.2 Genetic, environmental and hormonal abnormalities in SLE

Monozygotic twins have a higher rate of concordance (34%) than dizygotic twins (3%) for SLE and more than eighty genes have been identified that increase SLE susceptibility. These include genes in the interferon pathway, including IRF5 (interferon regulatory factor 5), IFIH1 (interferon-induced helicase C domain- containing protein 1) and STAT4 (signal transducer and activator of transcription 4).25 The greatest genetic risk for SLE is conferred by deficiencies of complement pathway components C1q, C4A and B, and C2. Low complement activity contributes to defective phagocytosis of apoptotic material. Damaging mutations in the TREX1 gene, which encodes a 3’ repair endonuclease, causes accumulation of DNA. These single gene defects are, however, relatively rare, and susceptibility in most patients probably results from a combination of common variations in multiple genes. The most common genetic predisposition occurs at the major histocompatibility complex

5 (MHC) locus, with susceptibility loci including HLA-DR2, HLA-DR3 and HLA- DRB1.26 Other genes involved in immune regulation have been implicated in SLE. These include those which affect the function or survival of T or B cells (including PD-1 [programmed cell death protein 1], LYN and BLK [tyrosine kinases], TNFSF4 [tumour necrosis factor superfamily member 4], PTPN22 [protein tyrosine phosphatase, non-receptor type 22] and BANK-1 [B-cell scaffold protein with ankyrin repeats 1]), or are involved in immune complex clearance (FcγRIIa, ITGAM [integrin alpha M] and complement components). Other predisposing genes and microRNAs influence DNA methylation and hypomethylation, which can alter the apoptotic clearance rate or increase inflammatory cytokine levels, or autophagy, which may influence the survival of autoreactive B cells and plasma cell differentiation in SLE.27

A number of hormonal and environmental factors are thought to perpetuate SLE. Given the strong female skewing of the disease, female sex hormones have been implicated. Increased oestrogen, and decreased androgen, levels have been found in females with SLE, and inflammatory cytokine production is elevated in DCs, T cells and B cells exposed to oestrogens.1 Disease-susceptibility genes on the X chromosome have also been identified.28 UV light is a recognized trigger of disease, as it induces apoptosis in keratinocytes and release of pro-inflammatory cytokines. Infections are also postulated to contribute to SLE through molecular mimicry, for example between EBV (Epstein Barr virus) nuclear antigens and self-antigens. A number of medications, including the anti-tumour necrosis factor (TNF)-α mAbs used to treat other autoimmune diseases, can induce autoantibodies typical of SLE. While the development of overt disease is uncommon, these observations suggest that cytokine cross regulation can influence susceptibility to SLE.1

1.3. Current therapeutics in SLE

Treatment of SLE has traditionally involved non-specific anti-inflammatory or immunosuppressive medications. Non-steroidal anti-inflammatory drugs and the immunomodulatory agent hydroxychloroquine, which is a TLR7/TLR9 antagonist, are used for milder disease, and stronger immunosuppressants such as methotrexate, azathioprine, mycophenolate or cyclophosphamide are employed for major organ involvement. Corticosteroids are generally used to treat flares of disease, although are

6 often continued long term.29 However, this conventional approach to treatment is ineffective in many patients, and can be associated with many dose-limiting toxicities and undesirable side effects.30

1.3.1 B cells are a major therapeutic target in SLE

The B cell has been a major therapeutic target in SLE, due to its importance in producing pathogenic autoantibodies. There are two main therapeutic approaches employed to target B cells in SLE (Table 1-1). One involves targeting cells for depletion using mAbs directed against B lymphocyte markers such CD19, CD20 and CD22, which are expressed on immature and mature B cells.31 These cells are the precursors of plasma cells, which produce pathogenic autoantibodies. The second approach involves decreasing the survival of B cells through targeting B cell survival factors such as BAFF and APRIL.

The only two biologic agents to have entered clinical use in SLE are both B cell targeted mAbs. The first of these is rituximab, a mAb targeting B cells for depletion, via binding to CD20, which is expressed on all stages of B cells up to and including memory B cells. Despite failing to meet primary endpoints in two phase 3 trials of renal32 and non-renal33 lupus, rituximab remains part of the therapeutic armamentarium for refractory disease, due to apparent efficacy in earlier phase and post marketing studies.34, 35 Clinical trials involving rituximab continue, with a long- term phase 3 trial utilising remission in lupus nephritis as an endpoint (RING, trial number NCT01673295), and another with concomitant mycophenolate use in a steroid minimising regimen (RITUXILUP, NCT01773616) currently recruiting. The second mAb, belimumab, which targets BAFF/BLyS, was the first drug in fifty years to be specifically approved for use in SLE (by the US FDA in 2011). However, the benefits of belimumab in two phase 3 trials were modest.36, 37 It was not evaluated in more severe disease, as initial trials excluded severe lupus nephritis and CNS lupus. Such trials are now underway, with a clinical trial of belimumab in lupus nephritis due to be completed in 2019 (NCT01639339). In Australia, belimumab is only accessible via clinical trial or compassion access.

7 Table 1-1: B cell targeted therapies in SLE

Trial number Target Drug Name Progress and references

B cell depleting agents

Phase 3 trials in renal and non- 32, 33 renal lupus completed Anti-CD20 mAb Rituximab NCT01673295, (endpoints not met). Phase 3 NCT01773616 trials in lupus nephritis ongoing

Phase 3 completed – endpoints Anti-CD22 mAb Epratuzumab 38 not met

Anti-CD20 mAb Phase 2 trial in lupus nephritis (modified for enhanced Obinutuzumab NCT02550652 recruiting cytotoxicity)

Anti-CD20 mAb and Rituximab and Phase 3 trials in lupus nephritis NCT02260934, anti-BAFF mAb Belimumab currently recruiting NCT02284984

Anti-CD19 mAb XmAb5871 Phase 2 trial currently recruiting NCT02725515

B cell survival inhibitors

Phase 3 trials completed (severe lupus nephritis and CNS lupus 36, 37 Anti-BAFF mAb Belimumab excluded) NCT01639339 Phase 3 trials in lupus nephritis underway

Phase 3 trials completed – one of two studies met primary Anti-BAFF mAb Tabalumab endpoint, however decision 39 made by drug company not to pursue development further

Phase 2/3 trials terminated Anti-TACI fusion Atacicept prematurely due to safety 40 protein concerns

Anti-BAFF NCT01395745, Blisibimod Phase 3 trials recruiting peptidobody NCT02514967

8

More recently, several mAbs that had encouraging outcomes in phase 2 trials were reported to have failed in phase 3. These include epratuzumab, a mAb targeting CD22 on B cells38 and tabalumab, an anti-BAFF mAb.39 Trials of atacicept, a fusion protein which blocks the activity of two B cell survival factors, BAFF and APRIL, by binding to their common cell surface receptor TACI (transmembrane activator and calcium- modulating ligand interactor) were terminated prematurely in phase 2/3 due to safety concerns; however, the data showed a trend to benefit for the higher dose tested.40 Some of these failures may have been due to issues with study design, including the use of different primary endpoints and the influence of background therapies such as steroids and other immunomodulators.41

Studies of B cell directed agents continue, with blisibimod, an anti-BAFF peptidobody (in which high affinity BAFF binding peptides are fused to an IgG Fc fragment), progressing to phase 3 trials (NCT01395745 and NCT02514967). An anti- CD19 Fc-engineered mAb, XmAb5871, is currently recruiting in a phase 2 trial (NCT02725515). This antibody binds to both CD19 and FcγRIIb, a receptor that inhibits B cell activation. XmAb5871 decreased B cell proliferation and costimulatory molecule expression ex vivo in SLE patients and healthy donors, and increased the survival of mice engrafted with peripheral blood mononuclear cells (PBMCs) from an SLE patient.42 Interestingly, the phase 2 trial of XmAb5871 aims to address the problem of high background immunosuppression in SLE trials. SLE patients with moderate to severe, but non-organ threatening, disease have their ineffective background immunosuppressives discontinued during screening, and intramuscular steroids are used to treat symptoms. Other B cell targeted agents have had modifications made to improve efficacy, such as obinutuzumab, an anti-CD20 mAb engineered for enhanced cytotoxicity, which is currently recruiting for a phase 2 trial in lupus nephritis (NCT02550652).

Therapeutic approaches targeting long-lived memory plasma cells are gaining interest, as these cells reside in survival niches in the bone marrow and inflamed tissues, where they contribute to disease chronicity by secreting autoantibodies independently of antigen contact.43 Conventional immunosuppressive agents and traditional B cell

9 targeting therapies do not affect long-lived memory plasma cells. Presently, complete depletion of this cell type can only be achieved with an immunoablative regimen, such as anti-thymocyte globulin (ATG). This approach, in the context of autologous haematopoietic stem cell transplantation, which reconstitutes a tolerant immune system, has some evidence for its success in SLE.44 Proteasome inhibitors provide partial but more selective depletion of plasma cells compared to immunoablative regimens. Bortezomib, which is already utilized for treatment of multiple myeloma, a disease of malignant plasma cells, drives plasma cells with high rates of immunglobulin (Ig) synthesis into apoptosis due to the accumulation of unfolded proteins.45 Bortezomib therapy resulted in near complete plasmablast and plasma cell depletion in NZB/W mice, with a reduction in anti-dsDNA antibodies and nephritis, and an increase in survival.46 Peripheral blood and bone marrow plasma cells were also decreased by bortezomib in a pilot study in human SLE, and treatment led to a decrease in serum anti-dsDNA antibody levels and clinical disease activity scores.47 A phase 2 trial of bortezomib in SLE is currently underway (NCT02102594). Other potential ways of modulating plasma cells in SLE, such as by disrupting the interaction of CXCR4-CXCL12 that is crucial for homing of plasma cells to the bone marrow, or by targeting cell surface molecules such as CD38, are still in pre-clinical stages of development.43, 48, 49

The use of more than one therapeutic agent may be a more effective approach to B cell inhibition, with some data from mouse models supporting the combined strategy of B cell depletion and inhibition of B cell survival. Post rituximab flares of disease activity characterised by high anti-dsDNA antibody levels have been associated with increases in circulating BAFF levels, and increased plasmablast numbers in the B cell pool.50 The use of an anti-CD20 mAb, together with a plasma-cell depleting agent51 or anti-BAFF agent52 in NZB/W F1 mice resulted in greater improvements in disease compared to B cell depletion alone. In the human setting, there have been case reports of successful combined use of rituximab and belimumab,53 and clinical trials utilizing this approach to B cell inhibition in lupus nephritis (CALIBRATE, NCT02260934 and NCT02284984) are currently underway.

10 However, although there has been some success with B cell directed agents, these have not, to date, provided a definitive solution to SLE, and there is increasing interest in several non-B cell related therapeutic targets in this disease.

1.4. Type I interferon - a promising therapeutic target in SLE

Type I IFNs, and in particular IFNα, have emerged as key pathogenic cytokines in SLE. In humans, type I IFNs comprise at least thirteen IFNα subtypes, in addition to IFNβ, IFNε, IFNκ and IFNω. Type I IFNs have antiviral, antiproliferative and immunomodulatory effects. Signaling through the canonical type I IFN pathway is initiated upon binding to the ubiquitously expressed type I IFN receptor (interferon α/β receptor - IFNAR) - a receptor complex consisting of two transmembrane proteins, IFNAR1 and IFNAR2, activating two cytoplasmic tyrosine kinases, JAK1 (Janus kinase 1) and TYK2 (tyrosine kinase 2). Following IFNAR binding, STAT1 and STAT2 proteins undergo JAK-mediated tyrosine phosphorylation. Together with IRF9, these form the ISGF3 (interferon-stimulated gene factor 3) transcription factor complex, which translocates to the nucleus and binds to IFN-stimulated response elements (ISREs) in the promoter regions of IFN-inducible genes.25

Type I IFNs induce an array of biological effects that can augment autoimmunity through altering the function of key effector cells such as B cells, T cells and DCs (Figure 1-2). For example, in vitro, IFNα promotes the differentiation of autoreactive B cells into Ig-secreting plasma cells,54 and upregulates the expression of BAFF and APRIL on myeloid derived DCs (mDCs),55 promoting B cell survival. IFNα in SLE serum induces mDC differentiation when cultured with CD34+ haematopoietic precursors56 and monocytes57 in vitro, and upregulates MHC class II, CD80 and CD86, thereby enhancing T cell costimulatory capacity.54 IFNα-primed naïve CD8+ T cells have been shown to undergo proliferation and acquire effector functions in C57BL/6 mice,58 and type I IFN also stimulates mDCs to induce differentiation of naïve T cells into helper T cells.54 Additionally, IFNα causes human Treg inactivation in vitro by downregulating intracellular cAMP and negatively regulating TCR signaling,59 and stimulates the generation of lymph-node resident Tfh (T follicular

11 helper) cells in mice.60 IFNα has also been shown to prime neutrophils for apoptosis in response to autoantibodies, promoting the release of NETs.61

Neutrophil Tfh cell

Monocyte

pDC NETs Type I IFN

BAFF mDC

APRIL Immune complexes B cell T cell

Autoantibodies

Figure 1-2: Effects of type I interferon which promote autoimmunity.

Type I interferon is produced by pDCs upon stimulation with self nucleic acids contained in immune complexes or neutrophil extracellular traps (NETs). It induces monocyte differentiation into dendritic cells, and myeloid dendritic cells to upregulate MHCII and costimulatory molecules to activate autoreactive T cells. BAFF and APRIL expression are upregulated, which promotes autoreactive B cell survival and proliferation. Type I IFN also stimulates the generation of lymph node resident Tfh cells (follicular T helper cells) and induces autoreactive B cells to differentiate into autoantibody producing plasma cells. Neutrophils are primed by type I IFN to undergo apoptosis, causing the release of NETs. Figure from Oon et al, Clin Trans Immunol 2016.5

12 Observational data studies suggested a link between IFNα and SLE, with IFN therapy for malignancy and viral hepatitis in humans sometimes inducing an SLE-like syndrome.62-64 Numerous findings from both animal models and human studies subsequently confirmed a central role for this cytokine in SLE. As early as the late 1970s, increased serum levels of type I interferon (mainly IFNα) were shown to be associated with SLE.65 These findings were confirmed in subsequent studies of both adult and paediatric SLE, with ~50-90% of patients demonstrating elevated serum IFNα levels detected with various methods including ELISA (enzyme-linked immunosorbent assay), sandwich immunoradiometric assay, and assessment of antiviral activity in response to viral challenge following incubation of SLE serum with a cell line or cell cultures.66-71 Some studies have found an association of elevated type I IFN levels with certain disease manifestations, conventional markers of disease activity and other serum cytokine levels. These have included the association of elevated type I interferon levels with cutaneous lupus or vasculitis, C3 levels, anti-dsDNA antibody levels and serum IL-10 levels.70, 71 The importance of type I IFN in SLE has also been reflected at a transcriptional level, discovered with the advent of high throughput transcriptional profiling techniques, with a type I IFN gene signature found to be present in 50-70% of adult SLE, and the majority of paediatric SLE patients.72, 73 This signature correlates with increased disease activity and can be modulated by treatment.73 In addition, elevated serum levels of IFN- upregulated cytokines in human SLE have been found,74 and genome wide association studies have identified susceptibility loci in the IFN signaling pathway.75 IFNα promoted disease, and IFNAR deficiency ameliorated disease in NZB/W lupus prone mice.76-78

IFNα (rather than the other type I IFNs) may be the major contributor to SLE pathogenesis: IFN-inducible gene expression upregulated by SLE patient serum in healthy donor PBMCs was neutralized by treatment with either an anti-IFNα mAb or an anti-IFNAR mAb;79 and SLE serum-induced IFN-upregulated gene expression from a cell line was neutralized by anti-IFNα, but not anti-IFNβ (or anti-IFNγ) mAbs.80 Compared to IFNα, there is relatively less data regarding the role of the other type I IFNs in SLE. IFNβ therapy for multiple sclerosis induces genes found in the SLE IFN gene signature,81 and there is evidence that IFNβ also contributes to the SLE

13 gene signature.82 IFNω transcripts were found to be elevated in SLE patients compared to healthy controls in microarray studies,83 and anti-IFNω autoantibodies have been found in SLE patients.84 Collectively, these studies demonstrate activation of the type I IFN pathway, particularly IFNα, in both human and murine SLE and raise the possibility of therapeutic blockade of this pathway in SLE.

Several anti-type I IFN therapeutics have undergone evaluation in clinical trials. The anti-IFNα mAbs, sifalimumab and rontalizumab, have completed phase 2 trials, as has anifrolumab, an anti-IFNAR mAb, which antagonises all type I IFN subtypes.85-87 The results of these phase 2 trials have been promising, with reduction of clinical disease activity measures and suppression of the IFN gene signature. The trials revealed an acceptable safety profile, although increased herpes zoster reactivation was observed. Interestingly, a small study in Japanese patients indicated that neutralization of IFNAR, rather than IFNα alone, may be more efficacious, with superior suppression of a 21 IFN gene signature seen with anifrolumab compared to sifalimumab.88 To date, only anifrolumab has progressed to a phase 3 trial, which is currently recruiting patients (NCT02446899).

Alternate therapies that target type I IFN include the anti-IFNα monoclonal antibody, ASG-009, which was well tolerated and effective in neutralizing a 27 IFN gene signature in a phase 1 trial,89 and an IFNα kinoid (IFN-K) vaccine composed of IFNα2b coupled to a carrier protein, that induces polyclonal anti-IFNα neutralizing antibodies. Recently reported results of a phase 1/2a trial of IFN-K showed induction of anti-IFNα antibodies that were associated with decreased expression of both IFN- induced and B cell activation-associated gene transcripts, without significant adverse events.90 A larger phase 2b trial is currently recruiting (NCT02665364).

However, as with B cell targeted therapeutics there is a population of SLE patients who have not apparently responded to therapeutics targeting type I IFN. It may be that blockade of common components of the different IFN signaling pathways, and thereby inhibition of multiple IFN types, would be more effective. However, as SLE is a highly heterogeneous disease, biomarkers that predict which patients may benefit from particular therapeutic approaches is an important objective. For example, those

14 with higher expression of a panel of IFN-inducible genes at baseline were found to have a greater response rate to anifrolumab in a phase 2 trial.87

Interestingly, there is evidence suggesting that targeting of the interferon pathway early in the course of disease may be more effective. In pre-autoimmune BXSB mice, the development of autoantibodies, hypergammaglobulinemia and glomuleronephritis was decreased with pDC depletion, which was associated with decreased IFN- inducible gene transcription.91 Another study showed that an anti-IFNα/β receptor blocking antibody had a protective effect, but only in young BXSB mice.92 Additionally, a rise in anti-RNP autoantibodies and proteinuria was prevented in young MRL/lpr mice that had been treated with prophylactic IFN receptor blockade.92 It may be that the best results from interferon targeting therapeutics will occur in patients with early disease, due to the prevention of key processes leading to B cell activation and autoantibody production.

1.4.1 Other interferon types contribute to SLE pathogenesis

Although the strongest evidence exists for the contribution of type I IFN to SLE, other IFN types may contribute.

Type II interferon, or IFNγ, signals through a different receptor complex, the IFNγ receptor (IFNGR), which is expressed by most cell types. Activation of IFNGR leads to phosphorylation of STAT1 homodimers and subsequent expression of genes containing IFNγ-activated sites.48 Although the receptor for IFNγ is distinct to type I IFNs, the downstream signaling pathways overlap (Figure 1-3).

Like type I IFN, the administration of IFNγ in humans can induce an SLE-like disease,93 and there are a number of murine, and in vitro human studies, that support a pathogenic role for this cytokine in SLE. Administration of IFNγ to NZB/W F1 mice augmented disease, whilst neutralization ameliorated disease and improved survival.94 Similarly, IFNγ receptor deletion inhibited autoantibody production and nephritis. In a different murine model of SLE, resulting from the genetic deletion of Lyn kinase, IFNγ deletion reduced BAFF production, myeloid proliferation and T cell

15 hyperactivation, reducing glomerular disease.94 In human SLE, elevated serum IFNγ levels and IFNγ transcripts in PBMCs have been observed compared to healthy controls.94 Both NK cells and T cells from SLE donors produce more IFNγ, which can induce BAFF production by monocytes.94 A recently published phase 1 clinical trial of an anti-IFNγ mAb, AMG811, showed it was well tolerated and reduced IFNγ- related gene expression.93

Type III IFNs Type I IFNs Type II IFN IFNλ1 (IL-29) IFNα IFNβ IFNγ IFNλ2 (IL-28A) IFNω IFNκ IFNε

IFNλ3 (IL-28B) IFN- IFN- IFN- IFN- IFN- IFN- IFNλ4 γR2 γR1 γR1 γR2 αR1 αR2

IFN- IL-10R2 IFNGR IFNAR λR1 IFNLR Jak2 Tyk2 Jak1 Jak2 Jak1 Tyk2 Jak1 Jak1

STAT1 STAT2 ISGF3 STAT1 STAT1 IRF9

Nucleus ISREs GAS

Figure 1-3: Overlapping type I, II and III IFN signaling pathways.

Type I, II and III IFNs signal via distinct receptors (IFNAR, IFNGR and IFNLR respectively) with signal transduction mediated through JAK/STAT activation. The downstream signaling pathways of the different interferons overlap, resulting in the production of interferon stimulated genes following activation of transcriptional response elements in the nucleus (ISRE – IFN stimulated response elements, and GAS - IFNγ activated sites). Figure from Oon et al, Clin Trans Immunol 2016.5

16

Type III IFNs are the most recently discovered IFN type and include IFNλ1 (IL-29), IFNλ2 (IL-28A), IFNλ3 (IL-28B) and IFNλ4.95 Unlike type I IFN, which is produced mainly by pDCs, type III IFNs are produced by a variety of cell types, including pDCs, Tregs, macrophages and hepatocytes. Type III IFNs signal through a heterodimeric receptor comprised of IFNλR1 (IL-28RA) and IL-10R2 subunits, with subsequent activation of the JAK/STAT cascade, similar to that seen with type I IFNs (Figure 1-3). Thus, type III IFNs may share biological activities with type I IFNs through the induction of the ISGF3 transcriptional complex, and have been postulated as a potential explanation for partial responses to type I IFN blockade.95 However, type III IFNs have a narrower range of effects, due to the limited expression of its receptor on epithelial, and some haematopoietic cells,95 and therefore may contribute less to the peripheral blood IFN gene signature in SLE.25

To date, there are limited data suggesting dysregulation of the type III IFN pathway in SLE. Serum IFNλ2 was elevated in a greater percentage of SLE patients compared to healthy donors,96 and serum IFNλ1 levels were associated with disease activity, the presence of anti-dsDNA antibodies, glomerulonephritis and arthritis.97 Elevated IFNλ2 mRNA transcripts were found in activated CD4+ T cells from lupus patients compared with healthy controls.96 Currently, there are no therapeutics that specifically target type III IFNs in clinical trials for autoimmune diseases, although the dual blockade of type I and type III IFNs, for example, by the depletion of pDCs (which produce both), may reduce type III IFNs. Blockade of the IFNAR or type I IFN itself may also reduce type III IFN, as reduced type III IFN production has been observed in IFNAR-deficient mice98 and type III IFN has been shown to be induced by type I IFN in vitro.99

1.4.2 Emerging strategies to modulate the type I IFN pathway

The therapeutics so far described inhibit only the type I IFNs or type II IFN. However, there is significant overlap in the signaling pathways of the different IFN subtypes and gene expression analyses suggest that the IFN signature is driven by both type I and type II IFNs in many patients,93 although, type III interferon may also

17 contribute. Therefore, the therapeutic targeting of IFN-producing cells, or inhibition of common components of the signaling cascade utilized by the different IFN types, such as the JAK-STAT pathway, may confer a therapeutic advantage over targeting individual IFN types alone, by more complete suppression of IFN-related processes. Such therapeutic strategies have been investigated in pre-clinical studies, and some have progressed to early phase clinical trials (Figure 1-4 and Table 1-2).

Self DNA/RNA

Immune Nucleic acid inhibitors complexes (recombinant DNase1, RSLV-132) IFNAR TLR7/9 inhibitors (DV-1179, IMO-3100, IMO-8400, CpG-52364) TLR7 TLR9 Tyk2Jak1 Jak1

JAK inhibition (Tofacitinib, Endosome GSK2586184, Baricitinib, R333) MyD88 Type I IFN MyD88 inhibitor STAT (ST-2825)

pDC BDCA2 IFN responsive cell Anti-BDCA2 mAb pDC depletion/survival (BIIB059) (ABT-199, bortezomib)

Stage of development Preclinical Phase I clinical trial Phase II clinical trial

Figure 1-4: Emerging strategies to target the type I IFN pathway.

Therapeutics targeting various aspects of the interferon pathway are in different stages of development, ranging from those that target the IFN producing cell, the pDC, to various parts of the IFN signaling machinery. Figure adapted from Oon et al, Clin Trans Immunol 2016.5

18 Table 1-2: Emerging strategies to target the type I IFN pathway

Trial Target Drug Name Progress number/references

TLRs

TLR7/9 oligonucleotide DV-1179 Phase 1b/2a – completed 100 inhibitor

TLR7/9 oligonucleotide IRS-954 Pre-clinical 101, 102, 103 inhibitor

Pre-clinical in SLE TLR7/9 oligonucleotide IMO-3100 Phase 2 completed in 104, 105 inhibitor psoriasis

Phase 1 trial in SLE TLR7/8/9 oligonucleotide IMO-8400 Phase 2 completed in 106, 107, 108 inhibitor psoriasis

TLR7/8/9 small molecule CpG-52364 Phase 1 – completed 109 inhibitor

DNA/RNA

RNase-Fc fusion protein RSLV-132 Phase 2a – recruiting NCT02660944, 110

Recombinant DNase 1 Phase 1b – completed 111

MyD88

MyD88 dimerization ST-2825 Pre-clinical 112, 113 inhibitor

JAK/STAT pathway

JAK1/3 inhibitor Tofacitinib Phase 1 – recruiting NCT02535689

NCT01777256, 114, JAK1 inhibitor GSK2586184 Phase 2 – terminated 115

JAK/SYK inhibitor R333 Phase 2 – completed 116

JAK2 inhibitor CEP-33779 Pre-clinical 117, 118

JAK1/2 inhibitor Baricitinib Phase 2 – recruiting NCT02708095

19 pDCs

Anti-BDCA2 mAb BIIB059 Phase 1 – completed NCT02106897, 119

Phase 1 studies of Venetoclax/ABT-199 ABT-199 completed in chronic NCT01686555, 120, Bcl-2 inhibitors (Venetoclax), lymphocytic leukemia 121 ABT-737 and SLE

Proteasome inhibitors Bortezomib Phase 2 – recruiting NCT02102594

1.4.3 TLRs as therapeutic targets

Several TLRs are relevant to the interferon signaling pathway in SLE. Immune complexes containing nucleic acids are internalized upon binding Fc receptors and stimulate IFN production by activating intracellular TLRs 3, 7/8 and 9.122 TLRs signal through two main pathways. All except TLR3 signal through the MyD88-dependent pathway, whereas TLR3 (and TLR4) signal through the TRIF (TIR-domain- containing adapter-inducing interferon-β)-dependent pathway. Recruitment of downstream signaling molecules, such as IRAK1/4 (interleukin-1 receptor-associated kinases 1 and 4) and TRAF6 (tumour necrosis factor receptor-associated factor 6), and the interferon regulatory factors (IRF3, IRF5 and IR57), subsequently leads to transcription of type I IFNs.122

As with the other components of the interferon signaling pathway, data from both human and murine studies support the role of TLRs in the pathogenesis of SLE. In humans, SLE PBMCs show upregulated TLR7 and TLR9 mRNA expression, which correlates with IFNα expression. Male BXSB lupus prone mice, which harbor the Y- linked autoimmune acceleration (Yaa) cluster that includes a TLR7 gene duplication, showed decreased autoantibody production when TLR7 signaling was ablated.123 Reduced IFNα and IL-6 levels were seen in murine TLR7-/- pDCs stimulated with ribonucleoprotein-containing immune complexes.124 Decreased autoantibody and immunoglobulin levels and lymphocyte activation were seen in the MRL/lpr murine lupus model lacking TLR7.125 Additionally, in the pristane-induced murine lupus

20 model, which is highly interferon dependent, TLR7 deficient mice developed lower autoantibody levels and less glomerulonephritis.126

Data regarding the pathological role of TLR9 in SLE are conflicting. Deletion of TLR9 in a number of TLR9-dependent murine lupus models, led to disease exacerbation, rather than abrogation.125, 127 In humans, despite increased TLR9 expression in DCs and B cells from SLE patients with severe disease, B cells are less activated and hyporesponsive to ODN-CpG (a TLR9 agonist) stimulation.128

TLR8 is phylogenetically similar to TLR7 and also recognizes ssRNA and synthetic ligands. There are few studies of TLR8 in SLE, with conflicting evidence to date regarding the contribution of TLR8 to SLE. For example, TLR8 deletion augmented disease in lupus prone mice through a TLR7-dependent mechanism.129 TLR8 has been postulated to contribute to the gender differences in SLE, because it is located on the X chromosome. The 564Igi murine model is an Ig-transgenic mouse strain in which B cells express an Ig receptor specific for the lupus antigen SSB/LA.130 In this model, decreased autoantibody production was seen in female mice with only one copy of the Tlr8 gene on a Tlr7/9-/- background.

The antimalarial drug hydroxychloroquine (HCQ), is a TLR7/8/9 antagonist. The activity of HCQ has been attributed to reduced endosomal acidification, which is required for TLR activation. More recent evidence suggests that HCQ binds directly to nucleic acids, causing structural modifications that prevent ligand binding to TLRs.131 HCQ is a mainstay of SLE treatment: its benefits include decreasing overall disease severity, preventing disease flares,132 increasing survival,133 altering lipid profiles favourably,134 and having anti-thrombotic effects.135 These benefits are seen without clinically significant immunosuppression, and vindicate the concept of therapeutically targeting TLR7 and TLR9.

There are a number of other therapeutics in development that target TLRs, or their downstream molecules, including oligonucleotides and small molecule inhibitors. Several oligonucleotides act as TLR antagonists. In a phase 1b/2a study, DV-1179, a TLR7/9 dual antagonist, was well tolerated, but did not achieve its pharmacodynamic endpoint of reducing IFNα-regulated genes.100 Pre-clinical studies with another dual

21 TLR 7/9 antagonist, IRS-954, showed inhibition of IFNα production by pDCs in response to DNA/RNA viruses and isolated SLE immune complexes102 and was effective in murine models.101 Interestingly, resistance to glucocorticoid-induced pDC death mediated by TLR7 and TLR9 was also reversed by IRS-954 treatment in lupus prone mice.103 Another compound, IMO-3100, was shown to not only inhibit IFNα, but TNF-α and IL-17 production from human PBMCs.104 A TLR7/8/9 antagonist, IMO-8400, showed efficacy in mouse models106 and is proceeding to a phase 1 trial in SLE.105 Both IMO-3100 and IMO-8400 have been well tolerated and interestingly, were effective in phase 2 trials in psoriasis, another disease in which IFN may play a role.107, 108

Small molecule inhibitors have the potential advantage of oral availability, and compounds have been designed to target TLRs and downstream signaling proteins, such as MyD88. The quinazoline derivative, CpG-52364, a small molecule inhibitor of TLR7/8/9, was shown to be safe, and more effective than HCQ in pre-clinical animal studies.109, 131 It has completed a phase 1 clinical trial in SLE (NCT00547014), although no results have been reported. The MyD88 dimerization inhibitor, ST-2825, interferes with recruitment of IRAK4 and IRAK1 via MyD88, and inhibits pro- inflammatory cytokine production and TLR9-induced B cell proliferation and differentiation.112, 113

1.4.4 Host nucleic acids as therapeutic targets

Nucleic acids may also activate TLR-independent pathways to stimulate type I IFN production.

One of these pathways involves the cytosolic RNA helicases RIG-I (retinoic-acid- inducible gene I), MDA5 (melanoma differentiation-associated gene 5), and LGP2 (laboratory of genetics and physiology 2), also known as RIG-I-like receptors (RLRs). Binding of RNA to RLRs leads to their association with adaptor protein IPS-1 (IFNβ promoter stimulator 1), located in the mitochondria, which activates TANK-binding kinase 1 (TBK1) and IκB kinase. Subsequently, IRF3, IRF7 and NFκB (nuclear factor

22 kappa-light-chain-enhancer of activated B cells) activation leads to type I IFN and pro-inflammatory cytokine production.136

The adaptor protein STING (stimulator of interferon genes) mediates signal transduction following sensing of cytosolic DNA. Its downstream signaling pathway overlaps with that of the RLRs, as it translocates to perinuclear regions to interact with TBK1.

Variants or mutations in components of these signaling pathways have been identified as predisposing factors to the development of SLE in murine models, and in humans.137-140 However, so far, attempts to decrease DNA and RNA levels therapeutically have met with inconclusive results. Recombinant DNase1 slowed the progression of disease in a murine lupus model.141 However, a phase 1b human study showed no change in relevant serum markers, although the drug was well tolerated.111 Tlr7 x RNase double transgenic mice (which have higher concentrations of serum RNase) had increased survival compared to Tlr7 Tg mice, associated with reduced T and B cell activation and less IgG and C3 deposition in the kidneys.110 A phase 1 trial of an RNase-Fc fusion protein, RSLV-132, has been completed, following pre-clinical studies showing efficacy in degrading circulating immune complexes, thereby preventing renal IFN production and kidney damage.142 A phase 2a study of RSLV- 132 has commenced (NCT02660944).

1.4.5 JAK/STAT inhibition as a therapeutic strategy

There are four JAK family members – JAK1, JAK2, JAK3, and TYK2, each of which is involved in the signaling cascade of various cytokine receptors. Therefore, a theoretical advantage of modulating the JAK/STAT pathway in SLE is the potential to inhibit other pathogenic cytokines, such as IL-6.143 Small molecule inhibition of the JAK/STAT pathway has already been successful in other autoimmune diseases.144-146

In SLE, genome wide association studies have linked TYK2 and STAT4 to SLE.147 Evidence from murine models shows that JAK2 inhibitors prevented or improved established disease. The administration of trophostin AG490 in MRL/lpr mice

23 decreased expression of IFNγ, as well as serum dsDNA levels, proteinuria, T cell and macrophage infiltrates and deposition of IgG and C3 in the kidneys.148 Another JAK2 inhibitor, CEP-33779, prevented the development of nephritis in mice and was superior to dexamethasone, and cyclophosphamide, in treating established nephritis.117, 118 In this model, mice treated with the JAK2 inhibitor had improved survival, reduced proteinuria, decreased anti-dsDNA antibodies and a decrease in autoantibody producing plasma cells in the spleen. Importantly, several pro- inflammatory cytokines, including IL-4, IL-6, IL-12, IL-17A and TNF-α were decreased after treatment with CEP-33779. These pro-inflammatory cytokines have also been implicated in SLE, with altered serum levels in SLE patients.149, 150

The first JAK inhibitor developed for autoimmune disease treatment in humans was tofacitinib, which inhibits JAK1, JAK3 and to a lesser extent JAK2.147 Tofacitinib has been shown to have an acceptable safety profile and is effective in treating rheumatoid arthritis,144 a disease in which type I IFN also plays a pathogenic role.151 Tofacitinib is approved for clinical use in rheumatoid arthritis and is currently undergoing a phase 1 clinical trial in SLE (NCT02535689).

A phase 2 trial (NCT01777256) of a specific JAK1 inhibitor, GSK2586184, in SLE was terminated early due to lack of efficacy, and development of this drug in SLE has been halted.114 In this trial, two patients who had received the drug also developed the DRESS syndrome (a rare but serious idiosyncratic drug reaction causing rash, eosinophilia and systemic symptoms) and severe, but reversible, abnormalities in liver function.115 Although mild liver function abnormalities are a recognized side effect of other JAK inhibitors, such as tofacitinib, DRESS has not previously been reported with JAK inhibitors. The development of a JAK/SYK inhibitor, R333, has also been terminated,116 after failing to meet its primary endpoint of a 50% decrease in active skin lesions in a phase 2 trial of treatment in discoid lupus (NCT01597050).

Other data supporting the potential benefit of JAK inhibition in IFN-driven diseases include studies in patients with rare, monogenic interferonopathies. A compassionate use study of baricitinib (a JAK1/2 inhibitor) in CANDLE (chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperatures) showed

24 decreased disease manifestations and steroid requirements.152 In vitro, JAK inhibitors - tofacitinib, baricitinib and ruxolitinib (a JAK1/2 inhibitor) - reduced constitutive upregulation of phosphorylated STAT molecules in lymphocytes from patients with SAVI (STING-associated vasculopathy with onset in infancy), and a dose dependent blockade of some interferon-responsive genes was seen.153 A phase 2 trial of baricitinib in SLE is currently recruiting (NCT02708095).

1.4.6 pDCs as a therapeutic target

Although type I IFN can be produced from a number of different cell types including lymphocytes, macrophages, fibroblasts and endothelial cells, the main producer (on a per cell basis) is the pDC,154 a rare and specialized dendritic cell type that is capable of rapid production of type I IFN. This usually occurs in response to viral infection, with viral nucleic acids triggering endosomal TLR7 and TLR9 activation. In SLE, TLR7 and TLR9 are instead triggered by self nucleic acids contained in immune complexes or NETs.155

Many in vitro human studies have indirectly implicated the pDC in SLE pathogenesis. pDCs have been mostly found to be decreased156-160 in SLE peripheral blood, although there are fewer studies of unaltered161 or increased162 pDC numbers compared to healthy donors. These changes are often associated with more active SLE disease. Some of these studies have shown a concomitant infiltration of pDCs into nephritic kidneys,156, 157 suggesting that the decrease in pDCs may be explained by their migration into sites of active inflammation. Apart from the kidney, increased numbers of pDCs have also been found in cutaneous lupus lesions,163-165 supporting the concept of pDC migration to sites of disease activity, explaining their decreased presence in SLE peripheral blood. pDCs have also been found to be activated by DNA-containing immune complexes, which is mediated via TLR9.166, 167

Recently, experimental murine lupus models have clarified a central role for pDCs in SLE pathogenesis.91, 168 Haploinsufficiency of Tcf4, which encodes the transcription factor E2-2 that is critical for pDC development from bone marrow progenitors, impairs the innate immune function of pDCs in mice and humans.169 In one study,

25 Tcf4 haploinsufficiency ameliorated disease in two different lupus mouse models.168 In the Tlr7-tg mouse, which overexpresses Tlr7, global and DC-specific Tcf4 haploinsufficiency abolished splenomegaly and myeloid cell expansion and decreased anti-RNA autoantibody levels. In the second model, B6.Sle1.Sle3, which contains the genomic regions of two susceptibility loci from the lupus prone NZM2410 strain crossed onto a C57BL/6 background,170 there was a significant decrease in anti-DNA antibody levels and glomerulonephritis. Transient depletion of pDCs (with diphtheria toxin) in other murine models showed beneficial effects on autoantibody production and the development of glomerulonephritis.91, 171

Therapeutic targeting of pDCs is still in early stage development. A mAb targeting BDCA2, which is a pDC-specific cell surface receptor, increased internalization of BDCA2 and CD32 (FcγRIIa) in vitro, which resulted in inhibition of both TLR and SLE immune complex stimulated type I IFN production.119 This antibody, known as BIIB059, has recently completed recruitment in a phase 1 clinical trial (NCT02106897), although no results are yet available. Selective depletion of pDCs has been explored pre-clinically. Inhibition of Bcl-2, an anti-apoptotic protein, was shown to deplete pDCs, but not conventional DCs, in lupus prone NZB/NZW mice, and in vitro in human SLE samples, which decreased IFNα production. A decrease in lymphocyte numbers was also observed in the NZB/W F1 murine lupus nephritis model.172 Interestingly, the depletion of pDCs was enhanced by co-exposure to glucocorticoids.120 A phase 1 study of the Bcl-2 inhibitor ABT-199 (Venetoclax) has been completed in both SLE (NCT01686555) and chronic lymphocytic leukemia.121 In the phase 1 study in female SLE patients, Venetoclax was well tolerated and a decrease in peripheral blood lymphocytes was observed.173 Bortezomib, a proteasome inhibitor previously mentioned as a plasma cell depleter, has also been shown to affect plasmacytoid dendritic cells. It decreased production of IFNα in NZB/W F1 lupus prone mice, through inhibiting both pDC survival and their ability to produce IFNα in response to stimulation with a number different ligands, including TLR9 and TLR3.174 The mechanisms by which this occurs may be through altering intracellular trafficking of TLRs, and endoplasmic reticulum (ER) homeostasis. Bortezomib treatment was associated with a decrease in the spliced form of transcription factor XBP1, which rescues cells from apoptosis by maintaining ER homeostasis. It also

26 interfered with intracellular trafficking of TLR9 from the ER to endolysosomes.175 The efficacy and safety of bortezomib in SLE is currently being explored in a phase 2 trial (NCT02102594).

1.5 Other therapeutic targets in SLE

A number of other therapeutic agents, targeting various aspects of the immune system other than the B cell, or the IFN pathway, are currently being explored in SLE.

1.5.1 Anti–cytokine therapies in SLE

A number of cytokines have been found to be altered in SLE or implicated in its pathogenesis. A summary of the major anti-cytokine therapies in development for SLE is presented in Table 1-3.

Table 1-3: Anti-cytokine therapies in SLE

Trial number Target Drug Name Progress and references

TNF-α

Phase 1 - safe and well TNF-α inhibitor Infliximab 176 tolerated

Phase 2 in discoid lupus TNF-α inhibitor Etanercept NCT02656082 recruiting

IL-6

Anti-IL-6R mAb Tocilizumab Phase 1 177

Phase 1 in cutaneous lupus and SLE 178 Anti-IL-6 mAb Sirukumab Phase 2 in lupus nephritis NCT01273389 completed

Anti-IL-6R nanobody ALX-0061 Phase 2 recruiting NCT02437890

27 M-CSF, GM-CSF

Anti-M-CSF mAb PD-0360324 Phase 1 – negative result 179

No trials in SLE Anti-GM-CSF mAb Mavrilimumub Phase 2 trial in RA 180 completed

IL-23/Th17 pathway

No trials in SLE Secukinumab Anti-IL-17A mAb Phase 3 trials in psoriasis 181, 182 Ixekizumab and psoriatic arthritis

Anti-IL-23/IL-12 mAb Ustekinumab Phase 2 – recruiting NCT02349061

Anti-IL-21 mAb NCT0114-0006 Phase 1 – terminated NCT01689025

IL-10

Anti-IL-10 mAb BT063 Phase 2 – recruiting NCT02554019

MIF

Phase 1 – terminated in NCT01541670, Anti-MIF mAb Imalumab SLE, completed in solid NCT01765790, tumours 183

Phase 1b currently Anti-CD74 mAb Milatuzumab NCT01845740 recruiting

TWEAK

Two phase 2 trials in lupus NCT01499355, Anti-TWEAK mAb BIIB023 nephritis terminated due to NCT01930890 lack of efficacy

1.5.1.1 Controversial role for TNF-α in SLE

TNF-α inhibitors have become an integral part of the treatment paradigm in other autoimmune diseases such as rheumatoid arthritis, psoriatic arthritis and inflammatory bowel disease. The role of TNF-α in SLE, however, is controversial.

28 TNF-α levels have been reported to be elevated in the sera of lupus prone animals184, 185 and SLE patients,186 and are associated with disease activity.187, 188 Other studies suggest that TNF-α gene polymorphism contributes to SLE susceptibility.185 However, as mentioned earlier, therapeutic blocking of TNF-α can induce autoantibodies and rarely, an SLE-like syndrome, and has generally been avoided in the clinical setting of overlap between RA and SLE features. However, in a small pilot study, the TNF inhibitor infliximab was found to be safe and well tolerated in SLE patients with active disease.176 Etanercept, another TNF-α inhibitor, led to the induction of clinical remission in refractory lupus arthritis after twelve weeks in the majority of patients in a small observational study, and was relatively safe with only transient elevations in anti-dsDNA antibodies that coincided with systemic flares.189 A phase 2 clinical trial utilizing localized application of etanercept through intradermal injection into discoid lupus lesions, is currently recruiting (NCT02656082).

1.5.1.2 Therapeutic blockade of IL-6 in SLE

The pleiotropic cytokine IL-6 has also been implicated in the pathogenesis of SLE and has broad ranging effects on the immune system including promoting Ig production,190 and T cell activation.

Like TNF-α, elevated serum levels of IL-6 have been observed in murine models191, 192 and SLE patients,187, 193 correlating with active disease,187 and gene polymorphisms have been found to be associated with SLE susceptibility.194 Higher levels of urinary IL-6 in patients with lupus nephritis correlate with active renal inflammation and pathology.195, 196 The benefit of IL-6 blockade in SLE was suggested by the finding that IL-6 deficiency ameliorates disease activity in murine lupus models.197 IL-6 inhibition in humans has enjoyed success for the treatment of rheumatoid arthritis.198

Blockade of IL-6 and its receptor in SLE has been explored in early phase clinical trials. Tocilizumab, a humanized murine anti-IL-6R mAb, was well tolerated in a phase 1 trial apart from a transient dose-dependent neutropenia,177 and was able to reduce B and T cell activation, memory B cells, and autoantibody producing plasma

29 cells without affecting naïve B cell populations.199 A fully humanized anti-IL-6 mAb, sirukumab, has completed a phase 1 trial for cutaneous and systemic lupus patients, where it was generally well tolerated apart from mild leucopenia, neutropenia and thrombocytopenia.178 A phase 2 trial in lupus nephritis (NCT01273389) did not result in a median improvement in proteinuria, however around 15-20% of treated patients showed a notable reduction in proteinuria.200 However, a high frequency of serious adverse events was observed, with almost half of the patients experiencing one or more serious adverse events, the majority of which were infections.200 ALX-0061, an anti-IL-6R nanobody linked to anti-human serum albumin to increase the serum half life in vivo, is currently in a phase 2 clinical trial in SLE (NCT02437890).

1.5.1.3 M-CSF and GM-CSF in SLE

M-CSF (macrophage colony-stimulator factor) levels are elevated in SLE patient serum201 and in the urine in lupus nephritis,202 and increased production of M-CSF by renal mesangial cells contributes to proteinuria, local macrophage infiltration, and glomerular proliferation.203, 204 In a murine lupus model, therapeutic blockade of M- CSF with a selective M-CSF receptor kinase inhibitor, GW2580, prevented macrophage and T cell accumulation in the kidney, decreased local renal inflammatory activity, and reduced kidney pathology.205 However, a fully humanized anti-M-CSF mAb, PD-0360324, did not alter clinical outcomes or affect cell infiltration or activation in a trial conducted in subacute cutaneous lupus and discoid lupus, despite being shown to suppress circulating monocytes and tissue macrophage function.179

Serum levels of GM-CSF (granulocyte-macrophage colony-stimulator factor) have been shown to be increased in SLE patients compared to healthy controls,206 and there is other evidence of its potential pathogenic role. An increased frequency of GM-CSF secreting PBMCs in active SLE patients correlates with anti-dsDNA antibody levels.207 Additionally, GM-CSF can be produced by glomerular mesangial cells, with levels correlating with lupus nephritis.203 There are no current clinical trials of GM- CSF antagonism in SLE, although blockade of this pathway has been promising in rheumatoid arthritis,208 with a fully humanized anti-GM-CSF receptor mAb, mavrilimumab, achieving rapid clinical responses in a phase 2 trial.180

30

1.5.1.4 The IL-23/Th17 pathway in SLE

The IL-23/Th17 pathway has emerged as an important pathway in the pathogenesis of a number of autoimmune diseases, with success in clinical trials utilizing therapeutic blockade of IL-17 (secukinumab and ixekizumab) or its receptor (brodalumab) in psoriasis181, 182 and psoriatic arthritis.209, 210 These agents were generally well tolerated apart from mild to moderate candida infections, infrequent neutropenia and some serious infections, although brodalumab studies have been halted due to concerns about suicidal ideation and completed suicides.211

IL-17 is produced by Th17 cells, upon which IL-23 and IL-21 act to stabilize and amplify this Th cell subset. There is emerging evidence that cytokines involved in this pathway play a role in SLE pathogenesis. Elevated serum and urine levels of IL-17 and IL-23 have been found in SLE patients, which correlate with disease activity.212, 213 Circulating Th17 cells, IL-17A mRNA in PBMCs and urinary IL17A gene levels in lupus nephritis patients have also been shown to be elevated in SLE. IL-17A and IL- 23 expression in class IV lupus biopsies was also increased.214, 215

These cytokines augment the immune response by promoting pro-inflammatory cytokine and chemokine production - such as IL-6 and GM-CSF - from multiple cell types, and by recruiting inflammatory cells including monocytes, neutrophils and lymphocytes.214 B cell survival and differentiation into plasma cells is also augmented by these cytokines, and the activation of T cells and their infiltration into tissues is facilitated by upregulation of ICAM-1 (intercellular adhesion molecule-1) and MMP (matrix metalloproteinases).216, 217

Pre-clinical studies have indicated that blockade of this pathway may be therapeutically beneficial. IL-17 blockade in BXD2 mice, a model in which elevated IL-17A production leads to spontaneous splenic germinal center formation, reduced lupus manifestations and germinal center numbers.218 A decrease in the incidence of lupus nephritis is seen in IL-17A deficient mice,219 and IL-23 deficient mice exhibit significantly less intestinal damage than MRL/lpr lupus prone mice when subjected to ischemic reperfusion.220

31

Currently there are no trials of IL-17 blockade in SLE, however, ustekinumab, a humanized monoclonal antibody directed against IL-23 and IL-12, that is already used for the treatment of psoriasis, is currently recruiting in a phase 2 trial in SLE (NCT02349061). A phase 1 study of an anti-IL-21 antibody (named NNC0114-0006) in SLE was terminated (NCT01689025), with no results currently available, although is advancing into phase 2 trials in type 1 diabetes.221

1.5.1.5 IL-10 in SLE

Elevated serum levels of IL-10, an anti-inflammatory cytokine, have been observed in SLE, that correlate with disease activity.222 Anti-IL-10 mAb treatment in a murine lupus model induced by transferring PBMCs from lupus patients into SCID mice, which lack functional B and T cells, significantly decreased anti-dsDNA antibody levels.223 Improvement in disease activity, cutaneous lesions and arthritis was seen after a short duration of administration of an anti-IL-10 murine mAb (B-N10) to six lupus patients, which was safe and well tolerated.222 A proof of concept phase 2 study of a humanized anti-IL-10 mAb, BT063, in SLE is currently recruiting (NCT02554019).

1.5.1.6 Macrophage migration inhibitor factor (MIF) in SLE

Macrophage migration inhibitory factor (MIF) was one of the earliest pro- inflammatory cytokines to be identified, and is produced by most cells of the immune system, including monocytes, macrophages, DCs, B cells, T cells, basophils, neutrophils, eosinophils and mast cells.224 It has a broad range of immunomodulatory functions, regulating multiple signal transduction pathways. It signals through its receptor CD74 (the cell surface form of the MHC class II invariant chain), which forms a signaling complex with accessory protein CD44, leading to downstream Src- family kinase activation and phosphorylation of ERK (extracellular signal regulated kinase) and MAPK (mitogen-activated protein kinase).

Certain MIF polymorphisms have been found to be associated with SLE susceptibility,225, 226 and elevated serum MIF levels have been found in SLE patients,

32 correlating with higher SLE disease damage indices.227 In MRL/lpr lupus prone mice, MIF expression was increased in skin and kidney lesions; a survival benefit in Mif-/- mice was seen, associated with reduced renal and skin lesions, glomerular injury and proteinuria.228 In another study, treatment of lupus prone MRL/lpr or NZB/W F1 mice with MIF antagonist ISO-1 reduced glomerulonephritis, inhibited CD74+ and CXCR4+ leukocyte recruitment, and lowered levels of TNF-α and CCL2.229 Interestingly, this occurred without a change in autoantibody production or T and B cell activation, suggesting that MIF may confer its protective effects in SLE through regulation of the innate immune system.

MIF-targeting therapeutics are still in relatively early stages of development. A phase 1 study of an anti-MIF mAb in lupus nephritis was terminated (NCT01541670), with no results available. A phase 1 study of this drug for solid tumors was completed, where it was found to be well tolerated.183 A phase 1b study of subcutaneously administered milatuzumab (an anti-CD74 mAb) is currently recruiting (NCT01845740).

1.5.1.7 TWEAK in SLE

TNF-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily ligands which signals via binding to Fn14, a type I transmembrane receptor belonging to the TNF receptor superfamily.230 However, as it lacks a cytoplasmic death domain, its actions are not the same as those of TNF. TWEAK is expressed on a number of cells types including macrophages, DCs and NK cells, and regulates a number of biological processes including cell survival, proliferation, angiogenesis, and inflammatory cytokine induction.231, 232

Higher serum TWEAK levels in SLE patients in comparison to healthy controls has been observed,233 with levels found to be higher in those with lupus nephritis,234 lupus headache and vasculitis.233 Similarly, TWEAK expression in PBMCs in SLE patients with lupus nephritis was elevated compared to those without renal lupus.235 Fn14 knockout (KO) MRL/lpr mice have lower levels of urinary protein and histological evidence of glomerular and tubulointerstitial inflammation,236 and attenuated skin disease.237 Interestingly, a depressive-like illness, evidenced by elevated immobility in

33 a forced swim test and loss of preference for sweetened fluids, and impaired cognition, was ameliorated in Fn14 KO mice. This correlated with downregulated cerebral expression of RANTES, C3, CXCL11, CCL3 and MCP-1, as well as improved blood brain barrier integrity and decreased cerebrospinal fluid anti-dsDNA antibody concentrations.238

Two phase 2 trials of an anti-TWEAK mAb, BIIB023, in lupus nephritis were terminated early due to lack of efficacy (NCT01499355, NCT01930890). The findings from studies involving the Fn14 KO murine models suggest that targeting TWEAK may improve neuropsychiatric manifestations of SLE, a manifestation for which there are currently few specific therapeutics. However, a recent study in humans did not find significant differences in serum TWEAK levels between SLE patients with or without CNS disease.239

1.5.2 Targeting T cells in SLE

T cells are also crucial to the pathogenic process in SLE, providing support to B cells to stimulate them to differentiate, proliferate, mature and class switch autoantibody production. T cells are activated by interaction with APCs, including B cells, which requires the presentation of antigenic peptides on MHC to the T cell, and an antigen independent interaction involving costimulatory signals including CD40/CD40L, CD28 and CTLA-4 (cytotoxic T-lymphocyte antigen 4) and CD80(B7.1)/CD86(B7.2).240 There are now a number of T cell targeting therapeutics in development for SLE, which are summarised in Table 1-4.

Table 1-4: T cell targeted therapies in SLE

Trial number Target Drug Name Progress and references

T cell costimulatory blockade

CD80/86 costimulatory Abatacept Phase 2 in non renal lupus 241-243 signal CTLA-4/IgG1 Phase 2/3 in lupus nephritis

34 CD80/86 costimulatory RG2077 Phase 1/2 in lupus nephritis NCT00094380 signal CTLA-4-IgG4

CD40/CD40L IDEC-131 Phase 1 – negative results 244 costimulatory signal Anti-CD40L mAb

Open label study CD40/CD40L BG9588 terminated due to 245 costimulatory signal Anti-CD40L mAb thromboembolic events

CDP7657 CD40/CD40L Fab’ anti CD40L Phase 1 – completed 246 costimulatory signal fragment

ICOS/ICOSL AMG-557 Two phase 1 trials 247 costimulatory signal Anti-ICOSL mAb completed

ICOS/ICOSL MEDI-570 Phase 1 trial terminated NCT01127321 costimulatory signal Anti-ICOS mAb early

T regulatory cells

Tregs hrIL-2 Phase 2 trial recruiting NCT02465580

Tregs Autologous T regs Phase 1 trial recruiting NCT02428309

T cells

Phase 1 trial withdrawn SYK inhibitor R935788 NCT00752999 prior to recruitment

Quinolone-3- NCT01085084, Laquinimod Phase 2 studies completed carboxamide derivative NCT0185097, 248

Quinolone-3- Paquinimod Open label study effective 41 carboxamide derivative

U1 small nuclear Phase 2 trial completed – Forigerimod/ ribonucleoprotein mannitol formulation 249, 250 Lupuzor fragment effective

Sphingosine-1- Phase 1 in subacute KRP203 NCT01294774 phosphate analogue cutaneous lupus completed

Tfh cells (CXCR5) SAR-113244 Phase 1 completed NCT02321709

Calcineurin inhibitor Voclosporin Phase 3 study underway NCT03021499

35 1.5.2.1 Costimulatory blockade in SLE

Blockade of costimulatory interactions can dampen both T and B cell functions, and data from early trials of therapeutic agents that block these interactions has been promising.

Abatacept, a fully humanized, soluble fusion protein containing the extracellular domain of human CTLA-4 linked to IgG1, modulates the CD80/CD86:CD28 costimulatory signal. Already used for the treatment of rheumatoid arthritis, abatacept was found in post hoc analyses of a phase 2 clinical trial in non-life threatening non- renal lupus to decrease disease activity particularly in patients with SLE-associated arthritis.241 A phase 2/3 trial in class III/IV nephritis, when used in conjunction with mycophenolate mofetil and steroids, failed to meet its primary endpoints, however, improvement in levels of anti-dsDNA antibodies, complement levels and proteinuria was seen.242 When used in addition to cyclophosphamide and then azathioprine, in a phase 2 trial, abatacept did not improve the outcome of lupus nephritis at either 24 or 52 weeks.243 A CTLA-4-IgG4 Fc fusion protein, RG2077, has completed a phase 1/2 study in lupus nephritis (NCT00094380), with no results yet published.

Several drugs have been studied which impair the CD40:CD40L interaction. In a phase 2 trial, a humanized anti-CD40L mAb, IDEC-131, was no more effective than placebo;244 an open-label multiple dose study of another anti-CD40L antibody, BG9588, showed efficacy in reducing anti-dsDNA antibody levels, increasing C3 concentrations and reducing haematuria, however it was terminated early due to thromboembolic events.245 This phenomenon appears to be a consequence of the functional Fc portion, as a high-affinity PEGylated monovalent Fab’ anti-CD40L antibody fragment, CDP7657, retained pharmacological activity but without thromboembolic events in Rhesus monkeys.251 A phase 1 trial in healthy volunteers and SLE patients showed that CDP7657 was well tolerated, with no thromboembolic events occurring,246 providing encouraging results for further trials.

Another costimulatory pathway involving ICOS/CD278 (inducible T-cell costimulator) and ICOS ligand (ICOSL/CD275) may play a role in lupus pathogenesis, with in vitro and in vivo murine studies showing that blockade of this

36 pathway reduces antibody production and disease activity.252 Two phase 1 studies in SLE showed that AMG-557, a human anti-ICOSL mAb, selectively reduced anti- KLH IgG, with an acceptable safety profile.247 Development of anti-ICOS mAb, MEDI-570 was terminated early by the sponsor (NCT01127321), possibly to focus on development of AMG-557.

1.5.2.2 Altering Treg cell function in SLE

Although the precise defects found in different studies are conflicting, aberrations in the number and function of Treg cells have been observed in SLE,253 in addition to impaired IL-2 production by T cells, which is critical for the maintenance of Treg cells in the periphery.254 Treatment of five SLE patients with subcutaneous injection of low dose IL-2 for five days expanded the Treg population.255 An open-label study in forty SLE patients showed low dose IL-2 was well tolerated and led to improvements in clinical and immunological parameters, including an increase in Tregs, and decrease in effector Th cells following therapy.256 A single centre, double- blind, phase 2 trial of the administration of hrIL-2 is currently recruiting participants (NCT02465580). A phase 1 trial of the administration of autologous Tregs is also underway (NCT02428309).

1.5.2.3 T follicular helper cells in SLE

T follicular helper (Tfh) cells are a relatively recently identified CD4+ T cell subset, which express CXCR5 (which guides Tfh migration into B cell follicles), PD-1, ICOS (essential for Tfh differentiation) and CD40L. Tfh cells are specialized for providing help to B cells in germinal centres, assist in germinal centre formation and maintenance through expression of CD40L and the secretion of IL-21 and IL-4, and play a role in the selection of high affinity B cells. IL-21 also induces Tfh cell differentiation in an autocrine fashion.257, 258

Evidence for the involvement of Tfh cells in SLE pathogenesis was first seen in murine lupus models. As an example, Sanroque mice (roquinsan/san), which spontaneously develop a lupus-like disease due to impaired function of the post-

37 transcriptional repressor Roquin, are found to have excessive Tfh cells and germinal centre responses.259 Roquin has been shown to post-transcriptionally regulate the expression of ICOS,260 and OX40 (TNFRSF4, tumour necrosis factor receptor superfamily, member 4),259 a TNF receptor superfamily molecule highly expressed by Tfh cells. Due to limitations in access to secondary lymphoid organ samples, studies of Tfh cells in humans have mainly focused on studying circulating CD4+CXCR5+ Tfh cells, which are phenotypically and functionally considered the counterparts of germinal centre Tfh cells.261 Supporting evidence for the role of Tfh cells in SLE pathogenesis in humans are the findings of expanded circulating Tfh cells,262, 263 and increased serum IL-21 in SLE.264

A phase 1 trial of an anti-CXCR5 mAb, SAR-113244, has recently been completed in SLE (NCT02321709), although there are no results as yet available. Modulating the molecules which govern Tfh maturation and function, such as by targeting ICOS or CD40L with T cell costimulatory blockers, or targeting IL-21, may also beneficially alter Tfh cells in SLE.

1.5.2.4 Other T cell targeted therapeutics

Other T cell targeted therapeutics in SLE are mostly in early stages of development.

Tyrosine kinase inhibitors have previously been mentioned in the context of JAK/STAT pathway inhibition. Syk (spleen tyrosine kinase) is a cytoplasmic protein expressed in many immune cells including T and B cells, and has a role in modulating TCR signaling in T cells from SLE patients, wherein silencing of Syk was found to normalize the expression of several cytokines.240 However, there are no studies yet in human lupus, with a phase 1 trial of Syk kinase inhibitor R935788 withdrawn prior to enrolment (NCT00752999).

The quinolone-3-carboxamide derivatives (laquinimod and paquinimod), are other T cell targeted therapies in early stages of development. Developed initially for the treatment of multiple sclerosis, laquinimod decreases the activation of T cells via TLR4, and has completed two studies in lupus arthritis and nephritis (NCT01085084, NCT01085097). Although not powered to show a statistically significant difference,

38 an additive benefit was seen over standard of care in the improvement of renal function and proteinuria in lupus nephritis, with no major safety issues.248 Paquinimod (ABR-215757) improved arthritis, oral ulcers and alopecia in addition to standard of care in a small twelve-week open label study.41

Forigerimod/Lupuzor is a fragment of autoantigen U1 small nuclear ribonucleoprotein which inhibits T cell activation after encountering MHC-presented self peptides, possibly by acting as an altered peptide ligand for the T cell receptor. This results in a change to the autoreactive T cell phenotype and cytokines that are secreted.265, 266 A randomized trial of forigerimod in SLE showed efficacy compared with placebo,250 however a subsequent trial utilizing a different formulation (in trehalose rather than mannitol) failed to meet its endpoints in a phase 2b trial, which was similarly designed. It is postulated that this is because trehalose is a potent inducer of autophagy, opposing the inhibitory effect on autophagy that forigerimod is proposed to have.249

Sphingosine-1-phosphate is a lysosphingophospholipid which was seen to inhibit T cell proliferation in vitro.267 Fingolimod, a sphingosine-1-phosphate (SP-1) analogue, is already in use for the treatment of multiple sclerosis and has shown efficacy in a murine lupus model.268 KRP203, another SP-1 analogue, has completed a phase 1 trial in subacute cutaneous lupus (NCT01294774), with no published results as yet.

Voclosporin, a novel calcineurin inhibitor, decreases T cell activation and IL-2 production. It increased renal remission rates, and reduced proteinuria and non-renal disease activity in phase 2 trials.269, 270 A phase 3 trial is ongoing (NCT03021499).

1.6 Anti-IL-3Rα mAb - a novel way of targeting pDCs in SLE? pDCs highly express IL-3Rα (CD123) compared to most other peripheral blood cells.165, 271 CSL362 is a humanized therapeutic mAb that binds to CD123 and incorporates two mechanisms of action. It inhibits IL-3 binding to CD123, antagonizing IL-3 signaling in target cells.272, 273 Second, the Fc region of CSL362 has been mutated to increase affinity for CD16 (FcγRIIIa), thereby enhancing antibody- dependent cell-mediated cytotoxicity (ADCC). CSL362 can induce ADCC against

39 CD123+ acute myeloid leukemia (AML) blasts and leukemic stem cells in vitro, and reduces leukemic cell growth in murine xenograft models of human AML.273 A phase 1 clinical trial of CSL362 in AML has recently completed (NCT01632852). In this study, intravenous administration of CSL362 at doses ranging between 0.3 mg/kg – 9.0 mg/kg was shown to be safe, with the most common side effect being infusion reactions (≥10% of patients), which were manageable with supportive care (hydrocortisone premedication). CSL362 has progressed to a phase 2 clinical trial in AML as JNJ-56024473 (NCT02472145).

1.6.1 Basophils express CD123 and may play a role in SLE pathogenesis

Like pDCs, basophils are a relatively rare cell type in peripheral blood, representing <1% of circulating leukocytes, and also highly express CD123.274 Although well known for their involvement in allergic diseases and parasitic infections, a role for basophils in SLE has been proposed only relatively recently (Figure 1-5).

Homing to lymph nodes and spleen Activated basophil

CD62L Basophil Basophil MHC-II

Activation IL-4, IL-6, BAFF IL-4 Th2 cell

IgE containing B cell immune complexes IgE class switch

Figure 1-5: Potential role for basophils in SLE pathogenesis.

Peripheral blood basophils are activated by IgE-containing immune complexes, which causes upregulation of CD62L, allowing migration to secondary lymphoid organs. Activated basophils also upregulate MHC-II which enables them to present antigen. Activated basophils also upregulate IL-4 production, which promotes a Th2 phenotype and, with increased production of IL-6 and BAFF expression, promotes class switching of B cells to produce IgE autoantibodies. This perpetuates further basophil activation. Figure adapted from Davidson et al, Nat Med 2010.275

40 Basophils in SLE have been mostly found to be decreased in peripheral blood,276-278 which is postulated to be explained by their homing to secondary lymphoid organs after increasing CD62L expression, as increased basophils were found in the lymph nodes and spleen of SLE patients compared to healthy donors.276 However, unlike pDCs, increased basophils have not been found in cutaneous lupus lesions.279 Decreased basophils in peripheral blood were observed to correlate with active disease, as well as conventional measures of disease activity such as anti-dsDNA antibodies, IgG, C3 and C4 concentrations, leukocyte and platelet counts.277, 278 Interestingly, one study also found that patients with active SLE had significantly higher basophil counts after immunosuppressive treatment, when SLE was no longer active, than before treatment. These findings contrasted with another study which found that immunosuppressive treatment decreased basophil numbers in SLE patients.276

Basophils in SLE appear to be activated by IgE-containing immune complexes, displaying higher levels of activation marker CD203c, and upon activation may augment autoantibody production by eliciting a Th2 response and production of various pro-inflammatory cytokines including IL-4, IL-6 and the B-cell survival factor BAFF.275, 276 Basophil depletion has been shown to ameliorate nephritis in a murine lupus model.276 Therapeutic targeting of basophils in SLE is currently being explored in a phase 1 trial of an anti-IgE mAb, omalizumab (NCT01716312).

1.6.2 Therapeutic targeting of IL-3 may be beneficial in SLE

IL-3 is a multipotent haematopoietic growth factor that is a known maturation and survival factor for a variety of immune cells including pDCs,280 basophils,281 and B cells.282, 283 In humans, IL-3 signals through the interleukin-3 receptor (IL-3R), a heterodimer that comprises an IL-3 specific alpha chain (IL-3Rα) and a common beta chain, that is shared with the receptors for GM-CSF and IL-5.284

Although IL-3 has not been extensively studied in SLE, a study in the 1990s reported elevated serum IL-3 levels in active SLE patients.285 Studies performed in murine models of SLE have also identified a potential role for IL-3 in SLE pathogenesis. The

41 sera of the mice appeared to contain a factor with IL-3 activity, as shown by the ability of sera from MRL/lpr mice to promote proliferation of IL-3 dependent cell lines.286 A study in the C3H/gld murine lupus model revealed that this factor may have been serum IgG rather than IL-3.287 A separate study in the MRL/lpr model established hybridomas from mouse spleen cells that secreted monoclonal antibodies which supported the growth of IL-3 dependent cell lines. The antibodies inhibited the binding of IL-3 to the cell lines, and vice-versa, raising the possibility that the antibodies were directed against the IL-3 receptor, and acting in a stimulatory manner.288

More recently, a study in MRL/lpr mice more directly demonstrated the role of IL-3 in SLE pathogenesis. Plasma IL-3 levels increased during disease progression, and IL-3 was found to be produced by CD4+ T cells in the spleen and bone marrow, and CD8+ T cells from the spleen.283 Administration of IL-3 in the model exacerbated nephritis, and IL-3 blockade decreased autoantibody production and improved renal function and skin lesions, suggesting an important role for IL-3 in the progression of lupus nephritis and cutaneous lupus. In the Lyn-deficient murine lupus model, elevated IL-3 responsive progenitor cells were found in the spleen.197 IL-3 has also been observed to promote signaling and survival of Lyn-/- plasma cells, suggesting that it may play a role in supporting autoreactive plasma cells.289 These data indicate that IL-3 neutralization in SLE may be beneficial, although there are no current clinical trials exploring this approach in SLE. In other autoimmune diseases, pre-clinical testing of an anti-IL-3 mAb in a murine rheumatoid arthritis model decreased progression and severity of disease, and reduced pro-inflammatory cytokines IL-6 and TNF-α.290

1.7 Hypothesis and aims

The aim of this study was to investigate a pre-clinical rationale for the therapeutic use of anti-IL-3Rα mAb CSL362 in SLE, by exploring its effects on key cell types and cytokines involved in SLE pathogenesis. The study was conducted ex vivo on primary human cells derived from SLE patients, and healthy and autoimmune disease controls, and in vivo in cynomolgus macaques (cyno).

42

I hypothesized that CSL362, via two mechanisms of action - the ability to neutralize IL-3 signaling and thereby cell survival, and effecting ADCC against CD123+ cells – would deplete both pDCs and basophils. I also hypothesized that depletion of pDCs and basophils would decrease type I IFN production and autoantibody production (Figure 1-5).

survival survival IL-3

pDC Basophil ADCC ADCC

Type I IFN production CSL362

Autoantibody production and immune complex formation

Figure 1-6: Proposed mechanism by which CSL362 may have therapeutic efficacy in SLE.

CSL362 may decrease pDCs and basophils through ADCC and neutralization of IL-3, which is a survival factor for both of these cell types. Depletion of pDCs will lead to a decrease in type I IFN production, which, along with basophil depletion, will decrease autoantibody production and immune complex formation.

Another aim of this study was to further the understanding of the role of IL-3 in SLE, through measuring serum IL-3 levels in SLE patients and healthy controls, and correlating them with clinical information including disease activity, clinical manifestations and medication use. The presence of an ‘IL-3 gene signature’ in the peripheral blood of SLE patients was also explored using high throughput sequencing techniques.

43 Chapter 2: Subjects, clinical characterisation and sample collection

2.1 Subjects

2.1.1 Subject recruitment

Between September 2012 and November 2015, SLE patients and healthy and autoimmune disease control donors were recruited to participate in the study, from whom clinical information and biological samples were collected. SLE and autoimmune control donors were recruited mostly from patients attending Rheumatology clinics for their routine clinic reviews, with a minority recruited from the Nephrology and Dermatology clinics at The Royal Melbourne Hospital, a large tertiary referral and teaching hospital that provides services to northwestern metropolitan Melbourne, and regional and rural Victoria. Three donations from SLE donors were given when the donors were admitted to hospital with an SLE disease flare. Healthy control subjects were recruited from The Walter and Eliza Hall Institute’s Volunteer Blood Donor Registry. Samples collected from these donors were used for assays in Chapters 3 and 4.

2.1.2 Study approval

The study was conducted with approval from the Human Research Ethics Committees of The Royal Melbourne Hospital (2012.039) and The Walter and Eliza Hall Institute (12.05) for SLE, autoimmune and healthy donors recruited from The Royal Melbourne Hospital and The Walter and Eliza Hall Institute’s Volunteer Blood Donor Registry. All donors signed a written consent form prior to participation in the study.

44 2.1.3 Inclusion and exclusion criteria

All donors were aged ≥ 18 years old. SLE donors fulfilled the SLICC (Systemic Lupus International Collaborating Clinics) classification criteria for SLE291 or had biopsy proven lupus nephritis or cutaneous lupus. SLE donors were all receiving standard of care assessments and therapy at the clinic visits. Donors who were pregnant, or had an inter-current acute illness (for example, an acute inter-current infection), other than a flare of disease activity, were excluded from study participation.

2.2 Clinical characterisation of donors

2.2.1 SLE donor characteristics

In order to look for clinic-pathological correlations in SLE donors, information regarding their clinical status was collected. This included age, sex, ethnicity, disease duration, disease activity, evidence of organ damage, disease manifestations and medication use. This data was collected from face-to-face interviews and/or from review of donor medical records, by a single researcher (SO).

Routine pathology testing was undertaken at each donation, as part of routine clinical care to assess SLE disease activity, and to monitor for adverse medication effects. These tests included a full blood examination (FBE), urea, electrolytes and creatinine (UECs), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), anti-double- stranded DNA (dsDNA) antibody level, complement C3 and C4 levels, a urine microscopy for assessment of red blood cells, white blood cells and casts, and analysis of urinary protein levels. For all but two donations, these blood tests were performed at Melbourne Health Pathology, with measurement of the anti-dsDNA antibody assessed by the Farr assay. For the other two donations, the routine pathology tests were performed at St John of God Pathology, or Melbourne Pathology, with the anti-dsDNA antibody level at St John of God Pathology measured by Luminex assay.

45 Disease activity was measured using the SELENA-SLEDAI (Safety of Estrogens in Lupus Erythematosus National Assessment – SLE Disease Activity Index)292 (Appendix 1) and organ damage assessed by the SDI (Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index)293 (Appendix 2) at each donation. When information regarding specific SLEDAI or SDI components was not available, it was assumed to be absent.

Forty-two SLE donors contributed to the study, with seventeen donors contributing samples more than once over the three-year period, for a total of eighty-two donations. Different assays were performed on the donated blood at each visit. The SLE donors were a typically female predominant population, who were ethnically diverse and heterogeneous in terms of disease manifestations and current medications (Table 2-1).

46 Table 2-1: SLE donor characteristics (n = 42)

Age (mean, range) 37.7, 18 - 70 (years)

Gender (female, male) 86, 14 (%)

Disease duration (mean, median, range) 7.9, 7, 0.25 - 23 (years)

Ethnicity - Caucasian 57 (%) - Asian 33 - Middle Eastern 7 - African 3

SLEDAI (mean, median, range) 6, 4, 0 – 25

Disease manifestations (current or past) - Rash 64 (%) - Arthritis 74 - Cardiorespiratory 33 - Renal 33 - Neuropsychiatric 10 - Cytopenia 33 - Positive anti-dsDNA antibody 83 - Hypocomplementemia 23

Current medications - No immunosuppression 16 (%) - Steroids 57 - Hydroxychloroquine 69 - Sulfasalazine 2 - Methotrexate 7 - Azathioprine 19 - Mycophenolate 10 - Cyclosporin 2 - Rituximab 2

47

Most donors had no (0) or mild (1-5) to moderate (6-10) disease activity as measured by the SLEDAI (Figure 2-1).

A B

Figure 2-1: SLE donor disease activity.

Disease activity, as measured by the SELENA-SLEDAI, for (A) n = 42 different SLE donors (SLEDAI from first donation from each donor depicted if donor donated more than once) and (B) n = 82 different donation episodes, from the n = 42 different SLE donors. SLEDAI 0 = no activity, 1-5 = mild disease activity, 6-10 = moderate disease activity, 11-17 = high disease activity, 20+ = very high disease activity.294

48 Most of the SLEDAI disease activity was accounted for by positive anti-dsDNA antibody levels, hypocomplementemia, rash, alopecia, arthritis and mouth ulcers (Figure 2-2).

A

B

Figure 2-2: Positive SLEDAI components.

SLEDAI components which were present in (A) n = 42 different SLE donors (SLEDAI components from first donation for each donor depicted if participant donated more than once), and for (B) 82 different donation episodes for the 42 different donors.

49 Donors had low SDI scores (Figure 2-3A), ranging from 0 to 3, with the majority of SDI scores being either 0 or 1. Stroke and osteoporosis were the most common positive SDI components (Figure 2-3B).

A

B

Figure 2-3: Organ damage as assessed by SDI.

(A) Organ damage, as assessed by SDI, for n = 42 different SLE donors (SDI from last donation from each donor depicted if participant donated more than once). (B) SDI components present in n = 42 different SLE donors (data from last donation from each donor depicted if participant donated more than once). CABG = coronary artery bypass grafting, AMI = acute myocardial infarction, eGFR = estimated glomerular filtration rate.

50 2.2.2 Autoimmune disease donor characteristics

Twenty donors with autoimmune diseases other than SLE contributed to the study (Table 2-2).

Table 2-2: Autoimmune disease donor characteristics

Age (mean, range) 49, 27 - 89 (years) Gender (female, male) 80, 20 (%) Ethnicity - Caucasian 90 (%) - Asian 10 Autoimmune disease diagnoses - Rheumatoid arthritis 4 (number of donors) - Psoriatic arthritis 4 - Scleroderma 3 - Psoriasis 2 - Ankylosing spondylitis 2 - Seronegative inflammatory arthritis 1 - Primary Sjögren’s syndrome 1 - Polymyositis 1 - Granulomatosis with polyangiitis 1 - Minimal change renal disease 1 Medications - No immunosuppression 35 (%) - Steroids 20 - Hydroxychloroquine 10 - Methotrexate 25 - Azathioprine 15 - Leflunomide 5 - Rituximab 5 - Infliximab 10 - Adalimumab 5 - Golimumab 5

51

2.2.3 Healthy donor characteristics

Fifty-four healthy donors (defined as donors without any autoimmune diseases) were included in the study. SLE, autoimmune disease and healthy donors were matched for age and sex. The majority of donors were Caucasian, with a minority who were Asian, Hispanic, Middle Eastern, African and Aboriginal (Table 2-3). A medical history and current medication use was elicited from each healthy donor.

Table 2-3: Age, sex and ethnicity of healthy, SLE and autoimmune disease donors

Healthy SLE Autoimmune

Age in years - mean 40.5 37.7 49 - median 40 38.5 46.5 - range 20 – 68 18 - 70 27 - 89

Sex - female (%) 83 86 80 - male (%) 17 14 20

Ethnicity (%) - Caucasian 89 57 90 - Asian 7 33 10 - Hispanic 2 0 0 - Middle 0 7 0 Eastern - African 0 3 0 - Aboriginal 2 0 0

52 2.3 Sample collection and processing

In addition to blood taken for routine pathology testing (for SLE donors), additional blood was collected from each donor for research.

2.3.1 Blood sample collection and storage

Whole blood was collected into lithium heparin tubes (BD, Catalog number 367526) for whole blood and peripheral blood mononuclear cell (PBMC) assays. Serum was collected into SST II Advance tubes (BD, Catalog number 367958) for storage at - 80°C after centrifugation. PAX gene Blood RNA tubes (BD, Catalog number 762165) were frozen at -20°C for later RNA extraction.

2.3.2 Standard tissue culture

PBMCs were isolated from whole blood by Ficoll (GE Healthcare Life Sciences, Catalog number 17-1440-02) density centrifugation. All blood was processed within six hours of collection from the donor, and all PBMC assays undertaken on freshly processed (not frozen) cells.

Culture of PBMCs was undertaken in RPMI 1640 Media (Sigma Aldrich, Catalog number R0083) supplemented with 10% heat-inactivated fetal calf serum (HyClone GE Healthcare, Catalog number SH30084.03HI), 2mM GlutaMAX (Gibco Life Technologies, Catalog number 35050-0610) and 0.5% penicillin/streptomycin (Gibco Life Technologies, Catalog number 15140-122) at 37°C with 5% CO2, unless otherwise stated.

Details of specific assays performed on the samples collected are outlined in the relevant subsequent Chapters.

53 2.4 Discussion

SLE is a disease that mainly affects young to middle-aged women,295 which was reflected in the SLE cohort recruited for this study, and the healthy donors who were age and sex matched. The autoimmune disease donors were an older population, but with a similar female predominance as the SLE cohort. The majority of donors were Caucasian, however ethnicity was not matched precisely between disease and healthy groups.

This SLE cohort displayed the typical disease heterogeneity evident in other SLE cohorts, and as with other cohorts less severe disease manifestations such as rash and arthritis were more common than severe major organ involvement. The frequency of occurrence of different disease manifestations, and use of different immunomodulatory or immune suppressing medications, was not dissimilar to other SLE populations both locally, and internationally,296-298 apart from a lower rate of hypocomplementemia (23% in this cohort compared to ~ 60-70% in other cohorts).296, 297

Inclusion of donors with a variety of different autoimmune diseases, rather than donors with a single alternate autoimmune disease diagnosis, was undertaken in order to perform a preliminary assessment of the effect of CSL362 in a variety of different diseases, including those where there is evidence for a role for type I IFN in their pathogenesis. These autoimmune diseases include psoriasis, scleroderma, primary Sjögren’s syndrome, inflammatory myopathy, and rheumatoid arthritis.72, 151, 299-302

A strength of the collected clinical data was its collection by a single person, producing consistency across donors. One potential limitation of the data collection was that data drawn from medical records may have been incomplete, and where not explicitly stated whether present or not, a particular characteristic was presumed to be absent.

54 Chapter 3: Anti-CD123 monoclonal antibody, CSL362, alters key cells and cytokines implicated in systemic lupus erythematosus

3.1 Introduction

As outlined in Chapter 1, therapeutic targeting of a number of key cell types and cytokines involved in SLE may be possible with anti-CD123 mAb CSL362, through two mechanisms of action – neutralization of IL-3 signaling, and ADCC of CD123 expressing cells. I hypothesized that pDCs and basophils, which both highly express CD123, and are dependent upon IL-3 for survival, will be depleted by CSL362. Depletion of pDCs, which are the main producers of type I IFN, should decrease levels of a cytokine that plays a key role in SLE pathogenesis.

I examined the effects of CSL362 on primary human cells, derived ex vivo from SLE patients, and healthy and autoimmune controls, and in vivo in cynomolgus macaques. First, the expression of CD123 on pDCs and basophils, as compared to other peripheral blood cell types, was evaluated in SLE patients, healthy and autoimmune disease controls. Following this, the effect of CSL362 on pDCs and basophils, and other cell types including B cells, was explored, together with its effect on type I, II and III IFN. A targeted interferon gene signature comprising eleven IFN-inducible genes was developed, and the effect of CSL362 on this gene signature evaluated. Lastly, the effects of CSL362 on pDCs and basophils, and an IFN gene signature were examined in vivo in cynomolgus macaques.

55 3.2 Materials and methods

3.2.1 Human subjects, sample collection and standard tissue culture

Donors were recruited, blood samples collected, and standard tissue culture performed as outlined in Chapter 2. For RNASeq analysis of isolated pDCs (outlined in section 3.2.10), buffy coat samples were obtained from healthy donors at the Victoria Red Cross Blood Service.

3.2.2 Study approval – human and cyno

Approval was granted as outlined in Chapter 2 for the human studies. Additionally, buffy coat samples from the Victorian Red Cross Blood Service were collected with ethical approval granted from the Victorian Red Cross Human Research Ethics Committee (14-04VIC-10). Animal studies were conducted at Maccine Pty Ltd, Singapore, in accordance with standard operating procedures, and approved by the Institutional Animal Care and Use Committee (IACUC) of Maccine (259-2012, amendment 34).

3.2.3 Flow cytometry, antibodies and reagents

Most flow cytometry antibodies and reagents used were commercially available and are listed in Table 3-1. One antibody was not commercially available and was generated in-house at CSL Limited. This was an anti-CD123-PE antibody (HU01C2) that was used in the pDC and basophil staining panels for the cynomolgus macaque study. Dead cells were identified using Sytox Blue (Life Technologies, Catalog Number S11348). Flow cytometry data was acquired with a MACSQuant Analyzer (Miltenyi Biotec) or an LSR Fortessa (BD Biosciences) and analyzed with Flowjo software (Treestar). Cell sorting was performed with a FACSAria (BD Biosciences) or FACS Fusion (BD Biosciences).

56 Table 3-1: Antibodies and reagents

Catalog Application Reagent Company number CD123 expression Anti-CD123 PE BD Biosciences 555644 Lineage cocktail 1 – Lin1 pDCs, basophils (CD3, CD14, CD16, CD19, BD Biosciences 340546 CD20, CD56) FITC pDCs, basophils Anti-HLA-DR APC BD Biosciences 641393 pDCs, basophils Anti-HLA-DR APC-H7 BD Biosciences 561358 pDCs Anti-BDCA2 PE-Cy7 E-Bioscience 25-9818-42 pDCs Anti-BDCA2 APC Miltenyi Biotec 130-090-905 Basophils Anti-CCR3 AF647 BD Biosciences 561745 Basophils IgE FITC KPL 02-10-04 mDCs Anti-CD11c BV421 BD Biosciences 562561 mDCs Anti-CD11c APC BD Biosciences 340544 mDCs Anti-BDCA1 PE Miltenyi Biotec 130-098-007 mDCs Anti-CD123 BV510 BD Biosciences 563702 NK, T cells, monocytes Anti-CD3 PE-Cy7 BD Biosciences 557851 NK cells Anti-CD56 FITC BD Biosciences 340410 NK cells Anti-Nkp46 APC BD Biosciences 558051 NK cells Anti-CD107a APC-H7 BD Biosciences 561343 B cells Anti-CD19 PE-Cy7 BD Biosciences 557835 B cells Anti-CD19 APC BD Biosciences 555415 B cells Anti-CD20 FITC BD Biosciences 556632 B cells Anti-CD27 V500 BD Biosciences 561222 B cells Anti-CD38 BV421 BD Biosciences 562444 B cells Anti-CD138 AF700 Biolegend 352305 B cells Anti-CD138 PE Biolegend 356511 B cells Anti-CD38 PE-Cy7 BD Biosciences 335790 Monocytes, NK cells Anti-CD14 APC-H7 BD Biosciences 561384 Monocytes Anti-CD14 APC BD Biosciences 555399 Monocytes Anti-CD11b APC BD Biosciences 09546 Monocytes, granulocytes Anti-CD16 FITC BD Biosciences 555406 Granulocytes, eosinophils Anti-CD49d APC BD Biosciences 559881 T cells Anti-CD4 FITC BD Biosciences 555346 T cells Anti-CD8 APC BD Biosciences 555369 T cells Anti-CD8 APC-H7 BD Biosciences 561423

57 3.2.4 Measurement of cell surface CD123 expression

Whole blood (50-200 µL) was stained with antibody cocktails to delineate different peripheral blood cell types, each of which included anti-CD123 PE (Table 3-2, gating strategies shown in Appendix 3). Following red blood cell lysis with BD Lysing Solution (BD, Catalog number 349202), Quantibrite PE beads (BD, Catalog number 340495) were used to estimate the number of CD123 molecules on the surface of each cell type, using the MACSQuant Analyzer. This approach extrapolates CD123 expression from a standard curve which uses the geometric mean of CD123-PE fluorescence intensity for each cell population, and four known levels on the Quantibrite-PE beads.

Table 3-2: Defining cell surface markers for assessment of CD123 cell surface expression on peripheral blood cell types

Volume of Cell type Defining cell surface markers blood stained

pDC Lin1- HLA-DR+, BDCA2++ 150µL Basophils Lin1- CCR3+ 150µL mDCs Lin1- HLA-DR+, CD11c+ 100µL CD56dim NK cells NKp46+, CD3- CD56dim 50µL CD56+ NK cells NKp46+, CD3- CD56+ 50µL Naïve B cells CD19+ CD27- 200µL Memory B cells CD19+ CD27+ 200µL Plasmablasts CD19+ CD27++, CD20- CD38++ 200µL Classical monocytes CD3-, CD14++ CD16- 100µL Intermediate monocytes CD3-, CD14+ CD16+ 100µL Non-classical monocytes CD3-, CD14- CD16++ 100µL Neutrophils CD16+ CD49d- 50µL Eosinophils CD16- CD49+ 50µL CD4+ T cells CD3+, CD4+ CD8- 50µL CD8+ T cells CD3+, CD8+ CD4- 50µL

58 3.2.5 Evaluating the effect of CSL362 on pDCs, basophils, myeloid dendritic cells, and NK, B and T cell subsets ex vivo

PBMCs (1.0x106) were cultured with CSL362 or isotype control for 24 hours, with doses ranging from 0.001-0.1 µM. The percentage of viable (Sytox Blue-) pDCs, basophils, myeloid dendritic cells, CD56dim NK cells, CD56+ NK cells, naïve B cells, memory B cells, plasmablasts, monocytes, CD4+ T cells, and CD8+ T cells were determined on a MACSQuant Analyzer (Miltenyi Biotec) using the cell surface markers defined in Table 3-3, and gating strategies shown in Appendix 4.

Table 3-3: Defining cell surface markers for assessment of the effect of CSL362 on different peripheral blood cell types

Cell type Defining cell surface markers

pDC Lin1- HLA-DR+, BDCA2++ Basophils Lin1- CCR3+ mDCs Lin1-HLA-DR+, CD11c+ BDCA2-, BDCA1+ CD56dim NK cells CD14-, CD3- CD56dim CD56+ NK cells CD14-, CD3 CD56+ Naïve B cells CD19+ CD27- Memory B cells CD19+ CD27+ Plasmablasts CD19+ CD27++ Monocytes CD3-, CD14+ CD11b+ CD4+ T cells CD3+, CD4+ CD8- CD8+ T cells CD3+, CD8+ CD4-

CD123 was not used to define pDCs and basophils in these assays, due to variable shifts in the expression of this surface marker in the presence of CSL362.

A monocyte gating strategy delineating the three major monocyte subsets – classical (CD3-, CD14++ CD16-), non-classical (CD3-, CD14- CD16++) and intermediate (CD3-, CD14+ CD16+), was attempted, however CD16 proved an unreliable marker due to shifting expression in the presence of CSL362, presumably due to Fc binding.

59 Fab’CSL362, the Fab fragment of CSL362, binds to CD123, neutralizing IL-3 signaling, but does not affect ADCC as it lacks the Fc portion of CSL362. Fab’CSL362 was used in these assays to compare the effect of IL-3 blockade alone to that of ADCC on pDCs and basophils.

3.2.6 Evaluating the effect of CSL362 on NK cell activation ex vivo

PBMCs (0.5x106) were cultured with 0.01 µM CSL362, Fab’CSL362 and two isotype controls for 18-21 hours. The first isotype control (‘isotype control’) contained the same Fc portion as CSL362, which has been modified to increase affinity for CD16. The second isotype control (‘isotype 2’) contained an unmodified IgG1-Fc. All cultures contained a 1:1000 dilution of GolgiStop (BD Biosciences, Catalog number 554754) to promote intracellular recycling of CD107a. The percentage of viable (Sytox Blue-) CD107a+ NK cells (CD14-, CD3- CD56dim/CD56+) was analysed on a MACSQuant Analyzer (Miltenyi Biotec).

3.2.7 Evaluating the effect of CSL362, and two anti-IFNα mAbs, on TLR1- 9 agonist, or SLE serum-stimulated IFNα production and IFN- upregulated gene expression ex vivo

PBMCs (0.5-1.0x106) were cultured with 0.01 µM CSL362, Fab’CSL362, isotype control, or one of two recombinantly produced and purified anti-IFNα mAbs (KEGG DRUGS database numbers: D09668 and D09662) for 6-24 hours. Following this, in order to stimulate IFNα production and induce IFN-upregulated gene expression, PBMCs were cultured with either TLR1-9 agonists, (Human TLR1-9 agonist kit, InvivoGen, Catalog number tlrl-kit1hw) or 50% serum from SLE patients with low (3.4-7.1 IU/ml), medium (91.8-104.3 IU/ml) or high (>470 IU/ml) anti-dsDNA Ab levels (as measured by radioimmunoassay; normal range 0-4 IU/ml) for 18 hours.

60 The TLR agonists were used in the following concentrations: • TLR1 (pam3csk4) 1 µg/ml • TLR2 (HKLM) 1x108 cells/ml • TLR3 (Poly IC) 10 µg/ml • TLR4 (LPS) 10 µg/ml • TLR5 (flagellin) 2 µg/ml • TLR6 (FSL-1) 1 µg/ml • TLR7 (imiquimod) 2 µg/ml • TLR8 (ssRNA40) 2 µg/ml and • TLR9 (CpG C ODN 2395) 0.5 µM.

When SLE serum was used, fetal calf serum was omitted from standard culture medium.

At the end of the incubation time, culture supernatants were collected and frozen at - 20°C for later measurement of IFNα levels by ELISA.

Cell pellets were frozen at -80°C in Qiazol Lysis reagent (Qiagen, Catalog number 79306) for later RNA extraction.

3.2.8 Evaluating the effect of depleting pDCs and basophils on IFNα production ex vivo

PBMCs from healthy and SLE donors were depleted of pDCs (Lin1- HLA-DR+, BDCA2++ CD123++) or basophils (Lin1- CCR3+, CD123++) by sorting on a FACSAria (BD Biosciences). pDC-depleted and basophil-depleted PBMCs (1.0x106) were cultured for 18 hours with 0.5 µM CpG C (Invivogen, Catalog number tlrl-2395- 1) or 2 µg/ml imiquimod (Invivogen, Catalog number tlrl-imqs). At the end of the incubation time, culture supernatants were collected and frozen at -20°C for later measurement of IFNα levels by ELISA.

61 3.2.9 Extraction of RNA from PBMCs and whole blood for assessment of IFN-inducible gene expression

RNA extraction from PBMCs, and from whole blood stored in PAXgene tubes, was performed with the miRNeasy Mini Kit (Qiagen, Catalog number 217004). The Ambion TURBO DNA-free kit (Invitrogen, Catalog number AM1907) was used to remove contaminating DNA before RNA was converted to cDNA using the Superscript III First-Strand Synthesis SuperMix for qRT-PCR kit (Invitrogen, Catalog number 11752-050). All kits were utilized as instructed by the manufacturer.

Expression of a panel of eleven IFNα-upregulated genes (IFI44L, IFIT1, IFIT3, IRF7, ISG15, MX1, MX2, OAS1, OAS2, SERPING1 and XAF1) was determined using Taqman customized gene arrays (Applied Biosystems, Catalog number 4342247) analyzed on the 7900HT Fast RT-PCR System (Applied Biosystems).

3.2.10 RNA Sequencing of isolated pDCs

Healthy donor PBMCs from buffy coats were cultured for 18 hours with 0.5µM CpG C, 0.5 µg/ml imiquimod, or media alone for 18 hours. Following culture, cells were stained with Lin1 FITC and magnetically sorted using an EasySep FITC kit (Stemcell Technologies, Catalog number 18552) keeping only the negative unlabeled fraction. This Lin1- enriched fraction was then stained with Lin1, BDCA1, BDCA2, HLA-DR, CD11c, CD123 and propidium iodide (PI, BD, Catalog number 556463) and sorted using a BD FACS Fusion. pDCs were sorted as PI-, Lin1-, HLA-DR+, CD11c-, BDCA2+ and CD123+. Sorted pDCs were immediately stored in RNAprotect Cell Reagent (Qiagen, Catalog number 76526) and frozen at -80°C until RNA extraction using an RNeasy plus micro kit (Qiagen, Catalog number 74034). RNA was then submitted to the Australian Genome Research Facility for next generation sequencing on the Illumina HiSeq platform.

The data generated from this RNASeq have been deposited in NCBI’s Gene Expression Omnibus303 and are accessible through GEO Series accession number GSE79272 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE79272).

62

3.2.11 Evaluating the effect of CSL362, anti-IFNα mAbs and an anti- IFNAR mAb on TLR7 and TLR9 agonist-, and CD40L-induced, plasmablast expansion and proliferation ex vivo

PBMCs (0.5x106) were cultured with 1 µg/ml CSL362, one of two recombinantly produced anti-IFNα mAbs, an anti IFNAR mAb (anti-human interferon alpha/beta receptor chain 2 mAb, PBL Assay Science, Catalog number 21385-1) or isotype control, for 24 hours.

0.5 µM CpG C, CpG B/ODN2006 (InvivoGen, Catalog number tlrl-2006), CpG A/ODN 2216 (InvivoGen, Catalog number tlrl-2216), imiquimod or 0.5 µg/ml CD40L (R&D Systems, Catalog number 6245-CL-050) was then added to culture for 6 days to stimulate plasmablast expansion.

At the end of the incubation time, the percentage of viable (Sytox Blue-) naïve B cells (CD19+, CD27-), memory B cells (CD19+, CD27+) or plasmablasts (CD19+ CD27++, CD20- CD38++) were analysed on an LSR Fortessa (BD Biosciences). Additionally, culture supernatant was frozen at -20°C for later analysis of BAFF, and immunoglobulin levels.

To assess proliferation of the B cell subsets, cells were labeled with CFSE (Life Technologies, Catalog number C34554) prior to culture for 7 days, as described above.

3.2.12 Evaluating the effect of reconstituting CSL362-treated PBMCs with pDCs or basophils, or pDC and basophil conditioned medium, on TLR7- and TLR9-induced plasmablast expansion pDCs and basophils were isolated from healthy donor PBMCs using the Human Plasmacytoid Dendritic Cell Isolation II Kit (Miltenyi Biotec, Catalog 130-097-415) or the Human Basophil Isolation Kit II (Miltenyi Biotec, Catalog number 130-092- 662) respectively, on an AutoMACS ProSeparator (Miltenyi Biotec). Non-depleted

63 PBMCs (0.5x105) were treated with 1 µg/ml CSL362 or isotype control for 24 hours and then washed three times to ensure drug removal. Cultures were reconstituted with isolated pDCs or basophils, at varying concentrations, and stimulated with 0.5 µM CpG C, or 0.5 µM imiquimod, for 6 days. The percentage of viable plasmablasts (CD19+ CD27++, CD20- CD38++) was then analysed on an LSR Fortessa (BD Biosciences).

To produce conditioned media, isolated pDCs and basophils (1.5x105) were cultured with 0.5 µM CpG C, imiquimod, or media alone for 24 hours. Supernatants were then added to PBMCs (0.5x105) that had been pre-treated for 24 hours with 1 µg/ml of CSL362 or isotype control. 0.5 µM CpG C or imiquimod, or media alone was added to the culture for 6 days. The percentage of viable plasmablasts (CD19+ CD27++, CD20- CD38++) was then analysed on an LSR Fortessa (BD Biosciences). Conditioned media was also stored at -80°C for later analysis of cytokine levels by ELISA and Luminex.

3.2.13 Enzyme-linked immunosorbent assays

IFNα levels in supernatant and conditioned medium were quantified with the VeriKine Human IFN-α Multi-subtype ELISA kit (PBL Assay Science, Catalog number 41105).

BAFF and IL-3 levels in culture supernatant and conditioned medium were determined by the BAFF Quantikine ELISA kit (R&D Systems, Catalog number SBLYS0B) and the IL-3 Duo Set (R&D Systems, Catalog number DY203) respectively.

Type III IFN levels in supernatant were assessed with the DIY Human IFN lambda 3/1/2 (IL-28B/29/28A) ELISA kit (PBL Assay Science, Catalog number 61840).

Type II IFN levels were determined using the VeriKine Human Interferon Gamma ELISA Kit (PBL Assay Science, Catalog number 41500-1).

64 All kits were used as per manufacturer’s protocols. ELISAs were analysed on a Wallac Envision Multilabel Reader (Perkin Elmer, Catalog number 2104-0010).

3.2.14 Luminex and Bioplex assays

Levels of twenty-five cytokines in conditioned media were analysed using Milliplex Multiplex Assays (Merck Millipore, Catalog number HT17MG-14K-PX25) on a Luminex 200 analyser. The cytokines analysed were GM-CSF, IFNγ, MIP-3α, TNF- α, TNF-β, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17A, IL-17E/IL-25, IL-17F, IL-21, IL-22, IL-23, IL-27, IL-28A, IL-31, and IL-33.

Immunoglobulin levels (IgG1, IgG2, IgG3, IgG4, IgA and IgM) in culture supernatant were determined using the Bio-Plex Pro Human Isotyping Panel (Biorad, Catalog number 171A3100M), on a Luminex 200 analyser.

Luminex and Bioplex kits were used as instructed by the manufacturer.

3.2.15 Evaluating the effect of blockade of IFNα, IL-6 and TNF-α by neutralizing mAbs on plasmablast expansion restored by conditioned medium produced by stimulating pDCs with CpG C

PBMCs (0.5x106) were cultured with 0.5 µM CpG C for 6 days, following pre- treatment with 1 µg/ml of CSL362 for 24 hours. Conditioned medium from healthy donor pDCs that had been stimulated with CpG C was added to restore plasmablast expansion at the same time as the CpG, in addition to 50 µg/ml of either an anti-IFNα mAb, an anti-IL-6 mAb (R&D Systems, Catalog number MAB2061) or an anti-TNF- α mAb (Etanercept, Pfizer). The percentage of viable plasmablasts (CD19+ CD27++, CD20- CD38++) was then analysed on an LSR Fortessa (BD Biosciences).

65 3.2.16 Evaluating the effect of subcutaneously administered CSL362 on pDCs, basophils, and IFNα-inducible gene expression in cynomolgus macaques

Naïve cynomolgus monkeys were administered a single dose (1 mg/kg, 10 mg/kg or 30 mg/kg) of CSL362 subcutaneously. Peripheral blood was collected at various time points for analysis of CSL362 serum levels by ELISA, and pDC (Lin1- HLA-DR+, BDCA2+ CD123+) and basophil (IgE+ CD123+) numbers by flow cytometry on an LSR Fortessa (BD Biosciences). PBMCs were cultured with CpG C for 24 hours, after which expression of a panel of IFNα inducible genes (IFI35, IFIT1, IRF7, MX1, MX2, OAS1) were determined by quantitative PCR, on the 7900HT Fast RT-PCR System (Applied Biosystems).

3.2.17 Statistical analyses

Comparison between two groups was analysed with the Mann Whitney U test. A p value of < 0.05 was considered statistically significant. Spearman’s rank correlation was used to determine correlations between two variables. Statistical analyses were performed with GraphPad Prism Software (Version 6.0).

66

3.3 Results

3.3.1 pDCs and basophils have high CD123 expression and are selectively depleted by CSL362

Cell surface expression of CD123 was examined on peripheral blood cells from SLE (n = 34), autoimmune disease control (n = 20) and healthy control donors (n = 34). Of the cell subsets evaluated, pDCs and basophils had the highest CD123 expression (~40,000 and 20,000 receptors/cell respectively) (Figure 3-1), with expression being highest on pDCs in most donors. Expression on all other cell types examined was much lower and ranged from ~2,000 receptors/cell in mDCs, intermediate monocytes and eosinophils to < ~1000 receptors/cell in the other cell types. These results confirm that SLE patients have high expression of CD123 on pDCs and basophils relative to other cells.

CD123 expression was significantly higher in healthy donor basophils and eosinophils as compared to SLE donors (Table 3-4). Healthy donor classical monocytes had significantly lower expression of CD123 compared to autoimmune disease donors, although as the autoimmune disease group contained patients with a number of different diseases, it is difficult to ascertain from these data if this difference is of any physiologic significance.

There was no correlation of CD123 expression on pDCs or basophils with disease activity, as measured by the SLEDAI (r = 0.061 [95% CI -0.290 – 0.400], p = 0.732 for pDCs, and r = -0.19 [95% CI -0.500 – 0.170], p = 0.291 for basophils). There was also no statistically significant difference in CD123 expression on pDCs or basophils in SLE donors who were, or were not, taking immunosuppressive or immunomodulatory medications, however comparison was difficult as there were only three donors who were not taking any medications for SLE treatment.

67

Healthy 105 Autoimmune SLE 104

103

102 CD123 number per cell

101 pDCs CD4+ CD8+ Naïve mDCs CD56+ Memory Classical CD56dim Basophils Neutrophils Eosinophils Intermediate

classical Non Plasmablasts

NK cells B cells Monocytes T cells

Figure 3-1: CD123 expression is highest on pDCs and basophils in SLE, autoimmune and healthy donors.

CD123 expression on peripheral blood cells in SLE donors (n = 34) and healthy (n = 34) and autoimmune (n = 20) controls, as determined by flow cytometry using Quantibrite-PE beads. The number of CD123 molecules per cell is shown for each donor.

68

Table 3-4: CD123 expression on SLE, healthy and autoimmune donor cell types

CD123 expression, receptors/cell p value (median) Cell type Healthy AI SLE Healthy Healthy AI vs (n = 34) (n = 20) (n = 34) vs SLE vs AI SLE

pDCs 40407 39530 38751 0.978 0.908 0.908

Basophils 24635 20176 16441 0.002 0.156 0.114

mDCs 1825 1853 2122 0.063 0.010 0.250

CD56dim NK 205 238 230 0.910 0.979 0.880 cells

CD56+ NK 209 238 255 0.650 0.824 0.894 cells

Naïve B cells 376 330 317 0.050 0.324 0.613

Memory B 486 333 436 0.207 0.934 0.187 cells

Plasmablasts 542 505 448 0.158 0.413 0.653

Non classical 680 698 750 0.786 0.677 0.729 monocytes

Intermediate 1812 1685 1722 0.066 0.439 0.377 monocytes

Classical 385 435 315 0.055 0.026 0.360 monocytes

Neutrophils 627 660 474 0.100 0.126 0.770

Eosinophils 1263 1112 884 0.007 0.235 0.332

CD4+ T cells 217 194 196 0.119 0.429 0.664

CD8+ T cells 247 230 214 0.063 0.141 0.894

AI = autoimmune

69 Given the selective, high-level CD123 expression on pDCs and basophils, the ability of the anti-CD123 mAb, CSL362, to deplete these cell types, was next evaluated. An isotype control mAb (isotype control) and Fab’CSL362 (the Fab fragment of CSL362 which lacks the Fc portion but retains IL-3 neutralizing activity) were used as comparisons. At 24 hours, pDCs were potently and reproducibly depleted by CSL362, but not the isotype control or Fab’CSL362 (Figure 3-2A and B), demonstrating that ADCC, and not IL-3 neutralization, is the main mechanism of depletion. Basophils were less completely depleted than pDCs (Figure 3-2A and C), possibly due to higher circulating numbers and relatively lower CD123 expression.

SLE donors had significantly lower depletion of pDCs compared to healthy (mean percentage depletion of pDCs was 81.6 ± 3.2 in SLE donors, compared to 86.5 ± 5.4 in healthy donors, p = 0.012) and autoimmune donors (mean percentage depletion of pDCs in autoimmune donors was 91.2 ± 1.9, p value = 0.032). Depletion of basophils in SLE donors was also significantly lower than in healthy donors (mean percentage depletion of basophils was 49.9 ± 5.6 in SLE donors, compared to 67.8 ± 6.3 in healthy donors, p value = 0.014). This may reflect decreased CD123 expression on basophils and pDCs in SLE donors, as compared to healthy controls (Table 3-4).

70

A 250K No treatment CSL362 Fab'CSL362 Isotype control 200K

150K

100K

50K pDCs

!"# 0 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 SSC BDCA2 105 No treatment CSL362 Fab'CSL362 Isotype control

104

103 Basophils

102 0

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 CCR3 Lin1 B C

Figure 3-2: pDCs and basophils are depleted by CSL362.

(A) Representative flow cytometric analysis from SLE donors of viable pDCs (Sytox Blue-, Lin1- HLA-DR+, BDCA2++) and basophils (Sytox Blue-, Lin1- CCR3+) after 24 hour culture with media alone (no treatment), CSL362, Fab’CSL362 or isotype control. Percentage of viable (B) pDCs and (C) basophils, determined by flow cytometry, after 24 hour culture with 0.01 µM CSL362, Fab’CSL362 or isotype control compared to media alone in SLE (n = 30), healthy (n = 25) and autoimmune donors (n = 18). Data expressed as mean ± SEM, * p < 0.05 (Mann Whitney test).

71 A higher dose of Fab’CSL362 also depleted pDCs, but not basophils (Figure 3-3), reflecting the requirement of IL-3 for pDC survival.280 Basophils also rely on IL-3 for survival,304 however may not have been depleted at these doses compared to pDCs, due to their relatively higher circulating numbers in peripheral blood.

A

B

Figure 3-3: IL-3 blockade with Fab’CSL362 depletes pDCs, but not basophils, at higher doses.

Percentage of viable (A) pDCs (Sytox Blue-, Lin1- HLA-DR+, BDCA2++) and (B) basophils (Sytox Blue-, Lin1- CCR3+) following 24 hour culture with CSL362, Fab’CSL362 and isotype control at three doses (0.001 µM, 0.01 µM and 0.1 µM) compared to media alone. Data expressed as mean ± SEM, * p < 0.05 (Mann Whitney test), compared to equivalent dose isotype control, from SLE (n = 6), autoimmune (n = 6) and healthy (n = 7) donors.

Depletion by CSL362 was selective for cells expressing high levels of CD123 (pDCs and basophils), as cells that expressed lower CD123 levels (mDCs, monocytes, NK, B and T cell subsets) were not depleted (Figure 3-4).

72 Figure 3-4: No depletion of A B C CD123lo cells with CSL362 treatment.

Percentage of viable (Sytox Blue-) (A) mDCs, (B) CD56dim NK cells, (C) CD56+ NK cells, (D) monocytes, (E) naïve B cells, (F) D E F memory B cells, (G) plasmablasts, (H) CD4+ T cells or (I) CD8+ T cells, following 24 hour culture with CSL362 or isotype control at three doses (0.001, 0.01 or 0.1 µM) compared to no treatment. Data expressed as mean ± SEM, * p < G H I 0.05 (Mann Whitney test), compared to equivalent dose isotype control from SLE, autoimmune and healthy donors (n = 5 each).

73

3.3.2 CSL362 activates NK cells, via its modified IgG1-Fc

CSL362 has been engineered for enhanced ADCC through mutations in its Fc region that increase its affinity for FcγRIII, CD16, expressed on NK cells.273 Therefore the effect of CSL362 on NK cell activation (percentage of CD107a+ NK cells) was examined. Fab’CSL362 and the isotype control, and a second isotype control (isotype 2) with an unmodified IgG1-Fc, were used for comparison. CSL362 and the isotype control with the modified IgG1-Fc, but not Fab’CSL362 or isotype control 2, activated NK cells in SLE, autoimmune and healthy control donors (Figure 3-5), confirming that the modified IgG1-Fc mediates NK cell activation.

74 NK cells CD107a+

No treatment

CSL362 Fab’CSL362 Isotype control Isotype control 2

Figure 3-5: CSL362 activates NK cells, via its modified IgG1-Fc.

Fold change of % viable CD107a+ NK cells, determined by flow cytometry, after 18-21 hour culture with 0.01 µM CSL362, Fab’CSL362 or two isotype controls compared to media alone for SLE (n = 12), healthy (n = 12) and autoimmune (n = 11) donors. Isotype control contains the same modified Fc region as CSL362, isotype control 2 contains an unmodified IgG1-Fc. Data expressed as mean ± SEM, * p < 0.05 (Mann Whitney test).

75 3.3.3 Depletion of pDCs by CSL362 inhibits TLR7-, TLR9- and SLE serum- stimulated IFNα-production and IFNα-inducible gene expression

Given the potent depletion of pDCs by CSL362, the effect of CSL362 on TLR- stimulated IFNα production and IFN-inducible gene expression was next evaluated. Pre-treatment with CSL362 inhibited TLR7 (imiquimod) and TLR9 (CpG C) stimulated Color Key and Histogram IFNα production in both SLE and healthy donors (Figure 3-6A), whereas TLR4 (LPS) 250 Count

stimulated production was not significantly100 reduced by CSL362. 0

0 2 4 6 8 Value B

TLR1 IFI44L TLR1 IFIT1 TLR1 IFIT3 TLR1 IRF7 TLR1 ISG15 TLR1 MX1 TLR1 MX2 TLR1 OAS1 TLR1 OAS2TLR1 TLR1 SERPING1 TLR1 XAF1 TLR2 IFI44L TLR2 IFIT1 TLR2 IFIT3 TLR2 IRF7 TLR2 ISG15 TLR2 MX1 TLR2 MX2 TLR2 OAS1 TLR2 OAS2TLR2 A TLR2 SERPING1 TLR2 XAF1 TLR3 IFI44L TLR3 IFIT1 TLR3 IFIT3 TLR3 IRF7 TLR3 ISG15 TLR3 MX1 TLR3 MX2 TLR3 OAS1TLR3 TLR3 OAS2 TLR3 SERPING1 TLR3 XAF1 TLR4 IFI44L TLR4 IFIT1 TLR4 IFIT3 TLR4 IRF7 TLR4 ISG15 TLR4 MX1 TLR4 MX2 TLR4 OAS1TLR4 TLR4 OAS2 TLR4 SERPING1 TLR4 XAF1 TLR5 IFI44L TLR5 IFIT1 TLR5 IFIT3 TLR5 IRF7 TLR5 ISG15 TLR5 MX1 TLR5 MX2 TLR5 OAS1 TLR5 OAS2TLR5 TLR5 SERPING1 TLR5 XAF1 TLR6 IFI44L TLR6 IFIT1 TLR6 IFIT3 TLR6 IRF7 TLR6 ISG15 TLR6 MX1 TLR6 MX2 TLR6 OAS1 TLR6 OAS2TLR6 TLR6 SERPING1 TLR6 XAF1 TLR7 IFI44L TLR7 IFIT1 TLR7 IFIT3 TLR7 IRF7 TLR7 ISG15 TLR7 MX1 TLR7 MX2 TLR7 OAS1 TLR7 OAS2TLR7 TLR7 SERPING1 TLR7 XAF1 TLR8 IFI44L TLR8 IFIT1 TLR8 IFIT3 TLR8 IRF7 TLR8 ISG15 TLR8 MX1 TLR8 MX2 TLR8 OAS1 TLR8 OAS2 TLR8 SERPING1TLR8 TLR8 XAF1 TLR9 (CpG A) IFI44L TLR9 (CpG A) IFIT1 TLR9 (CpG A) IFIT3 TLR9 (CpG A) IRF7 TLR9 (CpG A) ISG15 TLR9 (CpG A) MX1 TLR9 (CpG A) MX2 TLR9 (CpG A) OAS1 TLR9 (CpG A) OAS2 TLR9 (CpGTLR9 A) SERPING1 (CpG A) TLR9 (CpG A) XAF1 TLR9 (CpG B) IFI44L TLR9 (CpG B) IFIT1 TLR9 (CpG B) IFIT3 TLR9 (CpG B) IRF7 TLR9 (CpG B) ISG15 TLR9 (CpG B) MX1 TLR9 (CpG B) MX2 TLR9 (CpG B) OAS1 TLR9 (CpGTLR9 B) OAS2 (CpG B) TLR9 (CpG B) SERPING1 TLR9 (CpG B) XAF1 TLR9 (CpG C) IFI44L TLR9 (CpG C) IFIT1 TLR9 (CpG C) IFIT3 TLR9 (CpG C) IRF7 TLR9 (CpG C) ISG15 TLR9 (CpG C) MX1 TLR9 (CpG C) MX2 TLR9 (CpG C) OAS1 TLR9 (CpG C) OAS2 TLR9 (CpGTLR9 C) SERPING1 (CpG C) TLR9 (CpG C) XAF1 Isotype CSL362

Isotype1 controlIsotype2 Isotype3 Isotype4 CSL362 1 ColorCSL362 2 KeyCSL362 3 CSL362 4 and Histogram 250 Count 100 0

0 2 4 6 8 Fold change overValue unstimulated

TLR1 IFI44L TLR1 IFIT1 TLR1 IFIT3 TLR1 IRF7 TLR1 ISG15 TLR1 MX1 TLR1 MX2 TLR1 OAS1 TLR1 OAS2 Figure 3-6: CSL362 potently and specifically inhibits TLR7- and TLR9-induced IFNα TLR1 SERPING1 TLR1 XAF1 TLR2 IFI44L TLR2 IFIT1 TLR2 IFIT3 TLR2 IRF7 TLR2 ISG15 TLR2 MX1 TLR2 MX2 TLR2 OAS1 TLR2 OAS2 TLR2 SERPING1 TLR2 XAF1 TLR3 IFI44L production and IFNα-inducible gene expression. TLR3 IFIT1 TLR3 IFIT3 TLR3 IRF7 TLR3 ISG15 TLR3 MX1 TLR3 MX2 TLR3 OAS1 TLR3 OAS2 TLR3 SERPING1 TLR3 XAF1 TLR4 IFI44L TLR4 IFIT1 TLR4 IFIT3 TLR4 IRF7 TLR4 ISG15 TLR4 MX1 TLR4 MX2 TLR4 OAS1 TLR4 OAS2 TLR4 SERPING1 TLR4 XAF1 (A) IFNα production, measured by ELISA, in response to TLR4, TLR7 and TLR9 stimulation TLR5 IFI44L TLR5 IFIT1 TLR5 IFIT3 TLR5 IRF7 TLR5 ISG15 TLR5 MX1 TLR5 MX2 TLR5 OAS1 TLR5 OAS2 TLR5 SERPING1 TLR5 XAF1 TLR6 IFI44L TLR6 IFIT1 TLR6 IFIT3 following CSL362 or isotype control pre-treatment of healthy and SLE donor (n = 3 each) TLR6 IRF7 TLR6 ISG15 TLR6 MX1 TLR6 MX2 TLR6 OAS1 TLR6 OAS2 TLR6 SERPING1 TLR6 XAF1 TLR7 IFI44L TLR7 IFIT1 TLR7 IFIT3 TLR7 IRF7 TLR7 ISG15 TLR7 MX1 peripheral blood mononuclear cells (PBMCs). (B) Expression of 11 IFN-inducible genes TLR7 MX2 TLR7 OAS1 TLR7 OAS2 TLR7 SERPING1 TLR7 XAF1 TLR8 IFI44L TLR8 IFIT1 TLR8 IFIT3 TLR8 IRF7 TLR8 ISG15 TLR8 MX1 TLR8 MX2 TLR8 OAS1 TLR8 OAS2 (IFI44L, IFIT1, IFIT3, IRF7, ISG15, MX1, MX2, OAS1, OAS2, SERPING1, XAF1), determined TLR8 SERPING1 TLR8 XAF1 TLR9 (CpG A) IFI44L TLR9 (CpG A) IFIT1 TLR9 (CpG A) IFIT3 TLR9 (CpG A) IRF7 TLR9 (CpG A) ISG15 TLR9 (CpG A) MX1 TLR9 (CpG A) MX2 TLR9 (CpG A) OAS1 TLR9 (CpG A) OAS2 TLR9 (CpG A) SERPING1 TLR9 (CpG A) XAF1 TLR9 (CpG B) IFI44L by qPCR, in response to TLR1-9 agonist stimulation following CSL362 or isotype control pre- TLR9 (CpG B) IFIT1 TLR9 (CpG B) IFIT3 TLR9 (CpG B) IRF7 TLR9 (CpG B) ISG15 TLR9 (CpG B) MX1 TLR9 (CpG B) MX2 TLR9 (CpG B) OAS1 TLR9 (CpG B) OAS2 TLR9 (CpG B) SERPING1 TLR9 (CpG B) XAF1 TLR9 (CpG C) IFI44L TLR9 (CpG C) IFIT1 TLR9 (CpG C) IFIT3 TLR9 (CpG C) IRF7 TLR9 (CpG C) ISG15 treatment in healthy donor PBMCs (n = 4). Data expressed as mean ± SEM, * p < 0.05 (Mann TLR9 (CpG C) MX1 TLR9 (CpG C) MX2 TLR9 (CpG C) OAS1 TLR9 (CpG C) OAS2 TLR9 (CpG C) SERPING1 TLR9 (CpG C) XAF1 Whitney test). Isotype1 Isotype2 Isotype3 Isotype4 CSL362 1 CSL362 2 CSL362 3 CSL362 4

76

There was robust induction of an IFN gene ‘signature’, comprising eleven previously described IFN-inducible genes73 (IFI44L, IFIT1, IFIT3, IRF7, ISG15, MX1, MX2, OAS1, OAS2, SERPING1, XAF1) in healthy donors after TLR3, TLR4, TLR7, TLR8 and TLR9 stimulation, but weak induction by TLR5 (Figure 3-6B). The TLR7- and TLR9-stimulated IFN gene signature was completely and selectively neutralized by CSL362.

As CSL362 specifically depletes pDCs and basophils, and pDCs selectively express TLR7 and TLR9,305 this data suggests that the decrease in IFNα production and gene expression is a direct effect of pDC depletion. This was confirmed by removing pDCs or basophils from peripheral blood mononuclear cells (PBMCs) by FACS-sorting. Removal of pDCs, but not basophils, markedly reduced TLR7- and TLR9-induced IFNα production (Figure 3-7). These data imply that other cell types are unable to replace TLR7- or TLR9-induced IFNα production in the absence of pDCs and importantly, also show that global IFNα production is not affected by CSL362, as cells other than pDCs can produce IFNα in response to TLR3, TLR4, TLR5 and TLR8 stimulation.

10,000 * * * * 1,000 pg/ml

α 100 10 IFN 1 TLR7 TLR9 pDC depleted PBMCs Basophil depleted PBMCs Non depleted PBMCs

Figure 3-7: Depletion of pDCs, not basophils, by CSL362 inhibits TLR7- and TLR9- induced IFNα production.

IFNα production, determined by ELISA, from healthy (n = 2) and SLE (n = 2) donor PBMCs that were depleted either of pDCs or basophils and stimulated with TLR7 (imiquimod) or TLR9 (CpG C) agonist. Data expressed as mean ± SEM, * p < 0.05 (Mann Whitney test).

77

Next, an IFN gene score was developed based on the targeted IFN gene ‘signature’ (described above) to assess drug efficacy. The panel of eleven IFN-inducible genes was incorporated into a single gene score to facilitate comparison between SLE and healthy donors. The derived IFN gene score was calculated as the average of the log2 fold change in expression of the 11 genes compared to that of a universal healthy control. The gene score for most healthy donors was close to zero, whereas SLE donors had an average gene score of ~3.0, representing an ~ 8-fold change (Figure 3-8), consistent with prior exposure to type I IFN in this population.

* 10

5

0

Gene score -5

-10 Healthy SLE

Figure 3-8: IFN-inducible gene expression is higher in SLE donors compared to healthy controls.

IFN-inducible gene expression, expressed as a single gene score, in SLE (n = 31) and healthy (n

= 35) donor whole blood. Gene score represents the average log2 fold change in expression of 11 IFN-inducible genes (IFI44L, IFIT1, IFIT3, IRF7, ISG15, MX1, MX2, OAS1, OAS2, SERPING1, XAF1) compared to a universal healthy donor. IFN-inducible gene expression determined by qPCR. Data expressed as mean ± SEM, * p < 0.05 (Mann Whitney test).

78

The effect of CSL362 on TLR7- and TLR9-induced IFNα production and the IFN gene score was subsequently examined in a population of SLE, autoimmune and healthy donors. The ability of TLR7 agonist imiquimod and TLR9 agonist CpG C to stimulate IFNα production ex vivo from donor PBMCs was variable, with CpG C being a more reliable, and robust, stimulus of IFNα production than imiquimod.

CpG C stimulated significant (defined as > 50 pg/ml) IFNα production ex vivo in all (eleven) healthy controls and (twelve) autoimmune disease controls, and all but one (of ten) SLE patients in whom the assay was performed. This TLR9-induced IFNα production was essentially negated in all donors with CSL362 pre-treatment, but not with Fab’CSL362 or isotype control (Figure 3-9A). CSL362 pre-treatment also reduced the IFN gene score as compared to isotype control (Figure 3-9B).

Imiquimod stimulated IFNα production in only three of nine SLE donors, four of eleven healthy donors and six of seven autoimmune donors. In these TLR7 responsive donors, CSL362 also potently inhibited IFNα production, as compared with Fab’CSL362 or isotype control treatment, (Figure 3-9C) with a trend towards a decrease in IFN- inducible gene expression following CSL362 treatment (Figure 3-9D).

79

A B

C D

Figure 3-9: CSL362 potently inhibits TLR7- and TLR9- induced IFNα production and IFN-inducible gene expression in SLE, autoimmune disease, and healthy donors.

(A) IFNα production from SLE (n = 9), autoimmune (n = 12) and healthy (n = 11) donor peripheral blood mononuclear cells (PBMCs) and, (B) IFN-inducible gene expression, expressed as a single gene score for SLE (n = 10) and healthy (n = 9) donor PBMCs stimulated with TLR9 agonist (CpG C), following CSL362, Fab’CSL362 ((A) only) or isotype control pre- treatment. (C) IFNα production from SLE (n = 3), autoimmune (n = 6) and healthy (n = 4) donor peripheral blood mononuclear cells (PBMCs) and, (D) IFN-inducible gene expression, expressed as a single gene score for SLE (n = 8) and healthy (n = 7) donor PBMCs stimulated with TLR7 agonist (imiquimod), following CSL362, Fab’CSL362 ((C) only) or isotype control pre-treatment. The IFN gene score represents the average log2 fold change in expression of 11 IFN-inducible genes (IFI44L, IFIT1, IFIT3, IRF7, ISG15, MX1, MX2, OAS1, OAS2, SERPING1, XAF1) for each treatment compared to no treatment. IFN-inducible gene expression determined by qPCR, IFNα production by ELISA. Data expressed as mean ± SEM, * p < 0.05 (Mann Whitney test).

80 Immune complexes or other components of SLE serum may contribute to IFNα production independently of TLR7 and TLR9 activation. Therefore, IFNα production induced by SLE serum which had varying levels of anti-dsDNA antibody (Ab) titres was evaluated. Serum with low (3.4-7.1 IU/ml), medium (91.8-104.3 IU/ml) or high anti-dsDNA (>470 IU/ml) Ab levels was used to stimulate IFNα production in healthy donor PBMCs. Serum from donors with a high anti-dsDNA Ab level intrinsically contained a small amount of detectable IFNα, and when cultured with healthy PBMCs was able to stimulate further IFNα production (Figure 3-10A). Importantly, the IFNα that was produced by serum stimulation was completely inhibited by CSL362 (Figure 3-10A). Serum from the donors with low and medium anti-dsDNA Ab levels was not able to stimulate detectable IFNα production when cultured with healthy donor PBMCs, however it was able to upregulate expression of IFN-inducible genes, and this expression was also decreased by pre-treatment with CSL362 (Figure 3-10B).

81 A

Color Key and Histogram 4 3 2 Count 1 0

10 12 14 16 Value B

IFI44LIFI44L IFIT1IFIT1 IFIT3IFIT3 IRF7IRF7 ISG15ISG15 MX1MX1 MX2MX2 OAS1OAS1 OAS2OAS2 SERPING1SERPING1 XAF1XAF1

Color Key and Histogram 4 mAb mAb 3 Media 2 Isotype Isotype BM4 S007 stim BM4 S008 stim No drug No stim No No CSL362 CSL362 Count CSL362 S007 stim CSL362 S008 stim No drug S007 stim No drug S008 stim 1

Medium Low 0 anti-dsDNA anti-dsDNA 10 12 14 16 log of geneValue expression Ab serum Ab serum 2

IFI44L

IFIT1

Figure 3-10: CSL362 inhibits SLE serum-induced IFNα production and IFN-inducible IFIT3 gene expression from healthy donor PBMCs. IRF7 ISG15

(A) IFNα production from healthy donor (n = 3) PBMCs pre-treated with CSL362 or isotype MX1 MX2 control, then stimulated with 50% SLE serum containing high anti-dsDNA antibody levels. The OAS1 level of IFNα detectable in the serum alone (no PBMC control), is shown in the ‘serum alone’ OAS2 condition. (B) IFN-inducible gene expression in a healthy donor’s PBMCs stimulated with 50% SERPING1 XAF1 SLE serum containing low or medium anti-dsDNA antibody levels, following CSL362 or isotype control pre-treatment. IFN-inducible gene expression determined by qPCR, IFNα BM4 S007 stim BM4 S008 stim production by ELISA. Data expressed as mean ± SEM. No drug No stim CSL362 S007 stim CSL362 S008 stim No drug S007 stim No drug S008 stim

82 3.3.4 CSL362 inhibits IFN-inducible gene expression more effectively than IFNα blockade alone, and inhibits type III IFN production

Anti-IFNα mAbs sifalimumab and rontalizumab have shown efficacy and tolerability in phase 2 clinical trials,85, 86 setting a benchmark for IFN targeting therapies in SLE. Using published DNA sequences and recombinant methods, two anti-IFNα mAbs were produced and purified in order to be able to compare the effect of CSL362 to the approach of blocking IFNα alone. Both anti-IFNα mAbs neutralized TLR7- and TLR9- stimulated IFNα, with > 90% neutralization at doses ≥ 10 µg/ml. However, CSL362 reduced IFNα more potently, with complete inhibition at equivalent doses (Figure 3- 11A). Although the anti-IFNα mAbs neutralized IFNα, these had minimal impact on reducing TLR-induced IFN-upregulated gene expression in the IFN gene ‘signature’, as compared to CSL362 (Figure 3-11B).

This may be because activated pDCs produce more than one type of IFN that may contribute to the IFN gene signature. Elevated levels of multiple type I IFN subtypes (IFNA, IFNB1, IFNW, IFNE), as well as type III IFN (IFNL1, IFNL2, IFNL3) levels were seen by RNA sequencing of isolated pDCs stimulated with TLR7 or TLR9 agonists (Figure 3-12). Type II IFN (IFNG) levels were however not differentially expressed.

83 A

Color Key and Histogram 15 Count 5 0

!5 0 5 10 ValueB

hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 TLR1hMX2 hOAS1 hOAS2 hSERPING1 hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 TLR2hMX2 hOAS1 hOAS2 hSERPING1 hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 hMX2 TLR3hOAS1 hOAS2 hSERPING1 hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 hMX2 TLR4hOAS1 hOAS2 hSERPING1 hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 hMX2 TLR5hOAS1 hOAS2 hSERPING1 hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 hMX2 hOAS1 TLR6hOAS2 hSERPING1 hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 hMX2 hOAS1 TLR7hOAS2 hSERPING1 hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 hMX2 hOAS1 TLR8hOAS2 hSERPING1 hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 hMX2 hOAS1 TLR9hOAS2 (CpG A) hSERPING1 hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 hMX2 hOAS1 TLR9hOAS2 (CpG B) hSERPING1 hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 hMX2 hOAS1 TLR9hOAS2 (CpG C) hSERPING1 hXAF1

! ! Color Key BM4 A B

CSL362 and Histogram SIFALIMUMAB RONTALIZUMAB mAb mAb 15 Isotype

CSL362 Anti-IFN Anti-IFN Count 5 0

!5 0 5 10 Value log2 fold change over unstimulated

hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 hMX2 hOAS1 hOAS2 hSERPING1 hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 hMX2 hOAS1 hOAS2 hSERPING1 hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 Figure 3-11: CSL362 more effectively inhibits TLR7- and TLR9-stimulated IFN hMX1 α hMX2 hOAS1 hOAS2 hSERPING1 hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 hMX2 hOAS1 production and IFN-upregulated gene expression compared to type I IFN blockade. hOAS2 hSERPING1 hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 hMX2 hOAS1 hOAS2 hSERPING1 hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 hMX2 hOAS1 (A) IFNα production in response to TLR9 (CpG C) or TLR7 (imiquimod) stimulation of healthy hOAS2 hSERPING1 hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 hMX2 hOAS1 hOAS2 hSERPING1 donor peripheral blood mononuclear cells (PBMCs) (n = 2), and (B) expression of 11 IFN- hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 hMX2 hOAS1 hOAS2 hSERPING1 hXAF1 hIFI44L hIFIT1 inducible genes (IFI44L, IFIT1, IFIT3, IRF7, ISG15, MX1, MX2, OAS1, OAS2, SERPING1, hIFIT3 hIRF7 hISG15 hMX1 hMX2 hOAS1 hOAS2 hSERPING1 hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 XAF1) in response to TLR1-9 stimulation in healthy donors (n = 4), following pre-treatment hMX1 hMX2 hOAS1 hOAS2 hSERPING1 hXAF1 hIFI44L hIFIT1 hIFIT3 hIRF7 hISG15 hMX1 hMX2 hOAS1 with CSL362, isotype control or two anti-IFNα mAbs (A and B). IFN-inducible gene expression hOAS2 hSERPING1 hXAF1 BM4 determined by qPCR, IFNα production by ELISA. Data expressed as mean ± SEM. CSL362 SIFALIMUMAB RONTALIZUMAB

84

Type I IFNA IFN

IFNB1 IFNW IFNE IL28A/IFNL2 Type IL28B/IFNL3 III 0 4 8 12 IL29/IFNL1 IFN

log2cpm TLR9 TLR7 Uns@mulated

Figure 3-12: Activation of pDCs with TLR7 and TLR9 agonist stimulation upregulates type I and type III IFN gene expression.

Expression of type I (IFNA, IFNB1, IFNW, IFNE) and type III (IFNL1, IFNL2, IFNL3) IFN subtypes in response to TLR9 or TLR7 stimulation in healthy donors (n = 6), assessed by RNA sequencing of isolated pDCs. Type II IFN (IFNG) was not differentially expressed.

85

The effect of CSL362 on TLR7- and TLR9-induced type III IFN (IFNλ1, IFNλ2 and IFNλ3) levels was then assessed in nine healthy, and six SLE donors. Three healthy, and two SLE, donors responded to TLR9 (CpG) stimulation with detectable (defined as > 50 pg/ml) type III IFN levels, as assessed by ELISA, which was reduced to negligible levels in all donors by CSL362 treatment (Figure 3-13A). In the three healthy, and three SLE donors that responded to TLR7 (imiquimod) stimulation, CSL362 treatment led to decreased production of IFNλ in these donors (Figure 3-13) that approached significance (p = 0.09). In contrast, there was no significant inhibition by CSL362 of TLR7- or TLR9-stimulated type II (IFNγ) production in these same donors (Figure 3- 13B).

A B

Figure 3-13: CSL362 inhibits TLR7- and TLR9- stimulated type III IFN (IFNλ), but not type II IFN (IFNγ) production.

TLR7- and TLR9-stimulated (A) IFNλ (TLR9 – n = 3 healthy, n = 2 SLE; TLR7 – n = 3 healthy and n = 3 SLE) and (B) IFNγ (n = 9 healthy, n = 6 SLE) production, following pre-treatment with CSL362 or isotype control. IFNλ and IFNγ production determined by ELISA. Data expressed as mean ± SEM, * p < 0.05 (Mann Whitney test).

86

3.3.5 CSL362 inhibits TLR7- and TLR9-induced plasmablast expansion and proliferation

Virally-activated pDCs have been found to promote CD40L-stimulated plasmablast differentiation through IFNα and IL-6.306 I found that TLR7 (imiquimod) and TLR9 stimulation of PBMCs was also able to expand the same plasmablast population, reaching maximal expansion after six days (Figure 3-14). Of the three TLR9 agonists used, CpG B and CpG C were more effective than CpG A in stimulating plasmablast expansion, confirming findings from pre-existing literature.307 CpG C, rather than CpG B, was chosen for subsequent assays to stimulate plasmablast expansion, as it had been used in previous assays to stimulate IFNα production.

The effect of CSL362 on TLR7- and TLR9-induced plasmablast (CD19+ CD27++, CD20- CD38++) expansion was then evaluated. Pre-treatment with CSL362, but not isotype control, inhibited plasmablast expansion in both healthy and SLE donors (Figure 3-15), although inhibition in SLE donors was less robust than in the healthy controls (Figure 3-16). This may be due to less effective depletion of pDCs by CSL362 in SLE donors, as previously seen in Figure 3-2.

Analysis of plasma cells (CD19+ CD27++, CD20- CD38++, CD138++) was attempted, however, even when stimulated with TLR7 or TLR9 agonist, this population was too small to allow subsequent meaningful comparison between treated samples.

87 Day 0 Day 1 Day 2 Day 3 Day 5 Day 6

5

4

1

2

3

CD27 CD19

6 1 unstimulated CpG A s l l

e 4 2 CpG B c

a CpG C m

s 3 Imiquimod plasmablasts a l p

2 IL-3 % 4 5

% viable 0

day 0 day 1 day 2 day 3 day 5 day 6 Day 0 Day 1 Day 2 Day 3 Day 5 Day 6

1. TLR9 - CpG B 4. TLR9 - CpG A 2. TLR 9 - CpG C 5. Unstimulated 3. TLR7 - Imiquimod

Figure 3-14: Expansion of plasmablasts is maximal after six days of stimulation with TLR7 and TLR9 agonists.

Representative data from one (of three) healthy donors showing plasmablast (CD19+ CD27++) expansion in response to stimulation with TLR9 agonists CpG A, B and C, and TLR7 agonist imiquimod at days 0, 1, 2, 3, 5 and 6.

88

A TLR9 TLR7 Isotype control CSL362 Isotype control CSL362

105 3.25 1.36 1.2 0.156 104

103

102 0

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 CD27 CD19

105 79.9 30.7 77.8 35.2 104 Plasmablasts 103

102 0

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 CD38 CD20 TLR9 TLR7 B Isotype control CSL362 Isotype control CSL362

105

104 4.49 2.31 0.631 0.292 103

102 0

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 CD27 CD19

105

104 81.4 41.7 50.4 21.5

103 Plasmablasts

102 0

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 CD38 CD20

Figure 3-15: CSL362 inhibits TLR7- and TLR9-induced plasmablast expansion.

Representative flow cytometric analysis from a (A) healthy and (B) SLE donor, of viable plasmablasts (Sytox Blue-, CD19+ CD27++, CD20- CD38++) after stimulation with TLR9 (CpG C) or TLR7 (imiquimod) agonists, following CSL362 or isotype control pre-treatment.

89

A B

Figure 3-16: Inhibition of plasmablast expansion by CSL362 is more effective in healthy donors compared to SLE donors.

Viable plasmablasts, expressed as a fold change compared to no treatment, following CSL362 or isotype control pre-treatment in the presence of (A) TLR9 (CpG C) or (B) TLR7 (imiquimod) stimulation, for SLE (n = 9) and healthy (n = 10) donors. Data expressed as mean ± SEM, * p < 0.05 (Mann Whitney test).

90

TLR9 stimulation induced a greater plasmablast expansion than TLR7 and a higher concentration of CSL362 was required to suppress this TLR9-stimulated response (Figure 3-17).

A B

Figure 3-17: TLR9 induces greater plasmablast expansion than TLR7.

Representative data from a healthy donor showing % of viable plasmablasts, determined by flow cytometry, after stimulation with CpG (TLR9) or imiquimod (TLR7), following pre- treatment with CSL362 or isotype control at varying doses (n = 2).

Using CFSE staining, cellular proliferation in response to TLR7 and TLR9 agonists was found to be inhibited by CSL362 in both memory B cells and plasmablasts (Figure 3- 18). This suggested that either pDCs or basophils provide a proliferative stimulus to B cells when activated by TLR7 or TLR9 agonists. Interestingly, the induction of naïve B cell proliferation by TLR9 stimulation was not inhibited by CSL362, suggesting that TLR9-induced proliferation of naïve B cells is a direct effect.

91

100 8.44 50.4 21.4 80 7.44 57 33.4 60

40

20

0 100 5.26 17.9 15.8 80 3.09 36.9 39.7 60

40

20

0

100 30.6 40.6 29.7 80 17.8 57.5 53.9 60

40

20

0 2 3 4 5 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10

Figure 3-18: CSL362 inhibits proliferation of memory B cells and plasmablasts, but not naïve B cells.

Representative flow cytometric analysis (from n = 2 healthy donors) of the proliferation of naïve and memory B cells, and plasmablasts, assessed by CFSE labelling, in the presence TLR9 (CpG) or TLR7 (imiquimod) agonist, following CSL362 or isotype control pre-treatment. Percentages of proliferating (CFSE negative) cells are shown for each condition.

92

3.3.6 pDCs activated by TLR7 and TLR9 stimulation produce IFNα and IL- 6, which promote plasmablast expansion

To determine the contribution of pDCs and basophils to plasmablast expansion, each cell type was separately reconstituted into CSL362 pre-treated cultures. Reconstitution with pDCs, but not basophils, was able to restore TLR9-induced plasmablast expansion (Figure 3-19).

To determine whether the reconstitution effect was direct, or via secreted factors, conditioned medium (CM) from pDCs was added to CSL362 pre-treated cultures. Conditioned medium from pDCs stimulated with TLR9 agonist (CpG) was able to restore plasmablast expansion (Figure 3-20A) and did so in a dose-dependent manner (Figure 3-20B), whereas CM from non-stimulated pDCs did not induce plasmablast expansion. These data suggested that a soluble factor or factors produced by CpG- activated pDCs promote plasmablast expansion.

Similar experiments were performed using TLR7 agonist imiquimod as a stimulus, however in these experiments, there was insufficient plasmablast expansion to allow a comparison of non-reconstituted and reconstituted cultures.

93 A CSL362 - + + + pDCs - - + - Basophils - - - + IC + - - -

105

104 11.2 7.12 10.1 1.29 103

102 0

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 CD27 CD19

105

104 92.4 61.7 94.6 60.7

103

102 0

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 CD38 CD20 B C

Figure 3-19: Reconstitution of pDCs, but not basophils, into CSL362 pre-treated cultures, restores plasmablast expansion.

(A) Representative flow cytometric analysis from a healthy donor, of viable plasmablasts (Sytox Blue-, CD19+ CD27++, CD20- CD38++) following reconstitution of pDCs or basophils into CSL362 or isotype control (IC) pre-treated, and TLR9 (CpG)-stimulated PBMC cultures. (B and C) Viable plasmablasts, expressed as a percentage compared to isotype control, as determined by flow cytometry, following reconstitution of (B) pDCs or (C) basophils into CSL362 pre-treated, and TLR9-stimulated PBMC cultures (n = 4 healthy donors). Data expressed as mean ± SEM, * p < 0.05 (Mann-Whitney test).

94 A CSL362 - + + Neg CM - + - CpG CM - - +

105 2.96 2.42 2.95

104

103

102 0

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 CD27 CD19

105

4 10 80.6 39.9 88.1

103

102 0

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 CD38 CD20

B

Figure 3-20: Conditioned medium from activated pDCs restores plasmablast expansion that is inhibited by CSL362.

(A) Representative flow cytometric analysis from a healthy donor of viable plasmablasts in PBMCs stimulated with TLR9 agonist (CpG), following CSL362 or isotype control pre- treatment. Cultures were supplemented with conditioned medium from isolated pDCs cultured with TLR9 agonist (CpG CM) or media alone (neg CM). (B) Viable plasmablasts, determined by flow cytometry, after reconstitution of CSL362 or isotype control pre-treated healthy PBMCs (n = 4) with increasing concentrations of pDC conditioned media (CpG CM or neg CM) and stimulation with TLR9 agonist. Data expressed as a percentage compared to media alone. Error bars represent mean ± SEM.

95 To identify the soluble factor or factors elaborated by stimulated pDCs that promote plasmablast expansion, Luminex and ELISA analysis of the CM was undertaken. Twenty-eight cytokines were analysed and of these, only IFNα, IL-6 and TNF-α were differentially elevated in TLR9-stimulated pDC CM (Figure 3-21) when compared to CM from TLR9-stimulated basophils or unstimulated pDC CM. Levels of BAFF, GM- CSF, IFNγ, MIP-3α, TNF-β, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12p70, IL- 13, IL-15, IL-17A, IL-17E/IL-25, IL-17F, IL-21, IL-22, IL-23, IL-27, IL-28A, IL-31 and IL-33 were negligible.

A B

C

Figure 3-21: IFNα, IL-6 and TNF-α are elevated in conditioned medium produced by stimulating pDCs with TLR9 agonist CpG C.

Levels of (A) IFNα (B) IL-6 and (C) TNF-α in conditioned medium from n = 5 healthy donors, as determined by ELISA (IFNα) and Luminex assay (IL-6 and TNF-α). pDC+ and pDC- represent conditioned media from isolated pDCs stimulated (+) or not (-) with CpG C. Baso+ and baso– represent the same for stimulated (+) or not (-) basophils. Levels of BAFF, GM-CSF, IFNγ, MIP-3α, TNF-β, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL- 17A, IL-17E/IL-25, IL-17F, IL-21, IL-22, IL-23, IL-27, IL-28A, IL-31 and IL-33 were negligible.

96 Furthermore, inhibition of IFNα and IL-6, but not TNF-α, by neutralizing mAbs, prevented restoration of plasmablast expansion with TLR9-stimulated pDC CM (Figure 3-22). These data indicate that IFNα and IL-6 produced by CpG-activated pDCs stimulate plasmablast expansion.

10

8

6

4

2 % viable plasmablasts 0 CpG mAb mAb α α No mAb No Neg CM CpG CM + IL6 mAbs anti-IL6 mAb anti-IL6 α No stimulation No anti-IFN anti-TNF CSL362 + CpG CSL362 anti-IFN

CSL362 + CpG + CpG CM

Figure 3-22: Inhibition of IFNα and IL-6, but not TNF-α, by neutralizing mAbs, prevents restoration of plasmablast expansion with TLR9-stimulated pDC CM, in CSL362 pre- treated cultures.

Viable plasmablasts, expressed as a percentage of live cells, as determined by flow cytometry, in TLR9 (CpG)-stimulated PBMCs, following CSL362 pre-treatment. Cultures were supplemented with CpG CM in the absence of neutralizing antibodies (no mAb) or presence of anti-IFNα, anti-IL-6 or anti-TNF-α antibodies (n = 3 healthy donors). Data expressed as mean ± SEM.

97 3.3.7 CSL362 inhibits TLR9-induced plasmablast expansion more effectively than IFNα or IFNAR blockade alone

Again, CSL362 was compared to two anti-IFNα mAbs, in this instance for the ability to inhibit plasmablast expansion stimulated by either TLR7 or TLR9 agonists, or CD40 ligand (CD40L). CD40L induces plasmablast expansion through a non-TLR mediated mechanism and does not activate pDCs or induce IFNα production. CSL362 inhibited TLR-induced, but not CD40L-induced, plasmablast expansion (Figure 3-23), again suggesting that CSL362 does not directly affect plasmablast expansion. Inhibition by CSL362 was associated with depletion of pDCs and basophils.

In contrast, the anti-IFNα mAbs did not significantly reduce plasmablast expansion or deplete pDCs (Figure 3-23). It is possible that the concentration of anti-IFNα mAbs used were not sufficient to neutralize all pDC-produced IFNα. This suggests that depleting pDCs more efficiently prevents TLR-induced plasmablast expansion than inhibiting IFNα alone, possibly because activated pDCs produce additional cytokines, such as IL-6, that promote plasmablast expansion, as demonstrated above.

98

CpG Imiquimod CD40L 200 120 200 160 90 160 120 120 60 80 80 * (% control)

(% control) 30 40 (% control) * 40 plasmablasts plasmablasts plasmablasts 0 0 0 CpG Imiquimod CD40L 250 250 200 200 200 160 150 150 120 pDC pDC

pDCs 100 100 80 (% control) (% control) (% control) 50 50 40 0 * 0 * 0 * CpG Imiquimod CD40L 150 150 150

100 100 100 50 50 50 basophils basophils basophils (% control) (% control) (% control) 0 * 0 * 0 * Isotype control Anti-IFNα mAb A CSL362 Anti-IFNα mAb B

Figure 3-23: CSL362 more effectively inhibits TLR7- and TLR9-, but not CD40L-induced plasmablast expansion than IFNα blockade alone.

Viable plasmablasts, pDCs and basophils, determined by flow cytometry, from healthy donors (n = 4), expressed as a percentage compared to media alone, following pre-treatment with CSL362, isotype control or two anti-IFNα mAbs (A and B) and stimulation with TLR9 agonist (CpG), TLR7 agonist (imiquimod) or CD40L. Data expressed as mean ± SEM, * p < 0.05 (Mann-Whitney test).

99 An anti-IFNAR mAb, anifrolumab, is currently recruiting patients for a phase 3 clinical trial (NCT02446899), and has been shown in a small study to be more effective in suppressing a 21 gene IFN gene signature compared with the anti-IFNα mAb sifalimumab.88 A commercially available anti-IFNAR mAb was compared with CSL362 for the ability to inhibit plasmablast expansion stimulated by imiquimod, CpG C and CD40L. CSL362 was more effective in inhibiting TLR9-induced plasmablast expansion than both IFNα blockade, or IFNAR blockade, alone (Figure 3-24). In this assay, imiquimod and CD40L did not stimulate sufficient plasmablast expansion to allow comparison between different treatments.

4 *

3

2

1 % viable plasmablasts

0

TLR9 TLR7 CD40L No mAb Anti-IFNα mAb A Anti-IFNα mAb B Anti-IFNAR mAb CSL362 Isotype control

Figure 3-24: CSL362 more effectively inhibits TLR9-induced plasmablast expansion compared with IFNAR blockade.

Viable plasmablasts expressed as a percentage of live cells, as determined by flow cytometry, from healthy (n = 3) and SLE (n = 2) donors, following pre-treatment with CSL362, isotype control, two anti-IFNα mAbs (A and B) or an anti-IFNAR mAb, and stimulation with TLR9 agonist (CpG), TLR7 agonist (imiquimod) or CD40L. Data expressed as mean ± SEM, * p < 0.05 (Mann-Whitney test).

100 To determine the effect of CSL362 treatment on BAFF and Ig production ex vivo, BAFF and Ig levels were examined by ELISA and Bioplex respectively, in culture supernatant following TLR7 and TLR9 stimulation, and CSL362 or isotype control pre- treatment. BAFF levels after 24 hours, or 7 days, of TLR7 and TLR9 stimulation were negligible and did not allow comparison between CSL362 and isotype control treated conditions. IgG1, IgG2, IgG3, IgG4, IgM and IgA levels were not significantly different between CSL362 and isotype control treated samples (Figure 3-25).

A B

Figure 3-25: No difference in immunoglobulin levels after CSL362 treatment.

IgA, IgG1-4 and IgM levels in culture supernatant of n = 6 donors (n = 3 SLE and n = 3 healthy), following CSL362 and isotype control pre-treatment, and (A) CpG and (B) imiquimod stimulation, after 7 days. Immunoglobulin levels determined by Bioplex assay. Data expressed as mean ± SEM.

101 3.3.8 Subcutaneous administration of CSL362 to cynomolgus macaques depletes pDCs and basophils in vivo and inhibits TLR9-induced IFNα- inducible gene expression

Finally, the in vivo effect of CSL362 on pDCs, basophils and IFN-inducible gene expression was examined when administered subcutaneously (sc), which is usually the preferred route of delivery of biological therapeutics in chronic diseases such as SLE, due to its convenience. CSL362 does not cross react with mouse CD123; however, there is a high degree of sequence homology between human and cynomolgus macaque (cyno) CD123, which is also highly expressed on cyno pDCs and basophils. In addition, CSL362 demonstrated similar affinity between human and cyno CD123 and FcγRs and comparable activity in cell-based assays.273

Naïve cynomolgus monkeys were treated with a single sc injection of CSL362 at varying doses (1, 10, 30 mg/kg). Maximal serum concentrations of CSL362 were detected at 48 hours (~12, 190 and 380 µg/ml at doses of 1, 10 and 30 mg/kg respectively). Serum CSL362 was maintained above 1 µg/ml at day 7 for the 1 mg/kg dose and above 30 µg/ml for 14 days at the higher doses (Figure 3-26A). pDC depletion was achieved as early as 6 hours after administration for the highest dose and was maximal between days 5-15 for all doses (Figure 3-26, B and C). Similarly, basophils were also depleted by sc administration of CSL362 (Figure 3-26, B and D). These effects followed the peak serum drug levels and were maintained for four weeks at the two highest doses for pDCs, and for two weeks for basophils (Figure 3-26, B-D).

The effect of in vivo pDC depletion by CSL362 on IFN-induced gene expression was examined by stimulating PBMCs, isolated at various timepoints after CSL362 administration, with CpG for 24 hours ex vivo. A decrease in IFN-inducible gene expression was observed following CSL362 administration (Figure 3-26E). The decrease in serum drug levels from day 22 onwards correlated with recovery of pDC numbers and an associated increase in CpG-stimulated IFNα-inducible gene expression.

102 The effect of CSL362 on stimulated IFNα production was examined by ELISA, however pre-dose IFNα levels were negligible, making comparison post dose difficult.

Importantly, sc administration of CSL362 was well tolerated at all doses and no overt toxicity was observed.

103

A C

D

B 104 Pre dose 6 Hr Day 36

103 pDCs 102 E 101 5.05 0.233 3.97 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

!"#$%& CD123 104 Pre dose 6 Hr Day 36 103 Basophils

2 10 0.225 0.029 0.112

101

100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 IgE CD123

Figure 3-26: Subcutaneous administration of CSL362 to cynomolgus macaques depletes pDCs and basophils, and inhibits TLR9-induced IFN-upregulated gene expression.

(A) Serum concentration of CSL362, determined by ELISA at various time points following subcutaneous administration of CSL362 (at doses of 1, 10 and 30 mg/kg) to cynomolgus macaques. (B) Representative flow cytometric analysis showing pDCs (Lin1- HLA-DR+, BDCA2+ CD123+) and basophils (IgE+ CD123+) at baseline, and at 6 hours and day 36 post single dose of CSL362. At various time points following subcutaneous administration of CSL362, peripheral blood (C) pDCs and (D) basophils as determined by flow cytometry and expressed as % of baseline (pre-treatment). (E) Expression of six IFN-inducible genes (IFI35, IFIT1, IRF7, MX1, MX2, OAS1), expressed as a single gene score, in peripheral blood mononuclear cells (PBMCs) cultured with TLR9 agonist (CpG), as determined by qPCR. The gene score represents the average log2 fold change in the 6 IFN-inducible genes over unstimulated control. Data expressed as mean ± SEM for n = 3 animals per dosing group.

104 3.4 Discussion

The data from this Chapter show that a humanized monoclonal antibody targeting the IL-3Rα (CSL362) has effects ex vivo on several key cell types and cytokines involved in SLE, and has biological activity in vivo in non-human primates after subcutaneous administration.

CSL362 potently depletes pDCs, leading to selective abrogation of TLR7-, TLR9-, and SLE serum-induced IFNα production and reduced expression of a panel of IFNα- inducible genes. Of note, others have shown that depletion of pDCs ameliorates disease in murine models of lupus,91, 168 suggesting that pDC depletion in human SLE may be an effective therapeutic strategy. CSL362 was also found to deplete basophils, although not as potently as pDCs, possibly due to their higher circulating numbers in peripheral blood, and relatively lower CD123 expression. Depletion of pDCs by CSL362 was shown to inhibit TLR7- and TLR9-induced plasmablast expansion through decreasing the production of IFNα and IL-6, which are elaborated by activated pDCs. The decrease in plasmablast expansion did not however correlate with a significant decrease in immunoglobulin production.

Although IL-3 is a known survival factor for both pDCs280 and basophils,304 pDC and basophil depletion by CSL362 was shown to be largely dependent upon ADCC and NK cell activation. NK cells in SLE have been found to have an activated phenotype but normal ADCC capability, despite reduced CD16 expression.308 Importantly, the data from this study show that NK cells derived from patients with SLE can still be activated through the modified IgG1-Fc portion of CSL362, which is critical to cell depletion through enhanced ADCC.

The data also suggests that depletion of pDCs, rather than blockade of type I IFN alone, may be a more effective strategy to inhibit IFN driven processes such as gene expression and plasmablast expansion in response to stimuli, such as immune complexes, that activate TLR7 and TLR9 in SLE. This may be because CSL362, by depleting pDCs, reduces the production of not only type I, but type III IFN. There is

105 some evidence of aberrant regulation of type III IFN in SLE, with elevated serum levels found as compared to healthy controls.96 Increased levels of type III IFN were correlated with disease activity, anti-dsDNA antibody levels, glomerulonephritis and arthritis.97 Although type III IFNs signal through a separate receptor complex to type I IFNs, they share common signaling pathways and as a result, have been postulated to be responsible for ongoing disease activity despite therapeutic type I IFN blockade.95 Inhibition of multiple IFN types may therefore confer therapeutic advantage in these situations.

SLE is characterized by auto-reactive antibodies, which reflect aberrant activation of B cells and differentiation into antibody-producing plasma cells. The data from this study suggests that pDC depletion as a therapeutic strategy may potentially combat two major pathogenic targets in SLE simultaneously, namely inhibiting IFNα production and B cell expansion. The data in this study shows that by depleting pDCs with CSL362, plasmablast expansion and proliferation in response to TLR7 and TLR9 agonists is inhibited, although in SLE donors this appears to be less robust than in healthy donors. Plasmablast expansion occurred in response to soluble factors released by activated pDCs – specifically, IFNα and IL-6, confirming the importance of IFNα and IL-6 in promoting plasmablast expansion found previously in a virally-activated, CD40L- dependent system.306 Specific depletion of pDCs in murine models of SLE has not been found to alter peripheral plasma cell numbers; however, germinal centres in the spleen were abolished and there was a reduction in anti-dsDNA and anti-RNA antibodies.91, 168 It is possible that depleting pDCs may differentially affect short-lived compared to long-lived plasma cells in different compartments. Interestingly, this study found that TLR9 agonist CpG C was a more robust ex vivo stimulus of plasmablast expansion than TLR7 agonist imiquimod, and that the concentration of CSL362 required to inhibit imiquimod-induced plasmablast expansion closely followed that required to deplete pDCs and basophils. This suggests that the contribution of pDCs and soluble factors produced by pDCs to TLR7-stimulated plasmablast expansion may be greater than for TLR9.

106 Three other compounds that target pDCs are currently in development as potential treatments for SLE. The first is an inhibitor of the pro-apoptotic protein Bcl-2, based on the hypothesis that increased levels of Bcl-2 in SLE may inhibit the apoptosis of auto- reactive lymphocytes. The Bcl-2 inhibitor ABT-199 (Venetoclax) has been shown in a murine model172 and in humans,173 to decrease lymphocyte numbers, and to deplete pDCs without depleting conventional DCs in mice and ex vivo in humans.120 It has so far been well tolerated in a phase I trial.173 The second drug is a monoclonal antibody targeting the pDC-specific cell surface marker BDCA2, which has been shown to inhibit TLR7- and TLR9-induced type I IFN production through internalization of BDCA2 and CD32. This mAb, BIIB059, has recently completed recruitment in a phase I trial (NCT02106897), although no study results are yet available. The third drug is a proteasome inhibitor, bortezomib, which was found to decrease production of IFNα in NZB/W F1 lupus prone mice, through inhibiting both pDC survival and their ability to produce IFNα in response to stimulation with a number of different ligands, including TLR9 and TLR3.174 The efficacy and safety of bortezomib in SLE is currently being examined in a phase 2 trial (NCT02102594).

The ability of CSL362 to deplete basophils and inhibit IL-3 signaling may be of additional benefit above its ability to target pDCs, distinguishing it from other agents targeting pDCs.

Basophils in SLE have been less studied compared to pDCs, but can be activated by IgE-containing immune complexes. Upon activation, basophils may augment autoantibody production by eliciting a Th2 response and production of the B-cell survival factor BAFF.275, 276 Basophil depletion alleviated nephritis in a murine lupus model276 and therapeutic targeting of basophils in SLE is currently being explored in a phase 1 trial of an anti-IgE mAb, omalizumab (NCT01716312).

IL-3 is a known maturation and survival factor for pDCs280 and basophils.281 In this study, it was found that IL-3 blockade with higher doses of Fab’CSL362 depleted pDCs. Although IL-3 has not been extensively studied in SLE, elevated serum IL-3 levels have been reported in active SLE patients.285 More recently, administration of IL-

107 3 in the MRL/lpr murine lupus model was found to exacerbate nephritis, and this was improved by IL-3 blockade, suggesting an important role for IL-3 in the progression of lupus nephritis.283 Therefore, the potential beneficial effects of CSL362 in SLE may extend to its ability to neutralize IL-3 in addition to depletion of pDCs and basophils.

The cell types and cytokines affected by CSL362 are involved in protection against a number of infectious agents – viruses (pDCs/IFN) and helminths and parasites (basophils, and IL-3 via its effect on increasing basophils and mast cells during parasitic infections).154, 309, 310 An increase in herpes zoster infection was seen in the phase 2 trials of the anti-IFNAR mAb anifrolumab,87 and the anti-IFNα mAb sifalimumab.86 Compared to the blockade of all type I IFNs, or IFNα, CSL362 specifically targets IFN produced via TLR7- and TLR9- stimulation, potentially leaving some residual IFN production through other TLRs, or non-TLR mediated pathways, which may translate to a decreased risk of viral infections. A phase 1 trial in AML (NCT01632852) using intravenously administered CSL362 in doses ranging from 0.3-12.0 mg/kg has recently completed.311 In that study, there were no increased infections despite rapid (≤ 6 hours post dose) and complete pDC and basophil depletion at all doses for a fortnightly dosing frequency, which was sustained for ≥ 15 days for doses ≥ 3mg/kg; however, a phase 2 trial will provide data regarding longer-term infection risk. In the current study, the first use of subcutaneous administration of CSL362 was evaluated in cynomolgus monkeys. This approach was taken because subcutaneous administration has a range of benefits in chronic diseases such as, for example, convenience of administration. Single, subcutaneously administered doses of CSL362 to cynomolgus macaques were well tolerated and the biological effects were reversible, with recovery of depleted pDCs and basophils, and of IFN-inducible gene expression, occurring in step with waning serum drug levels.

Collectively, the data from this Chapter provides a rationale for the further evaluation of CSL362 as a therapeutic agent in SLE in a clinical trial. Furthermore, in this study CSL362 was also shown to be effective ex vivo in depleting pDCs and reducing IFNα production in patients with a variety of type I IFN associated autoimmune diseases, including psoriasis, scleroderma, primary Sjögren’s syndrome, inflammatory

108 myopathy,299 and rheumatoid arthritis.151 As in SLE, IFN therapy has been associated with the development or worsening of scleroderma, and elevated type I IFN levels have been observed in scleroderma patient blood, as has increased expression of IFN- inducible genes and proteins in blood and skin. In a recent phase 1 trial in scleroderma, IFN receptor blockade showed promising results, with near complete inhibition of IFN- stimulated gene expression in peripheral blood and skin.302 In contrast, IFNα blockade had no clinical activity against plaque psoriasis,312 although IFN receptor (through which both IFNα and IFNβ signal) blockade may be more effective than IFNα blockade alone. Although there were only small numbers of donors with each particular autoimmune condition tested in this study, the data suggests further pre-clinical evaluation of CSL362 for these other IFN-driven diseases is worthwhile.

109 Chapter 4: Investigating a potential role for IL-3 in SLE through serum cytokine analysis and transcriptional profiling

4.1 Introduction

4.1.1 Potential role for IL-3 in SLE

IL-3 is a multipotential haematopoietic growth factor that contributes to the maturation and survival of a number of immune cell types. The role of IL-3 in SLE has been relatively poorly studied compared to other cytokines, such as type I IFN or BAFF. One study from the 1990s demonstrated elevated serum IL-3 levels in SLE patients compared to healthy controls.285 These levels were detected by an in-house generated ELISA, and correlated with a commercial kit for IL-3 detection. The study was conducted on a relatively small study population of sixteen patients, none of whom were receiving immunosuppressive treatment at the time of the study. It was noted that the two patients with the lowest platelet counts also had the lowest IL-3 levels, possibly because IL-3 acts as a megakaryocyte-promoting factor. A separate study found that pregnant SLE patients with the anti-phospholipid syndrome had decreased IL-3 levels.313

Murine models have identified a potential role for IL-3 in lupus pathogenesis. The sera from MRL/lpr mice was shown to contain a factor with IL-3 activity, which promoted proliferation of IL-3 dependent cell lines.286 A study in the C3H/gld murine model suggested that this factor may have been serum IgG rather than IL-3 itself.287 A different study in MRL/lpr mice established hybridomas from mouse spleen cells that secreted antibodies which supported the growth of IL-3 dependent cell lines. These antibodies were able to inhibit the binding of IL-3 to the cell lines and vice versa, raising the possibility that they were directed against the IL-3 receptor, and acted in a stimulatory fashion.288

110 A recent study in the MRL/lpr murine lupus model more directly demonstrated its role in SLE pathogenesis, with IL-3 administration exacerbating nephritis, and IL-3 blockade alleviating disease and decreasing auto-antibody production.283 Plasma levels of IL-3 increased during disease progression, and IL-3 was found to be produced by CD4+ T cells in the spleen and bone marrow, and by CD8+ T cells in the spleen.

These data raise the possibility of IL-3 blockade as a therapeutic strategy in SLE. Pre- clinical testing of an anti-IL-3 mAb in a murine rheumatoid arthritis model showed decreased progression and severity of disease, and a reduction in pro-inflammatory cytokines IL-6 and TNF-α,290 however this has not yet translated into a human therapeutic clinical trial.

The work in this Chapter aimed to further understanding of the role of IL-3 in human SLE, by measuring serum IL-3 levels in a cohort of SLE patients receiving standard of care therapy, and healthy subjects. The IL-3 levels were correlated with (i) patient clinical information, (ii) peripheral blood cell types that play a pathogenic role in SLE and that are affected by IL-3 (that is, pDCs, basophils, B cells and T cells), and (iii) serum cytokines known to be altered in SLE and/or altered by CSL362, such as type I IFN and BAFF. The presence or absence of an ‘IL-3 gene signature’ was also evaluated for in a separate cohort of SLE and healthy donors. Although mean IL-3 levels were not elevated in SLE patients compared to healthy controls, an ‘IL-3 gene signature’ distinguishing SLE from healthy donors was found. Additionally, a novel association between IL-3 and IFN was seen in SLE and healthy donors, both through serum cytokine analysis and transcriptional profiling. This association raises the possibility that those with an IFN gene signature may also benefit from the therapeutic targeting of IL-3 in SLE.

111 4.2 Material and methods

4.2.1 Human subjects and collection of biological samples and clinical information

Serum cytokine analysis, and quantification of peripheral blood cell types was performed on SLE patient samples from The Royal Melbourne Hospital, and healthy donor samples from The Walter and Eliza Hall Institute’s Volunteer Blood Donor Registry. These donors were recruited, and blood samples and clinical information collected, as outlined in Chapter 2 - SLE patients and healthy donors were age and sex matched. Other clinical details (disease activity, disease manifestations and drug treatments) of these SLE patients are as outlined in Chapter 2. All blood samples collected were processed and/or serum frozen within 6 hours of collection.

Whole blood gene signature analysis was performed on 31 SLE patients recruited from The Monash Medical Centre (Monash Lupus Clinic), a large tertiary referral and teaching hospital that provides services to southeastern metropolitan Melbourne, and regional and rural Victoria. Whole blood gene signature analysis was also performed on 28 age and sex matched healthy donors recruited from The Skin and Cancer Foundation of Victoria, between August 2014 and February 2015. Ethical approval for these studies was granted by the Monash Health HREC #15427L, and from the Skin and Cancer Foundation HREC #2012-05-812. Formal written consent was obtained from each donor prior to participation in the study. Whole blood from each donor was collected into PAX gene Blood RNA tubes (BD, Catalog number 762165), which were frozen at - 20°C for later RNA extraction.

4.2.2 Serum cytokine analysis

All assays were performed on stored (-80°C) serum samples.

112 Serum IL-3, IFNα, BAFF and type III IFN levels were quantified by ELISA on a Wallac Envision Multilabel Reader (Perkin Elmer, Catalog number 2104-0010).

Serum IL-3 levels were quantified by the IL-3 Duo Set (R&D Systems, Catalog number DY203).

Serum IFNα levels were analysed with the VeriKine Human Serum Multisubtype IFNα ELISA kit (PBL Assay Science, Catalog number 41110-1). This ELISA kit detects fourteen human IFNα subtypes.

BAFF and type III IFN serum levels were determined with the BAFF Quantikine ELISA kit (R&D Systems, Catalog number SBLYS0B) and the DIY Human IFN lambda 3/1/2 (IL-28B/29/28A) ELISA kit (PBL Assay Science, Catalog number 61840) respectively.

Serum levels of G-CSF, GM-CSF, Flt-3l, IFNγ, IL-2, IL-4, IL-5, IL-6, IL-13, IP-10 and TNFα were determined using Milliplex Multiplex Assay (Merck Millipore, Catalog number #HCTYMAG-60K-PX38) on a Luminex 200 analyzer.

All kits were utilized as per manufacturer’s protocol.

The lower and upper limit of detection for each assay were as follows: • IL-3 ELISA: 15.625 - 2000 pg/ml • IFNα ELISA: 12.5 - 1000 pg/ml • Type III IFN ELISA: 62.5 – 4000 pg/ml • BAFF ELISA: 62.5 – 4000 pg/ml • Luminex assays: 3.2 – 2000 pg/ml

Where a detected value was extrapolated to be lower than that of the lower limit of detection for the assay, a number half way between the lower limit of the assay, and zero, was assigned.314 Conversely, when a detected value was higher than the upper

113 limit of the assay, a value equal to the upper limit of detection for the assay was assigned.

4.2.3 Measurement of peripheral blood cell types (pDCs, basophils, B cells and T cells)

Altered peripheral blood pDC (Table 4-1), basophil (Table 4-2), and B cell (Table 4-3) numbers and frequencies in SLE have been described in several prior studies.

Table 4-1: Studies evaluating peripheral blood pDC numbers and frequency in SLE

Result Assay technique Gating strategy Reference

Decreased frequency Flow cytometry Lin1-, CD11c-, C123hi, 157 compared to healthy controls on PBMCs BDCA2+, BDCA4+

Decreased absolute Flow cytometry number/ml in active disease BDCA2+ 156 on whole blood compared to inactive disease

Decreased frequency and Flow cytometry Lin1-, HLA-DR+, absolute number compared to 158 on whole blood BDCA2+ healthy controls

Decreased frequency ELISpot using Not applicable 159 compared to healthy controls PBMCs

Decreased absolute numbers Flow cytometry CD123hi, CD11c-, 160 in active disease on whole blood CD16-, HLA-DR+

Unaltered frequency and Flow cytometry absolute number compared to BDCA2+, CD123hi 161 on PBMCs healthy controls

Increased frequency Flow cytometry Lin1-, BDCA2+, compared to healthy controls, 162 on PBMCs CD123+ highest in active disease

114

Table 4-2: Studies evaluating peripheral blood basophil numbers and frequency in SLE

Result Assay technique Gating strategy Reference

Decreased absolute numbers Automated blood cell in active disease compared to Not applicable 277 analyzer on whole blood inactive disease

Decreased absolute count and Manual count after dye frequency compared to Not applicable 278 staining, on whole blood healthy controls

Decreased absolute numbers Flow cytometry Not stated 276 in active disease

Manual count after dye Unaltered absolute count staining, on whole Not applicable 316 compared to healthy controls blood315

115 Table 4-3: Studies evaluating peripheral blood B cell numbers and frequency in SLE

Result Assay technique Gating strategy Reference

Decreased absolute number of memory and naïve B cells, Naïve B cells: CD19+, CD27- Flow cytometry increased plasmablast frequency in SLE patients compared with Memory B cells: CD19+, CD27+ 317 on PBMCs healthy controls. Plasmablasts: CD19int, CD27hi

Total B cells: CD3-, CD19+ Increased frequency of memory B cells and plasma cells, and Flow cytometry Naïve B cells: CD19+, CD27-, CD38-/+ decreased frequency of total and naïve B cells in SLE compared 318 on whole blood Memory B cells: CD19+, CD27+, CD38-/+ to healthy controls. Plasma cells: CD19+, CD27+, CD38++

Increased frequency of naïve B cells, and decreased frequency of memory B cells in SLE patients compared to healthy Naïve B cells: CD19+, IgD+, CD27- controls. Flow cytometry Memory B cells: CD19+, CD38-, CD24hi, 319 on PBMCs CD27+ Increased frequency of plasmablasts in active disease (SLEDAI Plasmablasts: CD19+, CD38hi, CD27hi >6) compared to healthy controls

Increased plasmablast and IgD-CD27- memory B cell Memory B cells: IgD-CD27- and frequency, and decreased IgD+CD27+IgM memory B cell Flow cytometry IgD+CD27+IgM 320 frequency in SLE compared to healthy controls Plasmablast gating strategy not stated

116

Peripheral blood CD4+ T cells have been reported to be decreased in SLE,321-323 whereas CD8+ T cells have been varyingly found to be increased in active SLE,324 or decreased in SLE patients.324

Although the overall conclusions concerning pDC, basophil, B cell or T cell numbers and/or frequency between studies have generally been consistent, it is difficult to compare results between studies, due to differences in patient populations (disease activity, manifestations and drug treatments) and the different assays and methods of analyses employed in the various studies. However, all of the studies have contained at least one wash, or re-suspension, step in the preparation of the cells for analysis, which may lead to inaccuracies in generating cell counts. In my study, analysis of pDCs, basophils, B cells and T cells was undertaken by lysis of fresh whole blood without any wash steps to ensure accuracy of measured cell counts.

Fresh whole blood (50-200µL) was stained with antibody cocktails for 15 minutes at room temperature in the dark, with the volume of blood allocated to each stain depending on the anticipated proportion of the analysed peripheral blood cell type. For example, the largest volume of blood was allocated to staining for the rarer pDCs, basophils and plasmablasts, whereas a smaller volume was allocated to more common T cells.

Following red blood cell lysis with BD Lysing Solution (BD, Catalog number 349202), cell counts and percentages of the different peripheral blood cell types were acquired on the MACSQuant Analyzer (Miltenyi Biotec) and analyzed with Flowjo software (Treestar). The absolute count/ml for each different cell type was calculated by the MACSQuant Analyzer, which is capable of drawing a defined volume of blood for analysis. The percentage of each cell type, as a percentage of total white blood cells (WBCs), was calculated using Flowjo software.

117 The different cell types were defined by the cell surface markers in Table 4-4. Gating strategies can be found in Appendix 5.

Table 4-4: Defining cell surface markers for peripheral blood pDCs, basophils, B cells and T cells

Cell type Defining surface markers Blood volume stained

pDC Lin1- HLA-DR+, BDCA2++ CD123++ 150µL

Basophils Lin1-, CCR3+ CD123++ 150µL

Naïve B cells CD19+ CD27- 200µL

Memory B cells CD19+ CD27+ 200µL

Plasmablasts CD19+ CD27++, CD20- CD38++ 200µL

CD4+ T cells CD3+, CD4+ CD8- 50µL

CD8+ T cells CD3+, CD8+ CD4- 50µL

4.2.4 Antibodies for flow cytometry

All antibodies used were commercially available and are listed in Table 4-5.

118 Table 4-5: Antibodies for flow cytometry

Catalog Application Reagent Company number

pDCs, basophils Anti-CD123 PE BD Biosciences 555644

Lineage cocktail 1 – Lin1 pDCs, basophils (CD3, CD14, CD16, CD19, BD Biosciences 340546 CD20, CD56) FITC

pDCs, basophils Anti-HLA-DR APC-H7 BD Biosciences 561358

pDCs Anti-BDCA2 PE-Cy7 E-Bioscience 25-9818-42

Basophils Anti-CCR3 AF647 BD Biosciences 561745

T cells Anti-CD3 PE-Cy7 BD Biosciences 557851

B cells Anti-CD19 PE-Cy7 BD Biosciences 557835

B cells Anti-CD20 FITC BD Biosciences 556632

B cells Anti-CD27 V500 BD Biosciences 561222

B cells Anti-CD38 BV421 BD Biosciences 562444

T cells Anti-CD4 FITC BD Biosciences 555346

T cells Anti-CD8 APC BD Biosciences 555369

4.2.5 Assessing alterations in gene expression in response to IL-3 stimulation in healthy donor whole blood

Whole blood from seven healthy donors was collected in lithium heparin tubes (BD, Catalog number 367526) and subjected to red blood cell lysis with ammonium chloride (Stem Cell Technologies, Catalog number 07850).

1x107 PBMCs were cultured with either 1 µg/ml of rhIL-3 (R&D Systems, Catalog number 203-IL) or media alone for 6 hours, or 24 hours. Cell pellets were then suspended in RNAprotect Cell Reagent (Qiagen, Catalog number 76526) and frozen at - 80°C until RNA extraction.

119

RNA was extracted with a RNA Protect Cell Mini Kit (Qiagen, Catalog number 74624) according to manufacturer’s instructions. RNA was then submitted to the Australian Genome Research Facility for next generation sequencing on the Illumina HiSeq platform (each sample was run in technical duplicate).

4.2.6 Analysis of whole blood gene expression in SLE and healthy donors

Whole blood (2.5ml) was collected into PAXgene RNA tubes (BD, Catalog number 762165) and frozen at -20°C until later RNA extraction. RNA was extracted with the PAX gene Blood RNA kit IVD (PreAnalytix, Catalog number 762174) according to manufacturer’s instructions. RNA was then submitted to the Australian Genome Research Facility for next generation sequencing on the Illumina HiSeq platform (each sample was run in technical duplicate).

4.2.7 Statistical analyses

Comparison of quantitative values was performed by the Mann-Whitney U test for unpaired data. Spearman’s rank correlation was used to examine relationships between two variables. Statistical analyses were performed with GraphPad Prism (Version 6.0). P values of less than 0.05 were considered significant.

4.2.8 RNASeq bioinformatics

Sequence reads were aligned to a reference genome using bowtie2/topHat (bowtie2 version /usr/loca/bioinf/bin/bowtie2-align version 2.1.0 and Tophat v2.0.8) from a reference genome downloaded from ‘http://tophat.cbcb.umd.edu/igenomes.shtml’. Gene symbols were converted to an Ensembl ID. Aligned and non aligned reads were then counted with htseq-count version 3 (0.5.3p9). All other bioinformatics analyses, generation of heat maps and Venn diagrams, were performed with R (version 3.0.2). A false discovery rate (FDR) of < 0.05 was considered statistically significant.

120 4.3 Results

4.3.1 Serum IL-3 levels in SLE patients and healthy controls

IL-3 levels in donor serum were quantified in a total of 86 donors (42 SLE donors and 44 healthy donors). The 44 healthy donors were a subset of the 54 healthy donors outlined in Chapter 2. Demographic and clinical data for these donors were as outlined in Chapter 2.

Twenty-five SLE donors and 22 healthy controls had detectable serum levels of IL-3. Mean serum IL-3 levels were not significantly higher in SLE patients (272 ± 77 [SEM] pg/ml, range 7.8-2000 pg/ml) compared to healthy donors (400 ± 97 pg/ml, range 7.8- 2000 pg/ml, p = 0.840) (Figure 4-1A). Interestingly, of the twenty-two healthy donors with elevated IL-3 levels, six had a history of asthma. Only one other healthy donor had a history of asthma. Of the other sixteen healthy donors with elevated IL-3 levels, thirteen had no active medical conditions, whilst the other three had a history of type II diabetes, depression, and osteoarthritis, respectively.

2500

2000

1500

1000 IL-3 pg/ml IL-3

500

0 Healthy SLE

Figure 4-1: Serum IL-3 levels in SLE patients and healthy controls.

Serum IL-3 levels, measured by ELISA, in SLE (n = 42) and healthy (n = 44) donors. Data expressed as mean ± SEM.

121 4.3.2 Correlations between serum IL-3 levels and measures of disease activity in SLE

Serum IL-3 levels have previously been found to correlate with higher disease activity in SLE.285 In my study, serum IL-3 levels were compared with a number of different measures of disease activity – the SLEDAI, complement levels C3 and C4, anti-dsDNA antibody levels, and inflammatory markers C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR).

There were no statistically significant differences in IL-3 levels between healthy donors or less active SLE patients, compared to SLE patients with higher disease activity (as defined by patients with a SLEDAI score of ≥ 6, low C3 or C4, elevated anti-dsDNA antibody level, or raised CRP or ESR) (Table 4-6). There was a trend towards a lower mean serum IL-3 level for the more active SLE patients compared to the less active, with the exception of the group with elevated anti-dsDNA antibody levels.

122 Table 4-6: Mean IL-3 levels compared by disease activity in SLE patients, and healthy controls

IL-3 level pg/ml: mean (SEM) p value: high p value : high disease High disease activity disease activity vs low measure SLE – low SLE – high activity vs disease disease activity disease activity healthy Healthy n n n activity

400.3 303.2 225.2 SLEDAI ≥ 6 44 25 17 0.885 0.669 (97.3) (114.8) (89.4)

400.3 382.2 122.6 Low C3 44 25 15 0.6364 0.266 (97.3) (119.4) (62.1)

400.3 309 248.6 Low C4 44 24 16 0.851 0.731 (97.3) (103.3) (130.2)

Raised anti-dsDNA 400.3 109.3 329.2 44 11 31 0.659 0.536 Ab level (97.3) (46.8) (101.1)

400.3 312.4 201.8 Raised CRP 44 29 11 0.838 0.563 (97.3) (102.9) (367.1)

400.3 375.0 243.4 Raised ESR 44 9 33 0.738 0.689 (97.3) (226.4) (77.4)

123 There was no significant correlation between serum IL-3 levels, and SLEDAI score, or levels of C3 or C4, anti-dsDNA antibodies, or CRP or ESR (Table 4-7).

Table 4-7: Correlation between serum IL-3 levels and disease activity measures in SLE patients

Correlation with IL-3 level Disease activity measure p value r 95% confidence interval

SLEDAI score -0.213 -0.491, 0.107 0.177

C3 level 0.176 -0.153, 0.470 0.470

C4 level 0.149 -0.180, 0.448 0.360

Anti-dsDNA antibody level -0.130 -0.428, 0.191 0.414

CRP level -0.059 -0.372, 0.267 0.720

ESR level -0.081 -0.388, 0.241 0.613

4.3.3 Correlations between serum IL-3 levels and disease manifestations and medication use in SLE

There was no statistically significant difference between mean serum IL-3 levels in healthy donors compared to SLE patients (with or without arthritis, haematuria, proteinuria, pyuria, rash, alopecia, mucosal ulcers, pleurisy, fever, thrombocytopenia, leukopenia or anemia (Table 4-8)). However, for many of these disease manifestations, there were very few numbers in the disease manifestation group, making comparison between groups difficult – the only manifestations with ≥ 5 patients were arthritis, proteinuria, rash, alopecia, leukopenia, anemia and anti-phospholipid antibody positivity. There was no correlation between serum IL-3 levels and platelet (r = -0.085, 95% CI -0.391 – 0.237, p = 0.597), white cell (r = 0.088, 95% CI -0.230 – 0.390, p = 0.578) or haemoglobin (r = 0.179, 95% CI -0.141 – 0.466, p = 0.257) levels in the SLE patients.

124 Table 4-8: Mean serum IL-3 levels in SLE patients by disease manifestation

IL-3 level pg/ml : mean (SEM) p value : with p value: with

Disease manifestation SLE – no SLE – with manifestation manifestation vs Healthy n n n manifestation manifestation vs healthy no manifestation

Arthritis 400.3 (97.3) 44 308.0 (95.4) 31 169.1 (117.9) 11 0.336 0.110

Haematuria 400.3 (97.3) 44 284.8 (80.1) 40 7.8 (0) 2 0.404 0.240

Proteinuria 400.3 (97.3) 44 296.5 (85.9) 37 97.8 (178.8) 5 0.239 0.140

Pyuria 400.3 (97.3) 44 284.8 (80.1) 40 7.8 (0) 2 0.404 0.240

Rash 400.3 (97.3) 44 284.9 (96.7) 31 234.2 (113.7) 11 0.483 0.393

Alopecia 400.3 (97.3) 44 277.4 (91.8) 34 247.3 (110.0) 8 0.553 0.441

Mucosal ulcers 400.3 (97.3) 44 263.7 (82.7) 38 237.2 (206.5) 4 0.559 0.407

Pleurisy 400.3 (97.3) 44 275.9 (78.5) 41 94.6 (0) 1 No p value as n = 1

Fever 400.3 (97.3) 44 284.8 (80.1) 40 7.8 (0) 2 0.404 0.240

Thrombocytopenia 400.3 (97.3) 44 287.2 (83.9) 39 162.8 (143.4) 3 0.992 0.999

Leukopenia 400.3 (97.3) 44 305.7 (85.7) 37 19.7 (7.6) 5 0.341 0.151

Anemia 400.3 (97.3) 44 255.1 (73.9) 36 370.8 (326.4) 6 >0.999 0.761

Anti-phospholipid antibodies 400.3 (97.3) 44 320.1 (98.5) 37 223.6 (194.8) 5 0.669 0.515

125 The SLEDAI definition of each disease manifestation was used to define the presence or absence of each disease manifestation, with the exception of the latter three (thrombocytopenia, leukopenia and anemia), which were defined by standard laboratory parameters.

Unlike the previous cohort of SLE patients in whom elevated IL-3 levels were found,285 all but six of this cohort of SLE patients were receiving immunomodulatory or immunosuppressive medications (Table 4-9). There were no statistically significant differences in mean IL-3 levels when healthy donors were compared with SLE patients who were or were not taking prednisolone, hydroxychloroquine, methotrexate, sulfasalazine, azathioprine, mycophenolate, cyclosporin, rituximab or any other medication. However, again, the analysis was complicated by small numbers in certain medication groups, with only one patient each taking sulfasalazine, cyclosporin, or rituximab.

126 Table 4-9: Mean serum IL-3 levels in SLE patients by medication use

IL-3 level pg/ml: mean (SEM) p value: taking p value : taking medication vs Medication medication vs not taking Healthy n SLE – not taking n SLE – taking n healthy medication medication medication 400.3 318.7 263.8 Any medication 44 6 36 0.822 0.953 (97.3) (206.6) (83.8) 400.3 157.4 357.3 Prednisolone 44 18 24 0.794 0.819 (97.3) (73.6) (120.9) 400.3 265.0 274.3 Hydroxychloroquine 44 12 30 0.759 0.893 (97.3) (125.5) (32.6) 400.3 293.7 62.2 Methotrexate 44 38 4 0.739 0.589 (97.3) (84.1) (32.6) 400.3 278.1 7.8 Sulfasalazine 44 41 1 NA* NA* (97.3) (78.4) (0) 400.3 237.5 396.6 Azathioprine 44 33 9 0.328 0.350 (97.3) (78.1) (221.2) 400.3 218.8 662.5 Mycophenolate 44 37 5 0.456 0.346 (97.3) (69.9) (371.6) 400.3 256.6 889.0 Cyclosporin 44 41 1 NA* NA* (97.3) (77.1) (0) 400.3 278.1 7.8 Rituximab 44 41 1 NA* NA* (97.3) (119.7) (0) NA* - no p value as there was only one patient in the medication group

127 4.3.4 Correlations between serum IL-3 levels and peripheral blood pDCs, basophils, B cells and T cells

IL-3 is secreted by activated T cells, basophils and mast cells, and is a known maturation and survival factor for a number of different cell types, including pDCs,280 basophils281 and B cells,282 which contribute to SLE pathogenesis and which were shown in Chapter 3 to be altered by the anti-CD123 mAb CSL362 ex vivo. These cell types (pDCs, basophils, B and T cells) in peripheral blood of SLE and healthy donors were evaluated by flow cytometry, and correlated with serum IL-3 levels in these donors.

Compared to healthy donors, pDCs, basophils, naïve and memory B cells and CD4+ T cells (evaluated both as count/ml and % of total WBCs) were significantly decreased in SLE donors (Figure 4-2A-E). CD8+ T cells (expressed as a % of total WBCs) were also decreased in SLE compared to healthy donors (Figure 4-2F). Plasmablasts were decreased in SLE patients compared to healthy donors, however this difference was not statistically significant (mean plasmablasts 444.4 ± 93.93/ml compared to 416.7 ± 161.9/ml, p value = 0.926; and 0.011 ± 0.019% compared to 0.008 ± 0.017%, p value = 0.439, in healthy and SLE donors respectively).

With a true ‘no wash’ technique employed to evaluate the cell types, these findings confirm previous studies that have shown decreased peripheral blood pDCs, basophils and CD4+ T cells156-160, 276-278, 321, 323 in SLE. Naïve and memory B cells have been varyingly found to be increased or decreased in previous studies; my study has found a decrease in both cell types. However it is difficult to compare between B cell studies as different gating strategies to delineate different types of B cells were employed in previous studies.317-320

128 A B Figure 4-2: Decreased pDCs, basophils, naïve and memory B cells, CD4+ and CD8+ T cells in SLE compared to healthy donors. Peripheral blood (A) pDCs (Lin1- HLA-DR+, CD123++ C D BDCA2++), (B) basophils (Lin1-, CCR3+ CD123++), (C) naïve B cells (CD19+ CD27-), (D) memory B cells (CD19+ CD27+), (E) CD4+ T cells (CD3+, CD4+ CD8-) and (F) CD8+ T cells (CD3+, CD4- E F CD8+), evaluated by flow cytometry and expressed as a % of total WBC (% WBC) or count/ml, in SLE (n=42) and healthy (n=44) donors. Data represents the mean ± SEM, * p < 0.05 (Mann Whitney test).

129 Paired serum IL-3 and peripheral blood cell count data was available for 34 SLE, and 35 healthy donors. In SLE patients, positive correlations were found between serum IL- 3 levels and pDCs (as % total WBC; r = 0.409, 95% CI 0.071 – 0.662, p = 0.016), and basophils (as % total WBC, r = 0.372, 95% CI 0.029 – 0.637, p = 0.030; and as a count/ml, r = 0.394, 95% CI 0.053 – 0.651, p = 0.021) (Figure 4-3). These correlations may reflect the reliance of pDCs and basophils on IL-3 as a survival factor.280, 281 Interestingly, these correlations were not found in the healthy donor population.

Figure 4-3: Correlation between serum IL-3 levels and pDCs and basophils in SLE patients. Correlation between serum IL-3 levels, as measured by ELISA, and peripheral blood (A) pDCs (as % total WBC) and basophils, as (B) % total WBC and (C) count/ml, as measured by flow cytometry, in SLE patients (n = 34). Correlations determined by Spearman R analysis.

130 4.3.5 Serum levels of IL-3 correlate with IFNα and type III IFN in SLE and healthy donors

Serum levels of BAFF, GM-CSF, IL-2, IP-10 and IL-13 were significantly different between SLE and healthy donors (Figure 4-4A-D).

BAFF levels were increased in SLE patients (mean BAFF level 979.2 ± 93.6 [SEM] pg/ml) compared to healthy donors (684.1 ± 42.7 pg/ml, p = 0.027), in keeping with previous studies.184 IP-10, a type I IFN upregulated protein, levels were also greater in SLE (556.7 ± 187.4 pg/ml) compared to healthy donors (125.9 ± 9.9 pg/ml, p < 0.0001); IFNα itself was increased in the SLE group (80.1 ± 32.8 pg/ml) when compared with the healthy donors (42.0 ± 24.1 pg/ml), however the difference was not statistically significant (p = 0.545) (Table 4-10).

GM-CSF levels were decreased in SLE (49.8 ± 13.8 pg/ml) compared to healthy donors (191.8 ± 73.3 pg/ml, p = 0.042), as were IL-2 levels (8.4 ± 2.7 pg/ml in SLE patients compared to 33.4 ± 12.8 pg/ml in healthy donors, p = 0.013). IL-13 levels were decreased in SLE patients (13.4 ± 5.4 pg/ml) compared to healthy donors (62.2 ± 37.6 pg/ml, p = 0.044), however this was largely due to a single outlier in the healthy group (Figure 4-4E). Decreases in type III IFN, IFNγ, G-CSF, Flt-3l, IL-4, IL-5, IL-6 and TNFα were also seen in the SLE patients, however these differences were not statistically significant (Table 4-10).

131 A B

E

C D

Figure 4-4: Altered serum levels of BAFF, GM-CSF, IL-2, IP-10 and IL-13 in SLE patients compared to healthy donors. Serum (A) BAFF (n = 30 for each group), (B) GM-CSF (n = 32 healthy, n = 37 SLE), (C) IL-2 (n = 32 healthy, n = 37 SLE), (D) IP-10 (n = 32 healthy, n = 37 SLE) and (E) IL-13 levels (n = 32 healthy, n = 37 SLE) in SLE and healthy donors, as determined by ELISA (BAFF) and Luminex (GM-CSF, IL-2, IP-10 and IL-13) assays. Data expressed as mean ± SEM, * p < 0.05 (Mann Whitney test).

132

Table 4-10: Cytokine levels in SLE compared to healthy donors

Healthy : SLE : SLE vs Cytokine Mean (SEM) n Mean (SEM) n p value healthy pg/ml pg/ml

42.0 80.1 IFNα 42 44 é 0.545 (24.1) (32.8)

627.1 469.6 Type III IFN 36 37 ê 0.732 (203.7) (159.3)

137.1 60.9 IFNγ 32 37 ê 0.151 (38.7) (11.8)

230.2 113.1 G-CSF 32 37 ê 0.955 (79.7) (24.96)

145.0 83.1 Flt-3l 32 37 ê 0.121 (40.4) (16.9)

8.7 5.7 IL-4 32 37 ê 0.221 (5.8) (2.0)

17.5 4.8 IL-5 32 37 ê 0.512 (11.6) (1.5)

15.3 4.5 IL-6 31 37 ê 0.123 (5.7) (0.8)

31.5 16.6 TNF-α 32 37 ê 0.735 (10.4) (2.3)

133 Serum IL-3 levels in SLE and healthy donors correlated with IFNα levels (r = 0.612, 95% CI 0.455 – 0.733, p < 0.0001) (Figure 4-5A), although the correlation was weaker in SLE (r = 0.376, 95% CI 0.072 – 0.616, p = 0.014) (Figure 4-5B) compared to healthy (r = 0.784, 95% CI 0.629 – 0.879, p < 0.0001) (Figure 4-5C) donors.

Figure 4-5: Serum IL-3 levels correlate with IFNα levels in SLE and healthy donors. Serum IL-3 and IFNα levels in (A) SLE and healthy (n = 86), (B) SLE only (n = 42) and (C) healthy only (n = 44), donors, as determined by ELISA. Correlations determined by Spearman R analysis.

134 An association was also found between IL-3 and type III IFN levels (r = 0.585, 95% CI 0.406 – 0.720, p < 0.0001), which was present in both SLE (r = 0.666, 95% CI 0.432 – 0.816, p < 0.0001) and healthy (r = 0.515, 95% CI 0.213 - 0.724, p = 0.001) donors (Figure 4-6).

Figure 4-6: Serum IL-3 levels correlate with type III IFN levels in SLE patients and healthy donors. Serum IL-3 and type III IFN levels in (A) SLE and healthy (n = 73), (B) SLE only (n = 36) and (C) healthy only (n = 37), donors, as determined by ELISA. Correlations determined by Spearman R analysis.

135

A weaker correlation was seen in both SLE and healthy donors, between IL-3 and IL-4 levels (r = 0.279, 95% CI 0.038 - 0.489, p = 0.021) (Figure 4-7A), and IL-3 and IL-5 levels (r = 0.255, 95% CI 0.012 - 0.469, p = 0.035) (Figure 4-7B). There were no statistically significant correlations between IL-3 and BAFF, IFNγ, GM-CSF, G-CSF, Flt-3l, IL-2, IL-6, IL-13, IP-10 or TNFα (Table 4-11).

Figure 4-7: Serum IL-3 levels correlate with IL-4 and IL-5 levels in SLE and healthy donors. Serum IL-3 and (A) IL-4 and (B) IL-5 levels, in SLE and healthy donors combined (n = 69; n = 37 SLE, n = 32 healthy), as determined by ELISA. Weak correlations (assessed by Spearman R analysis) were found between IL-3 and IL-4, and IL-3 and IL-5.

136

Table 4-11: Correlations between serum IL-3 levels and cytokines in SLE and healthy donors

Both SLE and healthy SLE only Healthy only

Cytokine Correlation: r p Correlation: r p Correlation: r p (95% CI) value (95% CI) value (95% CI) value

0.122 0.117 0.143 BAFF 0.277 0.459 0.384 (-0.105, 0.338) (-0.20, 0.415) (-0.190, 0.447)

0.053 0.123 0.004 IFNγ 0.669 0.468 0.985 (-0.193, 0.292) (-0.219, 0.438) (-0.355, 0.361)

-0.021 0.017 0.026 GM-CSF 0.866 0.920 0.887 (-0.263, 0.224) (-0.318, 0.348) (-0.336, 0.381)

0.015 0.103 -0.073 G-CSF 0.905 0.544 0.691 (-0.230, 0.257) (-0.238, 0.421) (-0.420, 0.293)

0.104 0.174 0.103 Flt-3l 0.397 0.304 0.576 (-0.144, 0.338) (-0.169, 0.479) (-0.265, 0.445)

0.056 0.177 -0.008 IL-2 0.648 0.294 0.966 (-0.190, 0.295) (-0.165, 0.482) (-0.365, 0.351)

-0.144 -0.167 -0.087 IL-6 0.240 0.330 0.636 (-0.376, 0.105) (-0.478, 0.181) (-0.432, 0.279)

0.069 0.173 -0.029 IL-13 0.573 0.306 0.874 (-0.178, 0.307) (-0.170, 0.479) (-0.383, 0.332)

0.070 0.115 -0.071 IP-10 0.569 0.450 0.698 (-0.177, 0.308) (-0.227, 0.431) (-0.419, 0.294)

-0.149 -0.084 -0.175 TNFα 0.224 0.621 0.340 (-0.378, 0.099) (-0.406, 0.256) (-0.501, 0.196)

137 Longitudinal data (> 3 measurements at different time periods, up to 21 months from the first donation, for a total of 33 measurements between the donors) on serum IL-3, IFNα, type III IFN and BAFF levels were available for seven SLE donors (Figure 4-8). Disease activity, as measured by the SLEDAI, remained relatively stable for these donors, with six of the seven donors having a 2-4 point difference in minimum and maximum SLEDAI scores (Figure 4-8A-F); the seventh donor had an 8 point different (Figure 4-8G).

Correlations between IL-3 and IFNα (r = 0.560, 95% 0.313 - 0.786, p = 0.0002) (Figure 4-9A), and IL-3 and type III IFN (r = 0.445, 95% CI 0.003 - 0.742, p = 0.043) (Figure 4-9B) were again observed, however there was no correlation between IL-3 and BAFF (r = 0.210, 95% CI -0.195 - 0.556, p = 0.290), or IL-3 and SLEDAI scores (r = 0.159, 95% CI -0.206 - 0.484, p = 0.378).

138 A B

C D

E F

G

Figure 4-8: Longitudinal serum IL-3, IFNα, IFNλ and BAFF levels in SLE patients. Serum IL-3, IFNα, IFNλ and BAFF levels, as measured by ELISA, in seven different SLE donors (each graph represents a separate donor), at various times following their first blood donation, and plotted in relation to disease activity (as measured by the SLEDAI).

139

Figure 4-9: Serial serum IL-3 levels correlate with IFNα and type III IFN in SLE patients over time. Serum IL-3 and (A) IFNα, and (B) type III IFN levels, in SLE donors (n = 7 donors; n = 33 paired measurements across time), as determined by ELISA. Correlations determined by Spearman R analysis.

4.3.6 An ‘IL-3 gene signature’ differentiates SLE and healthy donors

As mentioned in previous Chapters, the presence of a peripheral blood ‘IFN gene signature’ in 50-70% of SLE patients has been well established by previous studies.72, 73 My work identified a correlation between serum IL-3 and IFN, and so the possibility of an ‘IL-3 gene signature’ in SLE patients (as compared to healthy donors), was evaluated.

To search for a potential ‘IL-3 gene signature’, genes altered in response to IL-3 stimulation were determined. Lysed whole blood from seven healthy donors was cultured with, or without, IL-3 for 6 or 24 hours. These donors were additional healthy donors to those previously described in Chapter 2, with a more limited age range, but a similar female predominance. The mean age for these seven donors was 32.9 years (median 33 years, range 29 - 48 years). There were five females and two males. Four were Caucasian, two Asian and one of Aboriginal background. RNA was extracted from the red cell depleted whole blood pellets and RNASeq analysis performed on an Illumina HiSeq platform (100bp read length).

140 A total of 26,206 differentially expressed (12,850 upregulated, and 13,356 downregulated) genes were identified between the IL-3 stimulated, and non IL-3 stimulated samples (Figure 4-10). When a p value of < 0.05 was applied, and a limitation of at least a 2 fold change in gene expression was set, there were 794 differentially expressed genes (650 upregulated, and 144 downregulated) between the stimulated and unstimulated groups, inclusive of both timepoints. Of these, 152 were differentially expressed (141 upregulated, 11 downregulated) at both timepoints (Appendix 6), 592 at 24 hours only (471 upregulated, 121 downregulated) (Appendix 7) and 50 at 6 hours only (38 upregulated, 12 downregulated) (Appendix 8).

24 hours 6 hours

471 141 38 121 11 12

Upregulated 12850 Downregulated 13356

Figure 4-10: Numbers of differentially expressed genes between IL-3 stimulated, or non- stimulated lysed whole blood from healthy donors. Numbers of differentially expressed genes between IL-3 stimulated (at 6 hours or 24 hours) and non-stimulated lysed whole blood from healthy donors (n = 7), as analysed by RNASeq. Numbers enclosed within the circles represent the numbers of genes that were statistically significantly (p < 0.05) differentially expressed, with at least a 2 fold change in gene expression.

To then determine if there was an ‘IL-3 gene signature’ in the peripheral blood of SLE patients, RNA was extracted from whole blood of 31 SLE patients (Table 4-12), and 28 age and sex matched healthy controls, and RNASeq analysis performed on an Illumina HiSeq platform (100bp read length). These donors were a different cohort of donors to those in whom serum cytokine levels had been assessed, however, apart from being a slightly older population (mean 45.0 years, compared to 37.7 years in the serum cohort),

141 the characteristics of the SLE patients were very similar in terms of SLEDAI, clinical manifestations, and medications taken. A slightly lower percentage were anti-dsDNA antibody positive (52% in this cohort compared to 83% in the serum analysis cohort) and a greater percentage had hypocomplementemia (61% in this cohort compared to 23% in the serum analysis cohort) (Table 4-12).

Table 4-12: SLE donor characteristics (n = 31)

Age (mean, range) 45, 23 -74 (years)

Gender (female, male) 94, 6 (%)

Disease duration (mean, median, range) 13.7, 11, 1 - 46 (years)

Ethnicity - Caucasian 76 (%) - Asian 23

SLEDAI (mean, median, range) 4, 4, 0 - 21

Disease manifestations (current or past) - Rash 48 (%) - Arthritis 72 - Cardiorespiratory 29 - Renal 42 - Neuropsychiatric 3 - Cytopenia 52 - Positive anti-dsDNA antibody 52 - Hypocomplementemia 61

Current medications - No immunosuppression 7 (%) - Steroids 52 - Hydroxychloroquine 77 - Methotrexate 16 - Azathioprine 10 - Mycophenolate 23

142

Two hundred genes were found to be significantly (FDR < 0.05) differentially expressed (with a fold change of at least 2) between SLE and healthy donors (132 upregulated genes, and 68 downregulated genes). Thirty-five of these genes were also found to be differentially expressed after IL-3 stimulation at either 6 or 24 hours (Table 4-13).

These thirty-five genes were more highly expressed in all but three of the SLE donors, and only nine of the healthy donors (Figure 4-11), indicating the presence of an ‘IL-3 gene signature’ in most SLE donors.

143 Table 4-13: Differentially expressed genes in response to IL-3 stimulation, and between SLE and healthy donor whole blood

EnsemblID Symbol Chromosome Description

ENSG00000119922 IFIT2 10 Interferon induced protein with tetratricopeptide repeats 2 ENSG00000119917 IFIT3 10 Interferon induced protein with tetratricopeptide repeats 3 ENSG00000134321 RSAD2 2 Radical S-adenosyl methionine domain containing 2 ENSG00000185745 IFIT1 10 Interferon induced protein with tetratricopeptide repeats 1

ENSG00000128383 APOBEC3A 22 Apolipoprotein B mRNA editing enzyme catalytic subunit 3A

ENSG00000117228 GBP1 1 Guanylate binding protein 1 ENSG00000188313 PLSCR1 3 Phospholipid scramblase 1 ENSG00000172322 CLEC12A 12 C-type lectin domain family 12 member A ENSG00000136689 IL1RN 2 Interleukin 1 receptor antagonist ENSG00000120217 CD274 9 CD274 molecule ENSG00000079385 CEACAM1 19 Carcinoembryonic antigen related cell adhesion molecule 1 ENSG00000140379 BCL2A1 15 BCL2 related protein A1 ENSG00000138772 ANXA3 4 Annexin A3 ENSG00000123610 TNFAIP6 2 TNF alpha induced protein 6 ENSG00000010030 ETV7 6 ETS variant 7 ENSG00000149131 SERPING1 11 Serpin family G member 1 ENSG00000088827 SIGLEC1 20 Sialic acid binding Ig like lectin 1

144

ENSG00000179750 APOBEC3B 22 Apolipoprotein B mRNA editing enzyme catalytic subunit 3B ENSG00000251230 RP11-701P16.5 4 MIR3945 host gene ENSG00000231233 RP11-127L20.6 10 CFAP58 antisense RNA 1 ENSG00000260943 RP11-476D10.1 12 Novel transcript ENSG00000126787 DLGAP5 14 DLG associated protein 5 ENSG00000152766 ANKRD22 10 Ankyrin repeat domain 22 ENSG00000168062 BATF2 11 Basic leucine zipper AT-like transcription factor 2 ENSG00000225492 GBP1P1 1 Guanylate binding protein 1 pseudogene 1 ENSG00000169245 CXCL10 4 C-X-C motif chemokine ligand 10 ENSG00000197646 PDCD1LG2 9 Programmed cell death 1 ligand 2 ENSG00000173369 C1QB 1 Complement component 1, q subcomponent B chain ENSG00000186049 KRT73 12 Keratin 73 ENSG00000170486 KRT72 12 Keratin 72 ENSG00000185897 FFAR3 19 Free fatty acid receptor 3 ENSG00000079393 DUSP13 10 Dual specificity phosphatase 13 ENSG00000181634 TNFSF15 9 Tumour necrosis factor superfamily member 15 ENSG00000108691 CCL2 17 C-C motif chemokine ligand 2 ENSG00000242265 PEG10 7 Paternally expressed 10

145

IFIT2 IFIT3 RSAD2 IFIT1 APOBEC3A GBP1 PLSCR1 CLEC12A IL1RN CD274 CEACAM1 BCL2A1 ANXA3 TNFAIP6 ETV7 SERPING1 SIGLEC1 APOBEC3B RP11-7001P16.5 RP11.127L20.6 RP11-476D10.1 DLGAP5 ANKRD22 BATF2 GBP1P1 CXCL10 PDCD1LG2 C1QB KRT73 KRT72 FFAR3 DUSP13 TNFSF15 CCL2 PEG10 5 SLE donor 9 SLE donor 3 SLE donor 4 SLE donor 1 SLE donor 7 SLE donor 8 SLE donor 5 SLE donor 2 SLE donor 6 SLE donor 11 SLE donor 15 SLE donor 13 SLE donor 16 SLE donor 26 SLE donor 19 SLE donor 12 SLE donor 10 SLE donor 28 SLE donor 14 SLE donor 24 SLE donor 20 SLE donor 27 SLE donor 30 SLE donor 17 SLE donor 25 SLE donor 31 SLE donor 22 SLE donor 29 SLE donor 23 SLE donor 18 SLE donor 21 Healthy donor 9 Healthy donor 1 Healthy donor 7 Healthy donor 2 Healthy donor 6 Healthy donor 3 Healthy donor 4 Healthy donor 8 Healthy donor Healthy donor 11 Healthy donor 16 Healthy donor 25 Healthy donor 23 Healthy donor 13 Healthy donor 28 Healthy donor 15 Healthy donor 20 Healthy donor 21 Healthy donor 10 Healthy donor 19 Healthy donor 24 Healthy donor 22 Healthy donor 27 Healthy donor 18 Healthy donor 12 Healthy donor 13 Healthy donor 17 Healthy donor 26

Figure 4-11: Expression of IL-3 regulated genes in SLE and healthy donors Heatmap showing expression of IL-3 regulated genes in SLE (n = 31) and healthy (n = 28) donors, as determined by RNASeq analysis of donor whole blood (FDR < 0.05).

146 4.3.7 Presence of an ‘IL-3 gene signature’ correlates with an ‘IFN gene signature’ in SLE

Given the association of IL-3 and IFN seen in the analysis of serum cytokines, a possible association between IL-3 and IFN at a transcriptional level was then explored. A panel of IFN-inducible genes (EPST11, ISG15, HERC5, IFI44, OAS3, OAS1, LY6E, CMPK2, RSAD2, IFI44L, IFIT1, IFIT3, USP18, SIGLEC1, IFI27, OTOF) that differentiated most SLE from most healthy donors was determined by heirarchical clustering of the five hundred most variable genes in SLE donors (Figure 4-12). These genes constituted the ‘IFN gene signature’ for this cohort. Interestingly, three genes in the ‘IFN gene signature’ and ‘IL-3 gene signature’ overlapped – IFIT1, IFIT3 and SIGLEC1.

Genes in the IL-3 and IFN gene signatures were combined into a single gene score for each SLE patient and healthy donor, based on the principal component 1 from each gene signature. A strong correlation between IL-3 and IFN gene signature scores was found (r = 0.939, 95% CI 0.898 - 0.964, p < 0.0001) (Figure 4-13). This finding supports, at a transcriptional level, the association between these two cytokines that was found in the serum cytokine analysis of an independent SLE and healthy cohort.

147 EPST11

ISG15

HERC5

IFI44

OAS3

OAS1

LY6E

CMPK2

RSAD2

IFI44L

IFIT1

IFIT3

USP18

SIGLEC1

IFI27

OTOF SLE donor 3 SLE donor 2 SLE donor 1 SLE donor 7 SLE donor 4 SLE donor 8 SLE donor 5 SLE donor 6 SLE donor 9 SLE donor 11 SLE donor 13 SLE donor 19 SLE donor 20 SLE donor 30 SLE donor 29 SLE donor 26 SLE donor 12 SLE donor 27 SLE donor 10 SLE donor 16 SLE donor 17 SLE donor 14 SLE donor 25 SLE donor 28 SLE donor 22 SLE donor 31 SLE donor 17 SLE donor 23 SLE donor 18 SLE donor 21 SLE donor 15 SLE Donor 24 Healthy donor 1 Healthy donor 8 Healthy donor 3 Healthy donor 4 Healthy donor 5 Healthy donor 6 Healthy donor 9 Healthy donro 7 Healthy donor 2 Heathy donor 27 Healthy donor 11 Healthy donro 19 Healthy donor 26 Healthy donor 16 Healthy donor 14 Healthy donor 24 Healthy donor 28 Healthy donro 23 Healthy donor 10 Healthy donor 15 Healthy donor 21 Healthy donor 18 Healthy donor 25 Healthy donor 20 Healthy donor 22 Healthy donor 12 Healthy donor 13

Figure 4-12: Expression of IFN regulated genes in SLE and healthy donors Heatmap showing expression of IFN regulated genes in SLE (n = 31) and healthy (n = 28) donors, as determined by RNASeq analysis of donor whole blood, and hierarchical clustering of the five hundred most variable genes (based on standard deviation) in SLE donors (FDR < 0.05).

148

Figure 4-13 Correlation between IL-3 and IFN gene signature scores in SLE and healthy donors. IFN and IL-3 gene signature scores in SLE (n = 31) and healthy (n = 28) donors, as determined by RNASeq analysis of donor whole blood. IL-3 and IFN gene signature scores were determined by taking the first principal component of the panel of genes comprising each gene signature. Correlation determined by Spearman R analysis, p < 0.05.

149

4.4 Discussion

In this study a correlation between IL-3 and IFN was identified, both through serum cytokine analysis (in both cross-sectional and longitudinal measurements), and transcriptional profiling of donor whole blood, in two separate, but not dissimilar, cohorts of SLE and healthy donors. This correlation has not previously been described in SLE, healthy or other diseases and was evident even though mean IL-3 levels were not elevated in SLE patients as compared to healthy donors.

The correlation between IL-3 and IFN (type I and type III) may be partly explained by the survival effect of IL-3 on pDCs,280 which produce both these types of IFN; a correlation between serum IL-3 levels and peripheral blood pDCs was also observed in this study, and a number of genes in the ‘IL-3 gene signature’ are known to be involved in the IFN pathway. Recently, IL-3 has been found to enhance IFNα production by pDCs stimulated with RNA containing immune complexes (RNA-IC), in a dose dependent fashion ex vivo in healthy donors.325 It was not elucidated whether IL-3 increased IFNα production through enhancing pDC survival, or via another mechanism. GM-CSF also potently increased pDC-derived IFNα production.325 This phenomenon was shown not to be due to the proliferation of pDCs in the presence of GM-CSF. Supernatant from T cells activated with Dynabeads (CD3/CD28) from both SLE and healthy donors was also able to stimulate IFNα production from pDCs. Depletion of GM-CSF from the activated T cell supernatants, and blockade of the GM-CSF receptor, and shared β subunit of the GM-CSF and IL-3 receptors reduced the stimulatory effect of the T cell supernatants on pDC-derived IFNα production, whereas depletion of IL-3 and blockade of the IL-3 receptor alone did not, indicating that GM-CSF may play a more important role in activated T cell supernatants. In my study, a correlation between serum GM-CSF and IFNα was not observed (data not shown).

A weak correlation of IL-3 with IL-4, and with IL-5 was seen in this study. A potential explanation for this could be the survival effect of IL-3 on basophils, which have been postulated to contribute to disease pathogenesis by elaborating Th2 cytokines (such as

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IL-4 and IL-5) in SLE, when activated by IgE-containing immune complexes.276 A positive correlation between IL-3 and basophil numbers was also seen in my study.

Using a true ‘no wash’ technique, this study has confirmed the findings from a majority of studies showing that pDCs and basophils are decreased in SLE compared to healthy controls. Naïve and memory B cells were also decreased in SLE patients in this study, as compared to divergent findings of either increased or decreased numbers or frequencies of these cell types in the literature.317-320 However it is more difficult to compare B cells with other studies in the literature, particularly memory B cells, as gating strategies between studies have been more varied than for pDCs and basophils.

Analysis of correlations between IL-3 levels with patient clinical manifestations and medication use was limited by the relatively small study numbers, and the heterogeneous nature of the SLE population. A potential association of IL-3 with platelet levels, as postulated in a previous study,285 was not observed in my study. However, the original study was performed on a patient population who were not receiving any standard of care medications, as compared to my patient cohort who were almost all receiving immunosuppressive or immunomodulatory medications, which may have affected the analysis.

Mean serum IL-3 levels were not found to be elevated in SLE patients compared to healthy controls, with almost equal numbers of SLE patients and healthy controls having detectable IL-3 levels. It is interesting that six of the healthy controls with elevated IL-3 levels had a documented history of asthma. There is evidence that IL-3 contributes to the pathogenesis of asthma, with increased IL-3 levels found in bronchoalveolar lavage fluid after allergen challenge.326 IL-3 has been shown to support the survival of the main pathogenic cell type in asthma, the eosinophil.327 However, serum IL-3 levels have not previously been found to be elevated in asthmatics. Specific targeting of IL-3 in asthma has not been trialled, however a humanized monoclonal antibody targeting the common β chain (a component of the receptors for IL-3, IL-5 and GM-CSF) has been shown in vitro to inhibit the survival of eosinophils (a key effector cell in asthma) present in induced sputum from human allergic asthmatics undergoing

151 allergic bronchoprovocation.328 The results of a completed phase 2 clinical trial of this agent in mild atopic asthma are pending (NCT01759849).

Recent data has uncovered the molecular heterogeneity of SLE patients, stratifying patients into different groups related to patient genotypes.329 The presence of an IFN gene signature in many SLE patients is well known, and has been used to stratify for potential treatment efficacy in clinical trials of the anti-IFNα, and anti-IFNAR mAbs. Early results from these trials indicate that this may be a successful strategy, as patients with higher IFN gene signatures at baseline were found to have a better response to these therapies.87 The findings in this Chapter support the presence of different genotypes in SLE patients, with a novel finding of an association between IL-3 and IFN gene signatures, and raise the possibility that those with a dual IFN/IL-3 gene signature may derive greater benefit from the therapeutic use of the anti-CD123 mAb, CSL362, than with IFN blockade alone.

Collectively, these findings highlight the interrelationships between key pathogenic cell types and cytokines IL-3, pDCs, basophils and IFN in SLE, and lend weight to the potential benefit of targeting these pathogenic mediators with the anti-CD123 mAb CSL362. These findings also support the existence of different molecular phenotypes in SLE patients, raising the possibility of personalized medicine to stratify patients according to their molecular signatures. Such signatures may determine their potential response to, and guide choices about, particular treatments.

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Chapter 5: Overall discussion, conclusions and future directions

SLE is a disease for which there is unacceptable ongoing morbidity, and mortality, despite currently available treatments, and there is an urgent need for novel therapeutics for this condition. B cell targeted strategies have been the focus of early biologic therapeutic development for SLE, but these have had relatively modest efficacy to date. The IFN pathway has emerged as a promising therapeutic target, with anti-IFNAR and anti-IFNα mAbs showing efficacy and safety in later phase clinical trials. Not all patients have responded to these therapies however, and other ways of targeting the IFN pathway, such as through the main IFN producing cell, the pDC, are worthy of exploration.

The work of this thesis has revealed a novel therapeutic approach to targeting the IFN pathway in SLE, through the use of a humanized anti-IL-3Rα/CD123 mAb, CSL362. CSL362 both neutralizes IL-3 mediated signaling, and effects ADCC against CD123- expressing cells. In this study, CSL362 was shown to alter a number of key pathogenic cell types and cytokines in SLE, including the pDC and IFN. These effects were demonstrated ex vivo in SLE patients, and in vivo in cynomolgus macaques.

This study has confirmed that CD123 is highly expressed on pDCs in SLE, as it is in healthy donors, and that the anti-CD123 mAb can potently deplete pDCs ex vivo in a heterogeneous population of SLE patients, who are already receiving standard of care therapies and immunosuppressive agents (such as steroids, hydroxychloroquine, azathioprine, methotrexate and mycophenolate). These effects were also seen in vivo in cynomolgus macaques. Although CSL362 was not tested in this study in vivo in an SLE disease model, others have shown that pDC depletion ameliorates disease in murine SLE models,91, 168 suggesting that pDC depletion in human SLE may be an effective therapeutic strategy. In this study, by depleting pDCs, CSL362 markedly reduced type I IFN production, and expression of a panel of IFN-inducible genes, specific to TLR7 and

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TLR9 stimulation. These receptors in SLE are thought to be activated by immune complexes or nucleic acids contained in NETs. Type I IFN production and IFN- inducible gene expression was also found to be stimulated by serum from SLE patients with high anti-dsDNA (>470 IU/ml) Ab levels, which is generally reflective of higher disease activity. Importantly, CSL362 also decreased IFN production in response to this physiologically relevant stimulus. Importantly also, some IFN production remained intact, most likely through stimulation of other TLRs (specifically 3, 4, 5 and 8). IFN has antiviral effects, and blockade of the IFN pathway increased viral infections, including herpes zoster reactivation, in anti-IFNα and anti-IFNAR mAb trials; potently reducing, but not eliminating, IFN may have beneficial implications for the safety profile of CSL362, compared to global IFN blockade.

CSL362 was also demonstrated to have effects on B cells, which are a key pathogenic cell type in SLE through their production of auto-antibodies which form immune complexes with autoantigens, subsequently causing organ damage. By depleting pDCs, CSL362 inhibited plasmablast expansion, which was specific to TLR7 and TLR9 stimulation (as it was not seen with a non-TLR stimulus CD40L). Reduced plasmablast expansion was a result of decreasing IFNα and IL-6 production from pDCs that were activated following TLR stimulation, confirming the importance of IFNα and IL-6 in promoting plasmablast expansion found previously in a virally-activated, CD40L- dependent system.306 Pre-treatment with CSL362 did not alter Ig levels or BAFF production ex vivo in SLE patients in this study; however, depletion of pDCs in murine models of SLE has been associated with diminished splenic germinal centres, and a decrease in autoantibody production, without any change in peripheral plasma cell numbers.91, 168 This raises the possibility that pDC depletion may differentially affect short-lived compared to long-lived plasma cells in these different compartments.

The findings from this work also suggest that depletion of pDCs, rather than blockade of type I IFN alone, may be more effective in inhibiting IFN-driven gene expression, and plasmablast expansion, in response to stimuli, such as immune complexes, that activate TLR7 and TLR9. Although it is the main type I IFN producing cell, activated pDCs also produce other cytokines, such as IL-6 and type III IFN, and so a decrease in

154 the pDC population may have several beneficial effects in SLE. In this study, CSL362 pre-treatment also decreased TLR7- and TLR9-induced type III IFN production. Although the exact role of type III IFN in SLE pathogenesis is still uncertain, it shares a common downstream signaling pathway with type I IFN, and has been hypothesized to contribute to ongoing disease activity despite therapeutic type I IFN blockade.95 There are as yet no specific type III IFN targeting therapies in SLE, and agents that can decrease both type I and III IFN may confer therapeutic advantages.

There are three other therapeutic strategies currently in development for SLE that target pDCs. Bcl-2, a pro-survival protein, has been postulated to inhibit the apoptosis of autoreactive lymphocytes. The Bcl-2 inhibitor ABT-199 (Venetoclax) has been found to deplete pDCs, without also depleting conventional DCs, in mice and also ex vivo in human SLE.120 Venetoclax was also shown to decrease lymphocyte numbers in both the NZB/W F1 mouse model of lupus nephritis,172 and in a phase 1 single and multiple ascending dose clinical trial of the drug in female SLE patients.173 The second pDC targeting therapeutic is an anti-BDCA2 mAb, BIIB059. BDCA2 is a pDC-specific cell surface marker. Rather than depleting pDCs, BIIB059 inhibits TLR7- and TLR9- stimulated type I IFN production through internalization of both BDCA2 and CD32 upon binding. A phase 1 trial (NCT02106897) of BIIB059 in SLE has recently completed recruitment; however, results are not yet available. The third agent, bortezomib, is a protease inhibitor that has been shown to decrease production of IFNα in lupus prone mice, through inhibiting pDC survival and their ability to produce IFNα in response to stimulation with a number of ligands including TLRs.174 CSL362 differs from these pDC targeting agents, with its mechanism of action resulting in the depletion of not only pDCs, but also basophils, as well as blockade of IL-3. Both basophils and IL-3 have also been shown to contribute to SLE pathogenesis.

Although relatively less studied in SLE compared to other cell types such as the B cell or pDCs, there is evidence that basophils contribute to SLE pathogenesis. Basophils have been postulated to be activated by IgE-containing immune complexes, and once activated, upregulate CD62L to home to secondary lymphoid organs. In lymph nodes, basophils augment autoantibody production by eliciting a Th2 response and production

155 of the B cell survival factor BAFF.275, 276 These effects were observed in vivo in the Lyn-/- murine SLE model, where basophil depletion by the antibody MAR-1 decreased disease, with a reduction in ANA autoantibodies, splenic plasma cells and levels of pro- inflammatory cytokines IL-4 and IFNγ in the kidney.276 Therapeutic targeting of basophil-mediated responses in SLE is currently being explored in a phase 1 trial of omalizumab, an anti-IgE mAb (NCT01716312). The data from my study indicate that basophils in SLE also highly express CD123 as compared to other major cell types in peripheral blood, although less so than pDCs. Like pDCs, basophils were also markedly depleted by CSL362 pre-treatment ex-vivo in SLE patients, and in vivo in cynomolgus macaques. The depletion of basophils ex vivo was not as complete as for pDCs however, possibly due to relatively lower CD123 expression, and higher circulating numbers in peripheral blood. It should be noted that administration of CSL362 to macaques resulted in complete depletion of both pDCs as well as basophils in vivo. Cell depletion by CSL362 was relatively specific to the CD123hi pDCs and basophils, as other cell types that demonstrated far lower CD123 expression, such as mDCs, monocytes, NK cells, and B and T cell subsets, were not depleted by CSL362 treatment.

CSL362 has two mechanisms of action - neutralization of IL-3 signalling, and activation of ADCC against CD123 expressing cells. IL-3 is a survival factor for both pDCs,280 and basophils,281 and this was supported by data in this study showing a positive correlation between serum IL-3 levels and peripheral blood pDCs and basophils. The depletion of pDCs and basophils by CSL362 was mostly a result of ADCC by activated NK cells. However, there may be a smaller contribution to cell depletion from IL-3 blockade, with higher doses of the IL-3 blocking Fab fragment of CSL362 reducing pDCs, but not basophils, ex vivo in SLE patients. In SLE, NK cells have been previously found to have an activated phenotype, but normal ADCC capability.308 This study confirms that NK cells from a heterogeneous population of SLE donors, taking various immunomodulatory treatments, are still activated by the IgG1-Fc portion of CSL362, which has been modified for enhanced ADCC through binding to CD16 on NK cells.

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Although IL-3 blockade was not the predominant mechanism of depletion of pDCs and basophils by CSL362, there may be as yet undiscovered benefits of neutralizing IL-3 signaling in SLE, apart from its survival benefit for pDCs and basophils. There have been few studies specifically addressing the role of IL-3 in SLE, apart from a small study showing elevated serum IL-3 levels in active SLE patients,285 and a more recent study showing that IL-3 perpetuates lupus nephritis in the MRL/lpr murine lupus model.283 Another recent study found that administration of IL-3 ex vivo enhanced IFNα production by pDCs stimulated with RNA containing immune complexes in healthy donors.325 Whether this was through IL-3 enhancing pDC survival, or by another mechanism, was not elucidated in that study, but further experiments to explore these mechanisms would be interesting. Supernatants from activated T cells from SLE patients were also found to enhance IFNα production from pDCs in the same study, however it was GM-CSF, and not IL-3 that was the key component promoting this phenomenon. Although I have not found elevated serum IL-3 levels in SLE patients compared to healthy controls, a novel association between IL-3 and IFN (both type I and type III) has been uncovered in SLE (and healthy) donors. This association is supported at a transcriptional level, with a strong correlation between donors with an ‘IL-3 gene signature’ and an ‘IFN gene signature’. SLE patients are known to display molecular heterogeneity,329 and gene expression ‘signatures’ can be used to stratify patients according to their clinical profile. These gene signatures may also potentially be used to indicate responsiveness to different therapies. For example, the ‘IFN gene signature’ has been used to stratify patients in clinical trials of the anti-IFN targeted mAbs, and those with a higher IFN gene score at baseline responded better to the anti- IFNAR mAb in a phase 2 trial.87 The data from this work supports the presence of different genotypes in SLE patients, and raise the possibility that those with a dual IFN/IL-3 gene signature may derive greater benefit from the therapeutic use of the anti- CD123 mAb CSL362, rather than from IFN blockade alone.

Analysis of potential correlations between serum IL-3 levels, and patient clinical manifestations and medication use was limited by the relatively small study numbers, and the heterogeneous nature of the SLE population. Further studies in greater numbers of patients may reveal correlations that were not evident in this study.

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Previous studies of intravenously administered CSL362 in cynomolgus macaques and in human AML have shown in vivo efficacy for depleting pDCs and basophils. Importantly, this study has confirmed in vivo efficacy of CSL362 via the subcutaneous route in cynomolgus macaques for depleting pDCs and basophils, and decreasing an IFN gene signature. Subcutaneous administration of parenteral therapeutics is the preferred route in chronic diseases such as SLE.

The cell types and cytokines altered by CSL362 contribute to anti-viral (pDCs and IFN), anti-helminth and anti-parasitic (basophil and IL-3) and haematopoietic functions,290 raising the possibility of unacceptable side effects from the blockade or depletion of these cell types or cytokines. In this study, CSL362 was well tolerated in cynomolgus macaques when delivered as a single, subcutaneous dose, and its biological effects were reversible as serum levels of the mAb declined. The mAb was similarly well tolerated in the phase 1 study in AML, in which patients received multiple intravenous CSL362 doses (ranging from 0.3-12.0mg/kg), resulting in rapid (≤ 6 hours post dose) and complete pDC and basophil depletion at all doses for a fortnightly dosing frequency. This effect was sustained for ≥ 15 days with doses ≥ 3mg/kg. No increase in infection risk, or cytopenias, were seen in this study of AML, with the only dose- limiting toxicities being hypertension, and infusion reactions that were controllable with hydrocortisone premedication.311 A currently recruiting phase 2 trial in AML (NCT02472145) will provide further data regarding longer term infection risk, and a clinical trial in SLE will be required to exclude any side effects specific to SLE patients.

Ex vivo efficacy of CSL362 in depleting pDCs, and decreasing IFNα production, was also demonstrated in this study in patients with a variety of other autoimmune diseases, including those where IFN may play a role in pathogenesis. These conditions included scleroderma, psoriasis, primary Sjögren’s syndrome, inflammatory myopathy, and rheumatoid arthritis. IFN receptor blockade has shown promising early results in a phase 1 trial in scleroderma, with near complete inhibition of IFN-inducible gene expression in peripheral blood and skin.302 Microarray analysis showed that IFN receptor blockade was also associated with inhibition of T cell receptor and

158 extracellular matrix related transcripts from whole blood and skin.330 A phase 1 trial of an anti-IFNα mAb sifalimumab, showed no clinical efficacy in plaque psoriasis, though it may be that the anti-IFNAR mAb anifrolumab may be more effective, as has been shown in SLE.88 Only small numbers of patients with each condition were tested ex vivo in this study; however, the results raise the possibility that CSL362 may be of benefit in these other IFN-dependent conditions.

In summary, this work has identified the IL-3Rα/CD123 as a potential therapeutic target in SLE. The anti-IL-3Rα mAb, CSL362, has a unique mechanism of action, altering multiple pathogenic cell types and cytokines in SLE donors ex vivo, and in vivo in cynomolgus macaques (Figure 5-1). CSL362 depletes pDCs and basophils, inhibits TLR7- and TLR9-induced type I and III IFN production, as well as IFN-inducible gene expression and plasmablast expansion. It appears to be more potent in terms of these effects than type I IFN blockade alone. There may be an additional benefit from CSL362 by neutralization of IL-3 signaling in SLE, and the novel association between IL-3 and IFN found in this study raises the possibility of stratifying patients in clinical trials of CSL362 with the molecular signatures identified in this study. In addition to its potential benefit as a therapeutic in SLE and other IFN dependent diseases, CSL362 may provide a new tool to dissect the role and interactions of pDCs, basophils, plasmablasts and IL-3 in SLE and other human diseases. To date, there are no major safety signals with the use of CSL362 in vivo, although its efficacy and safety in SLE await the results of future clinical trials.

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survival survival IL-3

pDC Type Basophil III ADCC ADCC IFN

CSL362 Th2 cytokines, BAFF IL-6 Type I IFN

IL-3

B cell

Autoantibody production

Figure 5-1: Proposed mechanism by which anti-IL-3Rα mAb CSL362 may be therapeutically useful in SLE Anti-IL-3Rα mAb CSL362 depletes pDCs and basophils, largely through ADCC, although there is a small contribution from IL-3 blockade which impairs pDC survival. Depletion of pDCs leads to a reduction in type I IFN production, and IL-6 production, which decreases plasmablast expansion. pDC depletion also decreases type III IFN.

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Appendix 1

SELENA-SLEDAI score292

Weight Descriptor Definition

8 Seizure Recent onset. Exclude metabolic, infectious or drug cause.

Altered ability to function in normal activity due to severe disturbance in the perception of reality. Include hallucinations, 8 Psychosis incoherence, marked loose association, impoverished thought content, marked illogical thinking, bizarre, disorganized, or catatonic behavior. Exclude uremia and drug causes.

Altered mental function with impaired orientation, memory or other intelligent function, with rapid onset fluctuating clinical features. Include clouding of consciousness with reduced capacity to focus, Organic brain 8 and inability to sustain attention to environment, plus at least two of syndrome the following: perceptual disturbance, incoherent speech, insomnia or daytime drowsiness, or increased or decreased psychomotor activity. Exclude metabolic, infectious or drug causes.

Retinal changes of SLE. Include cytoid bodies, retinal hemorrhages, 8 Visual disturbance serous exudate or hemorrhages in the choroid, or optic neuritis. Exclude hypertension, infection, or drug causes.

8 Cranial nerve disorder New onset of sensory or motor neuropathy involving cranial nerves.

Severe persistent headache: may be migrainous, but must be non- 8 Lupus headache responsive to narcotic analgesia.

8 CVA New onset of cerebrovascular accident(s). Exclude arteriosclerosis.

Ulceration, gangrene, tender finger nodules, periungual infarction, 8 Vasculitis splinter hemorrhages, or biopsy or angiogram proof of vasculitis.

More than 2 joints with pain and signs of inflammation (i.e. 4 Arthritis tenderness, swelling or effusion)

Proximal muscle aching/weakness, associated with elevated creatine 4 Myositis phosphokinase/adolase or electromyogram changes or a biopsy showing myositis

4 Urinary casts Heme-granular or red blood cell casts

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>5 red blood cells/high power field. Exclude stone, infection or 4 Hematuria other cause.

>0.5gm/24 hours. New onset or recent increase of more than 4 Proteinuria 0.5gm/24 hours.

4 Pyuria >5 white blood cells/high power field. Exclude infection.

2 New Rash New onset or recurrence of inflammatory type rash.

2 Alopecia New onset or recurrence of abnormal, patchy or diffuse loss of hair.

2 Mucosal ulcers New onset or recurrence of oral or nasal ulcerations.

Pleuritic chest pain with pleural rub or effusion, or pleural 2 Pleurisy thickening.

Pericardial pain with at least 1 of the following: rub, effusion, or 2 Pericarditis electrocardiogram confirmation.

Decrease in CH50, C3 or C4 below the lower limit of normal for 2 Low complement testing laboratory.

Increased DNA >25% binding by Farr assay or above normal range for testing 2 binding laboratory.

1 Fever >38°C. Exclude infectious cause.

1 Thrombocytopenia <100,000 platelets/mm3

1 Leukopenia <3000 white blood cell/mm3. Exclude drug causes.

TOTAL SCORE Sum of weights next to descriptors marked present

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Appendix 2

SDI Damage Index286

Item Score

Ocular (either eye, by clinical assessment) Any cataract ever 1 Retinal change or optic atrophy 1

Neuropsychiatric Cognitive impairment (e.g. memory deficit, difficulty with calculation, poor concentration, 1 difficulty in spoken or written language, impaired performance level) or major psychosis Seizures requiring therapy for 6 months 1 Cerebrovascular accident ever (score 2 if >1) 1 Cranial or peripheral neuropathy (excluding optic) 1 Transverse myelitis 1

Renal Estimated or measured glomerular filtration rate < 50% 1 Proteinuria ≥ 3.5gm/24 hours or 1 End-stage renal disease (regardless of dialysis or transplantation) 3

Pulmonary Pulmonary hypertension (right ventricular prominence, or loud P2) 1 Pulmonary fibrosis (physical and radiograph) 1 Shrinking lung (radiograph) 1 Pleural fibrosis (radiograph) 1 Pulmonary infarction (radiograph) 1

Cardiovascular Angina or coronary artery bypass (CABG) 1 Myocardial infarction (AMI) ever (score 2 if >1) 1 Cardiomyopathy (ventricular dysfunction) 1 Valvular disease (diastolic, murmur, or systolic murmur >3/6) 1 Pericarditis for 6 months, or pericardiectomy 1

Peripheral vascular Claudication for 6 months 1 Minor tissue loss (pulp space) 1 Significant tissue loss ever (e.g. loss of digit or limb) (score 2 if >1 site) 1

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Venous thrombosis with swelling, ulceration, or venous stasis 1

Gastrointestinal Infarction or resection of bowel below duodenum, spleen, liver, or gall bladder ever, for cause 1 any (score 2 if >1 site) Mesenteric insufficiency 1 Chronic peritonitis 1 Stricture or upper gastrointestinal tract surgery ever 1

Musculoskeletal Muscle atrophy or weakness 1 Deforming or erosive arthritis (including reducible deformities, excluding avascular necrosis) 1 Osteoporosis with fracture or vertebral collapse (excluding avascular necrosis) 1 Avascular necrosis (score 2 if >1) 1 Osteomyelitis 1

Skin Scarring chronic alopecia 1 Extensive scarring or panniculum other than scalp and pulp space 1 Skin ulceration (excluding thrombosis) for >6 months 1

Premature gonadal failure 1

Diabetes (regardless of treatment) 1

Malignancy (exclude dysplasia) (score 2 if >1 site) 1

Damage (non reversible change, not related to active inflammation) occurring since onset of lupus, ascertained by clinical assessment and present for at least 6 months unless otherwise stated. Repeat episodes must occur at least 6 months apart to score 2. The same lesion cannot be scored twice.

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Appendix 3

Gating strategies for assessment of CD123 cell surface expression on peripheral blood cell types

All gating strategies included a singlet gate.

Panel Cell type Defining cell surface markers

A pDC Lin1- HLA-DR+, BDCA2++ B Basophils Lin1- CCR3+ C mDCs Lin1-HLA-DR+, CD11c+ D Classical monocytes CD3-, CD14++ CD16- D Intermediate monocytes CD3-, CD14+ CD16+ D Non-classical monocytes CD3-, CD14- CD16++ E CD56dim NK cells NKp46+, CD3- CD56dim E CD56+ NK cells NKp46+, CD3- CD56+ F Naïve B cells CD19+ CD27- F Memory B cells CD19+ CD27+ F Plasmablasts CD19+ CD27++, CD20- CD38++ G Neutrophils CD16+ CD49d- G Eosinophils CD16- CD49+ H CD4+ T cells CD3+, CD4+ CD8- H CD8+ T cells CD3+, CD8+ CD4-

200

201

202

Appendix 4

Gating strategies for assessment of the effect of CSL362 on peripheral blood cell types

All gating strategies included a singlet gate.

Panel Cell type Defining cell surface markers

A pDC Lin1- HLA-DR+, BDCA2++ B Basophils Lin1- CCR3+ C CD56dim NK cells CD14-, CD3- CD56dim C CD56+ NK cells CD14-, CD3 CD56+ D Monocytes CD3-, CD14+ CD11b+ E mDCs Lin1-HLA-DR+, CD11c+ BDCA2-, BDCA1+ F Naïve B cells CD19+ CD27- F Memory B cells CD19+ CD27+ F Plasmablasts CD19+ CD27++ G CD4+ T cells CD3+, CD4+ CD8- G CD8+ T cells CD3+, CD8+ CD4-

203

204

205

Appendix 5

Gating strategies for assessment of peripheral blood cell counts

All gating strategies included a singlet gate.

Panel Cell type Defining surface markers

A pDC Lin1- HLA-DR+, BDCA2++ CD123++

B Basophils Lin1-, CCR3+ CD123++

C Naïve B cells CD19+ CD27-

C Memory B cells CD19+ CD27+

C Plasmablasts CD19+ CD27++, CD20- CD38++

D CD4+ T cells CD3+, CD4+ CD8-

D CD8+ T cells CD3+, CD8+ CD4-

206

207

Appendix 6

Differentially expressed genes between IL-3 stimulated, and unstimulated lysed whole blood from n = 7 healthy donors, at both 6 and 24 hours.

24 hours 6 hours

EnsemblID Symbol Chr log fold log fold change over p value change over p value unstimulated unstimulated

ENSG00000138135 CH25H 10 8.635011343 3.37E-06 7.803628308 5.55E-07 ENSG00000144681 STAC 3 6.391315672 8.38E-09 6.639687191 8.24E-13 ENSG00000099998 GGT5 22 6.017206466 6.67E-07 6.076104059 2.81E-09 ENSG00000183748 MRC1L1 10 5.501504333 6.02E-06 3.518345649 1.14E-08 ENSG00000102970 CCL17 16 4.987365805 0.00172062 7.927242029 4.19E-08 ENSG00000172752 COL6A5 3 4.857854948 1.62E-07 6.172582069 8.14E-10 ENSG00000122641 INHBA 7 4.746664362 6.67E-07 6.201737839 6.91E-10 ENSG00000184040 TMEM236 10 4.695275434 1.12E-06 3.692527044 2.24E-08 ENSG00000120586 MRC1 10 4.687201436 1.46E-05 3.177775128 1.63E-08 RP3- ENSG00000235782 333A15.1 1 4.63807295 6.14E-07 4.301559689 2.68E-07 ENSG00000137491 SLCO2B1 11 4.133036591 0.000652159 1.554936695 0.000843069 ENSG00000185897 FFAR3 19 4.021152503 6.50E-06 3.193377611 0.000127347 ENSG00000150510 FAM124A 13 3.922391901 8.18E-07 4.379695864 4.61E-08 ENSG00000050628 PTGER3 1 3.894713984 1.35E-08 3.154265472 1.15E-08 ENSG00000133101 CCNA1 13 3.74127691 3.45E-07 1.250282011 0.003834078 ENSG00000114737 CISH 3 3.611640512 1.75E-08 2.690136446 8.16E-08 RP4- ENSG00000227630 781K5.8 1 3.497810932 4.66E-07 2.838028324 5.46E-07 ENSG00000134531 EMP1 12 3.462747639 8.28E-09 1.657117671 8.28E-06 ENSG00000120833 SOCS2 12 3.425982875 8.28E-09 2.643389968 8.11E-09 ENSG00000196611 MMP1 11 3.270395767 0.002138447 6.020391694 1.04E-07 ENSG00000154269 ENPP3 6 3.264153977 8.93E-08 4.057559946 1.68E-09 RP11- ENSG00000229191 168O16.1 1 3.258360317 0.00011971 3.245758292 6.73E-06 ENSG00000109099 PMP22 17 3.125211173 1.93E-08 1.05324921 0.000732451

208

24 hours 6 hours

EnsemblID Symbol Chr log fold log fold change over p value change over p value unstimulated unstimulated

ENSG00000158477 CD1A 1 3.081618689 0.003084028 6.335955543 2.81E-08 ENSG00000169194 IL13 5 2.984688864 9.53E-05 3.58732143 2.30E-06 ENSG00000121594 CD80 3 2.90123498 2.98E-07 3.845757272 1.68E-09 ENSG00000142224 IL19 1 2.887484183 8.28E-09 1.293930295 0.003878231 ENSG00000002726 ABP1 7 2.787559512 0.003472596 1.119145707 0.010805696 ENSG00000122877 EGR2 10 2.765492198 8.93E-08 2.393707443 7.01E-08 ENSG00000136689 IL1RN 2 2.757180462 7.48E-05 3.580071155 4.26E-07 ENSG00000138316 ADAMTS14 10 2.74878989 0.002219303 3.395672418 7.50E-07 ENSG00000142549 IGLON5 19 2.745771984 9.14E-06 5.317389648 1.14E-09 ENSG00000120162 MOB3B 9 2.671904839 8.28E-09 3.389964253 1.36E-11 ENSG00000127954 STEAP4 7 2.632704678 5.62E-05 2.256764232 2.75E-05 ENSG00000103855 CD276 15 2.585263325 0.005756361 1.693384699 7.50E-07 ENSG00000158715 SLC45A3 1 2.552545467 1.27E-07 1.535900823 6.74E-06 ENSG00000167236 CCL23 17 2.484881961 0.000467146 6.121557376 8.61E-11 RP11- ENSG00000253698 1259L22.1 5 2.466727333 4.51E-06 1.572175076 0.000812131 ENSG00000152672 CLEC4F 2 2.465133832 0.005821073 4.256399664 2.31E-05 ENSG00000166670 MMP10 11 2.462773541 0.015325739 8.006312874 9.68E-10 ENSG00000166068 SPRED1 15 2.451530431 4.52E-08 2.059587921 4.88E-08 ENSG00000134489 HRH4 18 2.426217349 7.04E-05 1.270997614 0.002560611 RP11- ENSG00000259225 1008C21.1 15 2.355447658 1.65E-05 5.504894483 1.20E-12 ENSG00000090659 CD209 19 2.33281113 0.005333069 2.194737742 1.32E-06 ENSG00000148459 PDSS1 10 2.328018018 1.87E-07 1.632691578 7.25E-07 CTD- ENSG00000267416 2319I12.2 17 2.32784618 0.000404034 2.276180764 2.93E-05 ENSG00000113070 HBEGF 5 2.284611571 5.51E-07 2.058543322 7.19E-07 ENSG00000249992 TMEM158 3 2.267805949 0.003267102 3.369893928 4.63E-06 ENSG00000174705 SH3PXD2B 5 2.224661454 1.12E-06 1.779399304 1.68E-07 ENSG00000134460 IL2RA 10 2.199280244 4.52E-08 1.337378292 2.47E-06 ENSG00000152766 ANKRD22 10 2.16565359 0.028913387 3.69529338 4.82E-06 ENSG00000115008 IL1A 2 2.153874133 0.000104589 3.380504136 2.15E-07 RP11- ENSG00000223401 211G3.2 3 2.148541208 2.69E-05 1.564440292 7.75E-05

209

24 hours 6 hours

EnsemblID Symbol Chr log fold log fold change over p value change over p value unstimulated unstimulated

ENSG00000111729 CLEC4A 12 2.100015321 0.000114603 2.297417275 5.22E-06 ENSG00000158270 COLEC12 18 2.097068517 0.036162973 1.462330222 0.000231606 ENSG00000128918 ALDH1A2 15 2.086208152 0.000478264 5.458190011 1.83E-10 ENSG00000143382 ADAMTSL4 1 2.080633306 1.62E-07 1.855097012 5.07E-08 ENSG00000158488 CD1E 1 2.069176722 0.003739858 5.046025485 2.41E-11 ENSG00000198142 SOWAHC 2 2.049378403 1.27E-07 1.648825983 4.35E-07 ENSG00000163735 CXCL5 4 2.01829873 0.000385823 6.955447614 2.75E-12 ENSG00000185291 IL3RA X 1.975323325 8.93E-08 2.967798821 6.34E-11 ENSG00000106178 CCL24 7 1.972525599 0.000574414 3.914447871 5.42E-10 ENSG00000120659 TNFSF11 13 1.965637201 1.77E-06 2.886516184 9.68E-10 ENSG00000099250 NRP1 10 1.947004524 5.78E-07 2.004156617 4.53E-08 ENSG00000158481 CD1C 1 1.90061872 0.000128538 3.595191583 4.57E-09 ENSG00000152953 STK32B 4 1.900369297 0.009880177 1.176048613 0.008301899 ENSG00000254521 SIGLEC12 19 1.891376784 1.33E-06 2.059480931 2.51E-08 ENSG00000070214 SLC44A1 9 1.852147289 1.27E-07 1.020771248 3.70E-05 ENSG00000198121 LPAR1 9 1.844337644 0.00017948 2.357513202 1.25E-08 ENSG00000205927 OLIG2 21 1.83909684 0.003597601 1.855844597 0.000286196 ENSG00000108691 CCL2 17 1.828059003 0.010182038 3.304541671 3.06E-06 ENSG00000169255 B3GALNT1 3 1.813937949 3.21E-05 1.663414733 2.57E-05 ENSG00000189221 MAOA X 1.810182969 0.001692875 2.834837979 7.09E-08 ENSG00000163736 PPBP 4 1.777976124 9.58E-06 6.731565154 5.26E-15 ENSG00000110848 CD69 12 1.762629712 6.73E-08 1.357891883 4.16E-07 ENSG00000185275 CD24P4 Y 1.750677195 1.34E-05 1.865434575 3.02E-07 ENSG00000125538 IL1B 2 1.733608328 0.000164169 3.048713085 1.33E-08 ENSG00000117009 KMO 1 1.732171569 1.75E-08 1.58271105 6.37E-09 CTB- ENSG00000264868 167B5.2 7 1.724628566 0.002630204 1.647055125 4.75E-05 ENSG00000163235 TGFA 2 1.724402282 2.52E-07 1.096417527 1.65E-05 RP11- ENSG00000251230 701P16.5 4 1.686208876 0.034610669 2.738399355 0.000132705 ENSG00000119686 FLVCR2 14 1.683471885 6.40E-08 1.671517446 4.93E-09 ENSG00000120262 CCDC170 6 1.666815887 0.012333129 1.472654129 0.000390775 ENSG00000158485 CD1B 1 1.665339593 0.016583094 7.649423247 1.10E-13

210

24 hours 6 hours

EnsemblID Symbol Chr log fold log fold change over p value change over p value unstimulated unstimulated

ENSG00000198959 TGM2 20 1.6590131 0.000268965 2.6575326 2.27E-07 ENSG00000198400 NTRK1 1 1.625572089 2.86E-07 3.041011618 1.83E-10 ENSG00000105376 ICAM5 19 1.62295048 0.00139127 1.24629318 0.000281734 ENSG00000104921 FCER2 19 1.587391525 0.00080203 3.67695967 6.66E-10 ENSG00000161955 TNFSF13 17 1.549610137 6.21E-05 1.73490801 2.69E-07 ENSG00000185338 SOCS1 16 1.528575178 8.26E-07 2.248043463 1.11E-09 ENSG00000134755 DSC2 18 1.519356801 0.001912311 1.059320344 0.003627449 ENSG00000029153 ARNTL2 12 1.517797925 0.00014809 1.88082967 1.42E-07 ENSG00000158163 DZIP1L 3 1.510024844 0.000682316 1.684180857 2.78E-06 CTD- ENSG00000261040 2319I12.1 17 1.50258218 0.008863245 2.531424662 2.43E-06 ENSG00000163814 CDCP1 3 1.488319273 0.000256342 2.023459239 1.49E-06 ENSG00000134780 DAGLA 11 1.462143688 0.00024244 1.024914291 0.000115554 ENSG00000105492 SIGLEC6 19 1.459592482 0.002388897 1.871332174 2.10E-06 ENSG00000109861 CTSC 11 1.444019596 2.11E-05 1.678662393 5.72E-07 ENSG00000106537 TSPAN13 7 1.442772964 1.27E-07 1.497580022 1.23E-08 ENSG00000175445 LPL 8 1.438263145 0.007583254 2.367419386 1.50E-08 ENSG00000198829 SUCNR1 3 1.431203744 0.025473947 1.896319679 0.000121495 ENSG00000079385 CEACAM1 19 1.412507655 7.04E-05 1.441812158 4.99E-06 ENSG00000140450 ARRDC4 15 1.409565158 4.26E-05 2.033069195 1.94E-08 ENSG00000152315 KCNK13 14 1.401004603 0.000185511 1.639968858 8.18E-07 ENSG00000154262 ABCA6 17 1.387880385 0.008592081 3.063255428 1.04E-09 ENSG00000255398 HCAR3 12 1.380063471 0.00628131 2.053766351 9.53E-06 ENSG00000188215 DCUN1D3 16 1.377790358 1.65E-07 1.95240595 5.42E-10 ENSG00000157557 ETS2 21 1.35432354 7.55E-05 1.54617618 2.02E-06 ENSG00000182782 HCAR2 12 1.310359551 0.00315327 1.957019115 5.11E-06 ENSG00000175898 S1PR2 19 1.295769442 7.65E-07 1.089115537 5.61E-07 ENSG00000100368 CSF2RB 22 1.282989443 0.000363492 1.815208834 7.25E-07 ENSG00000183087 GAS6 13 1.280832019 0.001592945 1.658566077 5.00E-07 ENSG00000151012 SLC7A11 4 1.275712418 0.002569818 2.709487581 3.30E-08 ENSG00000039068 CDH1 16 1.270435137 3.93E-07 1.874641302 1.66E-09 ENSG00000138166 DUSP5 10 1.267746265 1.59E-05 2.14968583 1.92E-09 ENSG00000138623 SEMA7A 15 1.25686385 1.03E-08 1.072169267 1.30E-08 ENSG00000116031 CD207 2 1.253111059 0.041683465 1.9390849 0.001788854

211

24 hours 6 hours

EnsemblID Symbol Chr log fold log fold change over p value change over p value unstimulated unstimulated

ENSG00000171236 LRG1 19 1.249927283 0.000921663 1.8257223 3.09E-06 ENSG00000175592 FOSL1 11 1.243288389 0.0069738 3.130944134 8.14E-08 ENSG00000177469 PTRF 17 1.242778174 0.00033278 1.301038115 9.86E-05 ENSG00000183696 UPP1 7 1.238753387 2.76E-07 1.68616102 8.06E-10 ENSG00000164741 DLC1 8 1.199592776 0.011679204 1.785849609 6.62E-06 ENSG00000174944 P2RY14 3 1.196428159 0.003881195 2.032556116 9.39E-07 ENSG00000136630 HLX 1 1.19531932 0.002630204 1.055416124 0.000805266 ENSG00000070915 SLC12A3 16 1.194953464 0.045703845 1.322576753 0.001565672 ENSG00000267534 S1PR2 19 1.177015488 0.000102286 1.077176302 1.43E-05 ENSG00000145491 ROPN1L 5 1.166972095 0.018832327 2.370193062 3.53E-06 ENSG00000166689 PLEKHA7 11 1.149310287 0.000167397 1.301440094 3.34E-07 ENSG00000176597 B3GNT5 3 1.144108779 3.40E-05 1.182518995 3.26E-06 ENSG00000184557 SOCS3 17 1.136789904 0.000394316 1.449424441 2.69E-06 ENSG00000102471 NDFIP2 13 1.135893495 3.26E-05 2.065293526 4.79E-09 ENSG00000031081 ARHGAP31 3 1.101350139 3.26E-05 2.043280699 1.16E-09 ENSG00000134243 SORT1 1 1.085524204 0.001142485 1.315681906 1.74E-05 ENSG00000178719 GRINA 8 1.066008902 0.001438105 1.273467867 2.99E-05 ENSG00000032444 PNPLA6 19 1.064692693 1.12E-06 1.145965004 8.16E-08 ENSG00000123685 BATF3 1 1.051029389 0.000362575 3.352209032 2.75E-12 ENSG00000135047 CTSL1 9 1.031367189 0.020065238 1.942688621 6.18E-06 ENSG00000140749 IGSF6 16 1.0281269 0.000130895 1.687116508 3.26E-08 ENSG00000106266 SNX8 7 1.014901721 5.30E-06 1.037940933 5.25E-07 ENSG00000137193 PIM1 6 1.007929446 1.33E-06 1.300397207 8.09E-09 ENSG00000104951 IL4I1 19 1.001911205 0.000362575 1.446464806 2.75E-07 ENSG00000179639 FCER1A 1 -1.014590398 0.000583603 -1.079615407 2.66E-05 ENSG00000166033 HTRA1 10 -1.2401828 0.000807028 -2.685075218 8.63E-09 ENSG00000170893 TRH 3 -1.344272631 0.003472596 -2.083066341 0.000373372 ENSG00000091181 IL5RA 3 -1.372717124 0.000917334 -1.643009253 4.00E-06 ENSG00000169330 KIAA1024 15 -1.432961694 0.000609848 -1.346161063 6.43E-05 ENSG00000129757 CDKN1C 11 -1.520519572 0.000175502 -1.369917025 0.000361169 ENSG00000077063 CTTNBP2 7 -1.55819813 0.001347618 -4.592076666 6.66E-10 ENSG00000087245 MMP2 16 -1.587782952 0.002248934 -2.314359305 9.66E-07 ENSG00000166147 FBN1 15 -1.941196406 6.68E-05 -1.495875703 0.000124677 ENSG00000197046 SIGLEC15 18 -1.996404244 0.041570575 -2.076297615 0.000252572 ENSG00000119457 SLC46A2 9 -2.262757089 8.03E-05 -1.121766182 0.007045864

212

Appendix 7

Differentially expressed genes between IL-3 stimulated, and unstimulated lysed whole blood from n = 7 healthy donors, at 24 hours

log fold Chromo- EnsemblID Symbol change over p value some unstimulated

ENSG00000181374 CCL13 17 8.323390334 1.86E-12 ENSG00000117594 HSD11B1 1 6.690881063 8.97E-09 ENSG00000108688 CCL7 17 6.562991214 6.25E-07 ENSG00000131435 PDLIM4 5 4.76952717 4.19E-08 ENSG00000205846 CLEC6A 12 4.414577438 6.17E-09 ENSG00000124102 PI3 20 4.309058243 1.89E-06 ENSG00000135472 FAIM2 12 4.039834712 5.41E-09 ENSG00000117525 F3 1 3.878982278 4.35E-07 ENSG00000123610 TNFAIP6 2 3.690801306 9.18E-06 ENSG00000133687 TMTC1 12 3.630456824 0.000124728 ENSG00000006074 CCL18 17 3.604486859 1.39E-06 ENSG00000166920 C15orf48 15 3.571540762 1.50E-07 ENSG00000184292 TACSTD2 1 3.483818979 0.002171683 ENSG00000164935 DCSTAMP 8 3.393725413 1.32E-08 ENSG00000124785 NRN1 6 3.313433649 4.68E-05 ENSG00000213694 S1PR3 9 3.307776472 5.46E-07 ENSG00000186583 SPATC1 8 3.297045032 4.13E-05 ENSG00000229754 CXCR2P1 2 3.276260107 3.34E-07 RP11- ENSG00000260943 476D10.1 12 3.269433238 6.05E-06 ENSG00000134668 SPOCD1 1 3.261252781 2.09E-05 ENSG00000102755 FLT1 13 3.258618553 6.91E-10 ENSG00000123700 KCNJ2 17 3.248238153 1.70E-05 ENSG00000267365 KCNJ2-AS1 17 3.246044323 0.000608966 ENSG00000008118 CAMK1G 1 3.242902192 0.00055523 ENSG00000183019 C19orf59 19 3.217799794 1.42E-10 ENSG00000137757 CASP5 11 3.197151218 4.11E-09 ENSG00000186081 KRT5 12 3.186862531 7.25E-06 ENSG00000165140 FBP1 9 3.142124442 2.72E-10

213

log fold Chromo- EnsemblID Symbol change over p value some unstimulated

ENSG00000259207 ITGB3 17 3.128939214 5.46E-07 ENSG00000184500 PROS1 3 3.124905701 1.92E-09 ENSG00000100985 MMP9 20 3.104365996 2.38E-06 ENSG00000091879 ANGPT2 8 3.094972156 1.41E-07 ENSG00000108700 CCL8 17 3.090393526 2.44E-07 ENSG00000106366 SERPINE1 7 3.036545638 7.89E-05 ENSG00000169429 IL8 4 2.255246446 2.20E-08 ENSG00000178175 ZNF366 5 2.246333835 1.75E-05 ENSG00000142910 TINAGL1 1 2.244390804 0.0011398 ENSG00000126262 FFAR2 19 2.22123709 5.04E-06 ENSG00000166523 CLEC4E 12 2.213027384 1.63E-06 ENSG00000174307 PHLDA3 1 2.200161814 3.52E-07 ENSG00000136052 SLC41A2 12 2.190862972 7.61E-10 ENSG00000085831 TTC39A 1 2.189229661 1.12E-08 ENSG00000125869 LAMP5 20 2.180240511 2.44E-07 ENSG00000101000 PROCR 20 2.166811793 7.77E-10 ENSG00000148926 ADM 11 2.153880364 5.96E-06 ENSG00000179294 C17orf96 17 2.147501527 4.15E-06 ENSG00000115828 QPCT 2 2.144539682 4.13E-08 ENSG00000183347 GBP6 1 2.143267608 1.61E-05 ENSG00000225342 AC079630.4 12 2.142454221 7.52E-06 ENSG00000170866 LILRA3 19 2.135315772 1.42E-10 ENSG00000139318 DUSP6 12 2.117732976 1.23E-08 ENSG00000139567 ACVRL1 12 2.108097265 1.96E-06 ENSG00000166265 CYYR1 21 2.074697222 3.35E-07 ENSG00000140279 DUOX2 15 2.048115776 5.74E-05 ENSG00000136830 FAM129B 9 2.04369324 3.79E-10 ENSG00000154065 ANKRD29 18 2.041410261 0.000947262 ENSG00000244405 ETV5 3 2.031101496 9.44E-06 ENSG00000178860 MSC 8 2.030514829 3.07E-06 ENSG00000117228 GBP1 1 2.028592687 1.50E-05 RP11- ENSG00000235531 383H13.1 8 2.01710435 4.15E-05 ENSG00000232810 TNF 6 2.00857741 7.92E-11 ENSG00000087842 PIR X 2.006012074 2.35E-06 ENSG00000173391 OLR1 12 1.994096124 1.39E-07 ENSG00000149798 CDC42EP2 11 1.986344276 0.000107475

214

log fold Chromo- EnsemblID Symbol change over p value some unstimulated RP11- ENSG00000259354 519G16.3 15 1.962592748 0.000777637 ENSG00000125744 RTN2 19 1.959526275 1.24E-06 ENSG00000140379 BCL2A1 15 1.956280487 3.82E-08 RP11- ENSG00000233029 439A17.9 1 1.954182151 7.25E-05 ENSG00000166803 KIAA0101 15 1.951285492 2.27E-05 ENSG00000138119 MYOF 10 1.945305555 2.20E-08 ENSG00000104783 KCNN4 19 1.942293425 9.30E-08 RP11- ENSG00000236700 557H15.2 6 1.928902212 9.83E-05 RP11- ENSG00000231233 127L20.6 10 1.92883615 0.000482019 ENSG00000163739 CXCL1 4 3.001254283 6.98E-11 ENSG00000049130 KITLG 12 2.972323773 2.95E-09 ENSG00000090530 LEPREL1 3 2.953742601 3.31E-07 ENSG00000225492 GBP1P1 1 2.875036933 6.49E-06 ENSG00000184371 CSF1 1 2.824446778 8.86E-08 ENSG00000229314 ORM1 9 2.7637973 0.002745569 ENSG00000254281 KB-1507C5.4 8 2.757360734 2.33E-05 ENSG00000173372 C1QA 1 2.742012474 5.14E-08 ENSG00000181649 PHLDA2 11 2.738609591 1.57E-05 ENSG00000089558 KCNH4 17 2.717197152 0.000636038 ENSG00000006075 CCL3 17 2.712042429 3.67E-08 ENSG00000250771 RP11-153M7.3 4 2.685724336 8.49E-08 ENSG00000250274 CTB-114C7.4 5 2.683749238 5.74E-07 ENSG00000169896 ITGAM 16 2.6507555 4.78E-11 ENSG00000120217 CD274 9 2.649153625 1.71E-06 ENSG00000163734 CXCL3 4 2.600012071 2.22E-08 ENSG00000205020 CCL4L1 17 2.522558067 5.41E-09 ENSG00000124107 SLPI 20 2.515681584 2.27E-05 ENSG00000101017 CD40 20 2.5061288 8.14E-10 ENSG00000168546 GFRA2 8 2.489876088 1.25E-07 ENSG00000258227 CLEC5A 7 2.462896316 8.63E-09 ENSG00000123689 G0S2 1 2.456673364 9.46E-06 RP11- ENSG00000267452 1018N14.5 17 2.453244793 3.34E-08 ENSG00000100336 APOL4 22 2.421258011 2.09E-05 ENSG00000180316 PNPLA1 6 2.413314091 0.00012081 ENSG00000166963 MAP1A 15 2.411111007 1.68E-09 ENSG00000124875 CXCL6 4 2.397836873 2.25E-05

215

log fold Chromo- EnsemblID Symbol change over p value some unstimulated

ENSG00000105855 ITGB8 7 1.718311964 0.001794023 ENSG00000196878 LAMB3 1 1.716349355 0.000239133 ENSG00000162433 AK4 1 1.70720366 5.66E-05 ENSG00000008394 MGST1 12 1.707014274 9.31E-06 ENSG00000175471 MCTP1 5 1.706103511 8.50E-05 ENSG00000176014 TUBB6 18 1.701487883 1.74E-09 ENSG00000165685 TMEM52B 12 1.695251949 1.09E-05 ENSG00000197262 CCL4L2 17 1.691169425 0.003972157 ENSG00000207808 MIR27A 19 1.689791525 0.000220175 ENSG00000183762 KREMEN1 22 1.68794048 1.45E-05 ENSG00000175352 NRIP3 11 1.682950166 9.84E-06 ENSG00000181634 TNFSF15 9 1.679168933 0.00145044 ENSG00000094804 CDC6 17 1.678612027 1.10E-07 ENSG00000183307 CECR6 22 1.677377292 5.71E-06 ENSG00000166926 MS4A6E 11 1.670119566 0.014914041 ENSG00000019169 MARCO 2 1.66970982 0.013745321 ENSG00000126787 DLGAP5 14 1.668111751 4.53E-05 ENSG00000188056 TREML4 6 1.666185094 0.000278827 ENSG00000136010 ALDH1L2 12 1.663798526 1.88E-06 ENSG00000100906 NFKBIA 14 1.661584412 2.07E-06 ENSG00000205021 CCL3L1 17 1.655451151 0.006138113 ENSG00000157193 LRP8 1 1.654855356 5.29E-08 ENSG00000137094 DNAJB5 9 1.653816972 3.64E-05 ENSG00000164023 SGMS2 4 1.650745619 1.76E-09 ENSG00000115107 STEAP3 2 1.650661773 0.000157263 ENSG00000136960 ENPP2 8 1.650603909 7.18E-05 ENSG00000110841 PPFIBP1 12 1.650306568 0.00014897 ENSG00000104267 CA2 8 1.648815269 1.97E-07 ENSG00000120051 CCDC147 10 1.64559589 0.000216713 ENSG00000227591 RP1-28O10.1 1 1.625377774 0.000105483 ENSG00000148180 GSN 9 1.624090295 7.83E-05 ENSG00000119326 CTNNAL1 9 1.62360291 1.23E-06 ENSG00000123360 PDE1B 12 1.615156874 1.55E-05 ENSG00000166002 C11orf75 11 1.614230958 6.91E-06 ENSG00000228278 ORM2 9 1.610792762 0.006332453 ENSG00000005961 ITGA2B 17 1.606008422 4.51E-05 ENSG00000198794 SCAMP5 15 1.60363895 1.53E-07

216

log fold Chromo- EnsemblID Symbol change over p value some unstimulated

ENSG00000074660 SCARF1 17 1.601151627 7.52E-06 ENSG00000185215 TNFAIP2 14 1.59894174 1.48E-05 ENSG00000165490 C11orf82 11 1.596869056 4.35E-06 ENSG00000197249 SERPINA1 14 1.591940596 7.44E-08 ENSG00000185022 MAFF 22 1.578044076 8.37E-07 ENSG00000129682 FGF13 X 2.391537287 1.05E-05 ENSG00000159640 ACE 17 2.378714317 1.94E-08 ENSG00000261269 RP11-389C8.2 5 2.326007739 1.97E-07 ENSG00000010030 ETV7 6 2.316850838 0.000107475 ENSG00000171848 RRM2 2 2.315131418 1.08E-07 ENSG00000168062 BATF2 11 2.315109563 0.00017491 ENSG00000134070 IRAK2 3 2.311929858 7.56E-06 ENSG00000197467 COL13A1 10 2.309234148 6.58E-05 ENSG00000101384 JAG1 20 2.309078518 7.06E-08 ENSG00000106341 PPP1R17 7 2.303914445 7.25E-07 ENSG00000137819 PAQR5 15 2.295931267 3.13E-05 ENSG00000074181 NOTCH3 19 2.284449964 4.78E-05 ENSG00000166527 CLEC4D 12 2.264530888 1.49E-05 ENSG00000173369 C1QB 1 2.261493952 9.53E-06 ENSG00000164266 SPINK1 5 1.926112943 0.03181989 ENSG00000138772 ANXA3 4 1.911478951 0.000363292 ENSG00000104320 NBN 8 1.90812545 5.04E-06 ENSG00000197646 PDCD1LG2 9 1.904023591 0.000192652 ENSG00000108244 KRT23 17 1.899551754 5.38E-05 ENSG00000100234 TIMP3 22 1.898731373 0.000324929 ENSG00000163687 DNASE1L3 3 1.89813155 7.48E-05 ENSG00000128342 LIF 22 1.885882043 1.01E-06 ENSG00000213889 PPM1N 19 1.863620228 4.98E-06 ENSG00000124762 CDKN1A 6 1.855550397 2.18E-08 ENSG00000172070 SRXN1 20 1.852954314 6.20E-07 ENSG00000090339 ICAM1 19 1.848887106 8.89E-07 ENSG00000142512 SIGLEC10 19 1.847304493 0.000225043 ENSG00000112096 SOD2 6 1.838261268 2.78E-05 ENSG00000221955 SLC12A8 3 1.837641291 2.64E-05 ENSG00000139572 GPR84 12 1.83178111 2.38E-05 ENSG00000187134 AKR1C1 10 1.830660963 0.001367862 ENSG00000196932 TMEM26 10 1.817221018 0.00053003

217

log fold Chromo- EnsemblID Symbol change over p value some unstimulated

ENSG00000241163 RP11-398A8.3 3 1.813389212 0.000187035 RP11- ENSG00000253214 1149M10.2 8 1.801810635 3.13E-05 ENSG00000158714 SLAMF8 1 1.801352813 7.79E-08 ENSG00000163710 PCOLCE2 3 1.795575086 0.002957016 ENSG00000261596 CTB-31N19.3 16 1.793493286 1.62E-06 ENSG00000184106 TREML3P 6 1.784055279 0.000122202 ENSG00000143554 SLC27A3 1 1.778244662 2.98E-06 ENSG00000121797 CCRL2 3 1.773711864 4.94E-07 ENSG00000075618 FSCN1 7 1.772442735 1.56E-07 ENSG00000129277 CCL4 17 1.770773172 6.01E-06 ENSG00000119917 IFIT3 10 1.766470344 0.000335275 ENSG00000101335 MYL9 20 1.76328186 0.000346436 ENSG00000102174 PHEX X 1.755336476 2.91E-06 ENSG00000116096 SPR 2 1.754424017 1.26E-06 ENSG00000169504 CLIC4 1 1.753432076 1.49E-07 ENSG00000169439 SDC2 8 1.751425724 1.61E-05 ENSG00000100024 UPB1 22 1.750250711 0.004754696 ENSG00000181631 P2RY13 3 1.747776493 2.10E-06 ENSG00000150048 CLEC1A 12 1.744391338 5.75E-06 ENSG00000143067 ZNF697 1 1.743061107 1.42E-07 ENSG00000198019 FCGR1B 1 1.737466801 0.000244991 ENSG00000145365 TIFA 4 1.736004858 3.23E-07 ENSG00000103569 AQP9 15 1.728851149 7.09E-08 ENSG00000168389 MFSD2A 1 1.577072324 0.000269828 ENSG00000118292 C1orf54 1 1.576122846 1.15E-08 ENSG00000124134 KCNS1 20 1.575456396 0.001244022 ENSG00000065833 ME1 6 1.574364764 5.04E-06 ENSG00000122861 PLAU 10 1.572849806 7.56E-06 ENSG00000172322 CLEC12A 12 1.559170793 2.83E-05 ENSG00000197632 SERPINB2 18 1.551108689 9.98E-05 ENSG00000150337 FCGR1A 1 1.542415204 0.000103647 RP11- ENSG00000255801 561P12.5 12 1.541901757 0.001235321 ENSG00000075391 RASAL2 1 1.541294605 1.02E-08 ENSG00000169116 PARM1 4 1.538295411 9.60E-08 ENSG00000136158 SPRY2 13 1.537465433 1.47E-06 ENSG00000226091 RP11-90D4.2 12 1.536206657 0.000165924 ENSG00000100448 CTSG 14 1.532025859 0.013065567

218

log fold Chromo- EnsemblID Symbol change over p value some unstimulated

ENSG00000167641 PPP1R14A 19 1.531839073 9.66E-06 ENSG00000136810 TXN 9 1.529236694 5.23E-08 ENSG00000211650 IGLV5-45 22 1.523734106 0.000264429 ENSG00000183010 PYCR1 17 1.520378861 1.11E-05 ENSG00000147650 LRP12 8 1.51955889 1.44E-06 ENSG00000159189 C1QC 1 1.519468846 0.000239141 ENSG00000125730 C3 19 1.514284633 0.001940958 ENSG00000075420 FNDC3B 3 1.506674907 5.46E-07 ENSG00000138678 AGPAT9 4 1.503009619 1.25E-05 ENSG00000239672 NME1 17 1.49817641 3.03E-06 ENSG00000125462 C1orf61 1 1.495170695 0.00249693 ENSG00000240310 RP11-24F11.5 3 1.492579433 0.006730741 ENSG00000052749 RRP12 10 1.474690088 1.50E-08 ENSG00000172965 AC068491.1 2 1.474133863 7.53E-05 ENSG00000072682 P4HA2 5 1.471231593 1.04E-05 ENSG00000165168 CYBB X 1.468502928 1.22E-05 ENSG00000233791 RP11-134P9.1 1 1.468377176 0.000264081 ENSG00000131203 IDO1 8 1.466450985 0.000126288 ENSG00000085733 CTTN 11 1.463732364 0.000194962 ENSG00000170396 ZNF804A 2 1.461814328 4.75E-06 ENSG00000233488 AC108463.3 2 1.457449485 1.51E-05 ENSG00000253958 CLDN23 8 1.457387431 0.000664639 ENSG00000154451 GBP5 1 1.452650046 0.000136915 ENSG00000140105 WARS 14 1.451076994 9.01E-07 ENSG00000168386 FILIP1L 3 1.450891028 1.76E-09 ENSG00000179750 APOBEC3B 22 1.446370065 1.21E-05 ENSG00000226644 RP11-128M1.1 20 1.444160336 0.000632828 ENSG00000037897 METTL1 12 1.442785893 1.85E-05 ENSG00000163624 CDS1 4 1.43575434 7.03E-06 ENSG00000225611 RP11-70C1.1 3 1.435709768 5.43E-05 ENSG00000198814 GK X 1.433418924 2.64E-07 ENSG00000155659 VSIG4 X 1.433402767 0.000675226 ENSG00000115598 IL1RL2 2 1.43242208 4.45E-05 ENSG00000181019 NQO1 16 1.430227423 1.23E-06 ENSG00000073008 PVR 19 1.423223675 2.63E-08 ENSG00000137393 RNF144B 6 1.420409909 0.000308175 ENSG00000166091 CMTM5 14 1.417490095 0.009067849

219

log fold Chromo- EnsemblID Symbol change over p value some unstimulated

ENSG00000148737 TCF7L2 10 1.413187628 0.000109122 ENSG00000165795 NDRG2 14 1.410641372 7.64E-05 ENSG00000179331 RAB39A 11 1.40925729 8.71E-07 ENSG00000123095 BHLHE41 12 1.408400102 3.51E-06 ENSG00000163823 CCR1 3 1.394777766 4.13E-08 ENSG00000117399 CDC20 1 1.389902643 0.000317649 ENSG00000169403 PTAFR 1 1.387394024 2.08E-05 ENSG00000211642 IGLV10-54 22 1.384321787 2.79E-05 ENSG00000071246 VASH1 14 1.383282214 4.74E-09 ENSG00000186431 FCAR 19 1.38253724 1.36E-06 ENSG00000124145 SDC4 20 1.378601137 8.63E-07 ENSG00000151726 ACSL1 4 1.376238761 5.89E-05 ENSG00000107130 NCS1 9 1.375775562 8.95E-05 ENSG00000102962 CCL22 16 1.372813925 1.97E-07 ENSG00000140961 OSGIN1 16 1.369786556 3.04E-06 ENSG00000132170 PPARG 3 1.367311501 0.002034359 ENSG00000189068 VSTM1 19 1.357639233 0.000185771 ENSG00000178726 THBD 20 1.351939389 8.35E-05 ENSG00000169245 CXCL10 4 1.351495778 0.005531255 ENSG00000204482 LST1 6 1.349273456 3.08E-08 ENSG00000164626 KCNK5 6 1.344789836 2.53E-08 ENSG00000196083 IL1RAP 3 1.342127188 1.41E-07 ENSG00000188313 PLSCR1 3 1.341191227 2.36E-06 ENSG00000118193 KIF14 1 1.341080017 0.000121382 ENSG00000204287 HLA-DRA 6 1.33895195 3.79E-10 ENSG00000102003 SYP X 1.336227074 2.26E-05 ENSG00000101057 MYBL2 20 1.335869997 5.83E-06 ENSG00000186074 CD300LF 17 1.326698449 9.80E-07 ENSG00000104312 RIPK2 8 1.323576436 6.71E-06 ENSG00000132965 ALOX5AP 13 1.32259593 4.97E-08 ENSG00000268734 CTB-61M7.2 19 1.322416695 8.14E-06 ENSG00000151490 PTPRO 12 1.322067137 5.77E-08 ENSG00000067113 PPAP2A 5 1.320702362 4.16E-05 ENSG00000149131 SERPING1 11 1.317338117 0.012498128 ENSG00000188820 FAM26F 6 1.316454234 0.007990343 ENSG00000105426 PTPRS 19 1.307115596 8.74E-08 ENSG00000107798 LIPA 10 1.306415366 0.000487568

220

log fold Chromo- EnsemblID Symbol change over p value some unstimulated

ENSG00000132514 CLEC10A 17 1.303117693 6.85E-05 ENSG00000022556 NLRP2 19 1.300117634 0.001512504 ENSG00000006327 TNFRSF12A 16 1.299839735 6.25E-07 ENSG00000136048 DRAM1 12 1.298203853 4.35E-07 ENSG00000160223 ICOSLG 21 1.29814914 5.17E-09 ENSG00000115956 PLEK 2 1.297675782 6.18E-07 ENSG00000163661 PTX3 3 1.291018648 0.00032926 ENSG00000187116 LILRA5 19 1.287991933 2.31E-06 ENSG00000105967 TFEC 7 1.285339404 7.70E-06 ENSG00000157017 GHRL 3 1.284716937 0.000947262 ENSG00000118113 MMP8 11 1.284629667 0.00077916 ENSG00000160883 HK3 5 1.284506695 2.47E-06 ENSG00000137331 IER3 6 1.280480434 3.23E-07 ENSG00000124731 TREM1 6 1.279352134 1.23E-06 ENSG00000145936 KCNMB1 5 1.279280486 1.09E-05 ENSG00000167207 NOD2 16 1.275836979 0.000327983 ENSG00000065911 MTHFD2 2 1.275295764 1.74E-09 ENSG00000162654 GBP4 1 1.274617604 3.18E-05 ENSG00000099377 HSD3B7 16 1.268191864 0.000627409 ENSG00000119862 LGALSL 2 1.267893076 6.71E-05 ENSG00000196923 PDLIM7 5 1.266143741 4.51E-07 ENSG00000145920 CPLX2 5 1.265136241 0.00633842 ENSG00000134321 RSAD2 2 1.261527977 0.008704879 ENSG00000072401 UBE2D1 10 1.259284987 4.62E-07 ENSG00000237264 FTH1P11 8 1.258368812 0.00090364 ENSG00000144802 NFKBIZ 3 1.257418254 3.59E-05 ENSG00000146376 ARHGAP18 6 1.250982633 1.99E-06 ENSG00000155629 PIK3AP1 10 1.248985917 2.79E-05 ENSG00000120337 TNFSF18 1 1.246239316 0.011270801 ENSG00000160179 ABCG1 21 1.235639761 1.22E-06 ENSG00000231027 AC079325.6 17 1.226007649 0.001228498 ENSG00000171812 COL8A2 1 1.22587046 4.75E-06 ENSG00000086300 SNX10 7 1.225534897 9.47E-05 ENSG00000162783 IER5 1 1.220970644 5.83E-06 ENSG00000186047 DLEU7 13 1.220751757 2.93E-05 ENSG00000167987 VPS37C 11 1.219393405 2.53E-08 ENSG00000139618 BRCA2 13 1.218296138 3.47E-10

221

log fold Chromo- EnsemblID Symbol change over p value some unstimulated

ENSG00000111452 GPR133 12 1.21823606 0.000570637 ENSG00000075399 VPS9D1 16 1.215684348 6.26E-06 ENSG00000237576 AC097495.2 2 1.214281375 0.001135274 ENSG00000267809 NDUFV2P1 19 1.213290841 3.09E-05 ENSG00000255587 RAB44 6 1.209136137 0.003496646 ENSG00000164211 STARD4 5 1.20664287 1.09E-05 ENSG00000137462 TLR2 4 1.195881258 5.50E-07 ENSG00000138061 CYP1B1 2 1.195011343 6.45E-06 ENSG00000237181 AC147651.4 7 1.194671075 1.48E-05 ENSG00000104918 RETN 19 1.192922101 0.000539587 ENSG00000167642 SPINT2 19 1.19140226 9.78E-07 ENSG00000171049 FPR2 19 1.190929597 5.29E-06 ENSG00000143153 ATP1B1 1 1.190280987 6.61E-05 ENSG00000114315 HES1 3 1.18803768 0.023916993 ENSG00000005102 MEOX1 17 1.186193968 6.60E-06 ENSG00000197122 SRC 20 1.185019646 1.56E-07 ENSG00000101336 HCK 20 1.183461631 0.000108053 ENSG00000136560 TANK 2 1.181978245 1.56E-07 ENSG00000182541 LIMK2 22 1.181362593 0.000215961 ENSG00000128383 APOBEC3A 22 1.180741316 0.000203607 ENSG00000143369 ECM1 1 1.179584012 0.000163353 ENSG00000137767 SQRDL 15 1.179294764 1.84E-06 ENSG00000132465 IGJ 4 1.179171186 2.38E-09 ENSG00000151715 TMEM45B 11 1.176350704 1.81E-07 ENSG00000018280 SLC11A1 2 1.175152868 3.01E-08 ENSG00000100767 PAPLN 14 1.174822369 3.81E-06 ENSG00000125753 VASP 19 1.172908031 7.22E-08 ENSG00000078401 EDN1 6 1.172882787 0.005697156 ENSG00000172243 CLEC7A 12 1.17186631 4.11E-09 ENSG00000130052 STARD8 X 1.171470224 1.14E-08 ENSG00000170017 ALCAM 3 1.170622911 8.63E-08 ENSG00000026103 FAS 10 1.168346603 0.000122089 ENSG00000143476 DTL 1 1.167242053 0.006187468 ENSG00000115590 IL1R2 2 1.165609713 0.00018761 ENSG00000103257 SLC7A5 16 1.164985412 1.03E-07 ENSG00000143537 ADAM15 1 1.164333601 5.09E-05 ENSG00000176641 RNF152 18 1.162098598 0.015146783

222

log fold Chromo- EnsemblID Symbol change over p value some unstimulated

ENSG00000183742 MACC1 7 1.15265416 0.000239062 RP11- ENSG00000224397 290F20.3 20 1.152325973 1.14E-08 ENSG00000167874 TMEM88 17 1.151398338 0.006008754 ENSG00000242048 RP13-452N2.1 7 1.150289619 0.001694477 ENSG00000203814 HIST2H2BF 1 1.148949076 0.005555664 ENSG00000143333 RGS16 1 1.145231872 0.001851179 ENSG00000181790 BAI1 8 1.141951304 0.011278499 ENSG00000135525 MAP7 6 1.140501669 0.000165397 ENSG00000110237 ARHGEF17 11 1.13880037 0.034143424 ENSG00000137936 BCAR3 1 1.132480428 4.35E-07 ENSG00000152413 HOMER1 5 1.130940873 0.000539872 ENSG00000165682 CLEC1B 12 1.130921453 0.002535937 ENSG00000168214 RBPJ 4 1.13035623 3.01E-09 ENSG00000176890 TYMS 18 1.128936388 0.001112276 ENSG00000134107 BHLHE40 3 1.128710771 2.25E-07 ENSG00000090376 IRAK3 12 1.126820616 0.001359723 ENSG00000105048 TNNT1 19 1.123497049 2.76E-05 ENSG00000254709 IGLL5 22 1.123225292 0.0009037 ENSG00000132334 PTPRE 10 1.122961338 3.91E-07 ENSG00000100558 PLEK2 14 1.122863898 0.000116489 ENSG00000170476 MZB1 5 1.122210533 1.69E-07 ENSG00000196126 HLA-DRB1 6 1.118141039 8.16E-08 ENSG00000146476 C6orf211 6 1.11737362 4.72E-07 ENSG00000025708 TYMP 22 1.116692768 1.22E-05 RP11- ENSG00000248810 362F19.1 4 1.116665562 0.035976903 ENSG00000164877 MICALL2 7 1.116051247 0.000141531 ENSG00000231507 RP11-134P9.3 1 1.110213759 0.006361359 ENSG00000116663 FBXO6 1 1.108285437 2.02E-05 ENSG00000229801 RP11-353N4.1 1 1.107655705 0.000346079 ENSG00000081041 CXCL2 4 1.107310733 0.000145312 ENSG00000240864 IGKV1-16 2 1.105002168 3.50E-05 ENSG00000241294 IGKV2-24 2 1.104152577 0.000944632 ENSG00000105835 NAMPT 7 1.102068741 4.96E-08 ENSG00000211671 IGLV2-8 22 1.101856886 6.18E-05 ENSG00000165914 TTC7B 14 1.100054298 2.04E-06 ENSG00000118523 CTGF 6 1.096958258 1.43E-06 ENSG00000130202 PVRL2 19 1.096866271 1.87E-06

223

log fold Chromo- EnsemblID Symbol change over p value some unstimulated

ENSG00000142657 PGD 1 1.095661846 6.40E-06 ENSG00000011422 PLAUR 19 1.09536117 1.32E-06 ENSG00000135604 STX11 6 1.094316908 0.000147977 ENSG00000188786 MTF1 1 1.093987408 2.43E-07 ENSG00000115415 STAT1 2 1.09086547 0.00358059 ENSG00000180155 LYNX1 8 1.08801608 0.007488747 ENSG00000015475 BID 22 1.085513861 4.99E-05 ENSG00000251136 RP11-37B2.1 8 1.08528967 0.000446513 ENSG00000196735 HLA-DQA1 6 1.083350441 3.12E-06 ENSG00000014914 MTMR11 1 1.083204464 0.001759952 ENSG00000211670 IGLV3-9 22 1.081689574 0.010407581 ENSG00000230107 CTA-126B4.7 22 1.080299186 0.002352729 ENSG00000105246 EBI3 19 1.077982582 2.29E-05 ENSG00000128283 CDC42EP1 22 1.077106918 4.70E-05 ENSG00000189171 S100A13 1 1.077044773 0.000625526 ENSG00000229644 NAMPTL 10 1.076201776 7.75E-08 ENSG00000150636 CCDC102B 18 1.075957605 0.000127293 ENSG00000099860 GADD45B 19 1.072720081 2.97E-06 ENSG00000137312 FLOT1 6 1.072066403 5.23E-07 ENSG00000107954 NEURL 10 1.070707337 0.00017854 ENSG00000116514 RNF19B 1 1.070258405 1.75E-05 ENSG00000137809 ITGA11 15 1.068502494 0.0021908 ENSG00000185745 IFIT1 10 1.067067857 0.009218343 ENSG00000136160 EDNRB 13 1.061016381 0.025240699 ENSG00000175063 UBE2C 20 1.060089658 0.012172651 ENSG00000267519 CTD-3252C9.4 19 1.057968247 0.000212154 ENSG00000154359 LONRF1 8 1.05714039 4.35E-07 ENSG00000162723 SLAMF9 1 1.054372571 0.0067782 ENSG00000196139 AKR1C3 10 1.052959015 0.001860229 ENSG00000167553 TUBA1C 12 1.05111681 1.63E-05 ENSG00000134072 CAMK1 3 1.050775455 0.000252497 ENSG00000139832 RAB20 13 1.050613778 3.18E-05 ENSG00000155307 SAMSN1 21 1.050424594 2.04E-07 ENSG00000135424 ITGA7 12 1.048811602 0.004305395 ENSG00000198369 SPRED2 2 1.048646173 4.45E-08 ENSG00000100911 PSME2 14 1.04810359 2.43E-05 ENSG00000101255 TRIB3 20 1.047198422 7.48E-05

224

log fold Chromo- EnsemblID Symbol change over p value some unstimulated

ENSG00000160791 CCR5 3 1.046696365 6.08E-08 ENSG00000234883 MIR155HG 21 1.045601749 2.86E-06 ENSG00000007968 E2F2 1 1.041039316 0.001157185 ENSG00000135698 MPHOSPH6 16 1.039996885 1.69E-07 ENSG00000173404 INSM1 20 1.037284401 0.002870482 ENSG00000197329 PELI1 2 1.036439311 5.57E-05 ENSG00000256515 CCL3L3 17 1.036412864 0.012047897 ENSG00000188906 LRRK2 12 1.033703863 0.000426329 ENSG00000169026 MFSD7 4 1.031072297 3.00E-06 ENSG00000112578 BYSL 6 1.028219645 5.52E-07 ENSG00000211652 IGLV7-43 22 1.027313793 0.000326197 ENSG00000119922 IFIT2 10 1.027193985 0.002496972 ENSG00000060558 GNA15 19 1.025922051 1.03E-05 ENSG00000113140 SPARC 5 1.023355355 0.000886877 ENSG00000112195 TREML2 6 1.021780827 0.000425778 ENSG00000164484 TMEM200A 6 1.02165202 0.000157895 ENSG00000125347 IRF1 5 1.021546965 0.000516686 RP11- ENSG00000203999 290F20.1 20 1.019486016 0.00033947 ENSG00000111424 VDR 12 1.018098846 1.65E-08 ENSG00000136826 KLF4 9 1.017648763 0.004222394 ENSG00000150977 RILPL2 12 1.013608914 0.000293816 ENSG00000108179 PPIF 10 1.013593421 3.50E-05 ENSG00000186792 HYAL3 3 1.013499737 0.000317649 ENSG00000116133 DHCR24 1 1.013459373 0.00099205 ENSG00000153885 KCTD15 19 1.01242982 2.75E-06 ENSG00000196123 KIAA0895L 16 1.012379905 2.87E-05 ENSG00000170425 ADORA2B 17 1.011403703 0.013987737 ENSG00000071205 ARHGAP10 4 1.010159287 1.78E-06 ENSG00000134802 SLC43A3 11 1.008529294 4.25E-06 ENSG00000172216 CEBPB 20 1.007340153 2.02E-07 ENSG00000073331 ALPK1 4 1.005900359 4.96E-05 ENSG00000102595 UGGT2 13 1.004415218 2.60E-05 ENSG00000115233 PSMD14 2 1.004407788 2.54E-07 ENSG00000128245 YWHAH 22 1.003980139 1.50E-08 ENSG00000211630 IGKV1D-13 2 1.00144768 0.006379377 ENSG00000231389 HLA-DPA1 6 1.001342615 2.96E-08 ENSG00000089041 P2RX7 12 1.001089736 7.34E-05

225

log fold Chromo- EnsemblID Symbol change over p value some unstimulated

ENSG00000158186 MRAS 3 1.000193969 8.90E-06 ENSG00000173114 LRRN3 7 -1.000288156 3.80E-09 ENSG00000075826 SEC31B 10 -1.004186323 0.000285202 ENSG00000186049 KRT73 12 -1.006923869 5.04E-05 ENSG00000070371 CLTCL1 22 -1.012114751 0.00010487 ENSG00000049323 LTBP1 2 -1.012562988 0.0020649 ENSG00000111344 RASAL1 12 -1.012730993 6.27E-06 ENSG00000099284 H2AFY2 10 -1.016906337 9.59E-08 ENSG00000139344 AMDHD1 12 -1.018163359 0.000459904 ENSG00000167261 DPEP2 16 -1.018527625 1.20E-06 ENSG00000115718 PROC 2 -1.019597135 0.002007334 ENSG00000173825 TIGD3 11 -1.020253688 1.78E-05 ENSG00000004777 ARHGAP33 19 -1.02115891 0.000395651 ENSG00000232229 RP11-248C1.2 10 -1.024612207 2.19E-05 ENSG00000258810 RP11-219E7.1 14 -1.024809448 0.001521913 ENSG00000135549 PKIB 6 -1.025883481 0.003842951 ENSG00000225828 RP4-811H24.6 1 -1.030409313 0.000176046 ENSG00000174697 LEP 7 -1.03538233 0.027947584 ENSG00000255026 RP11-326C3.2 11 -1.036232952 0.0049371 JMJD7- ENSG00000168970 PLA2G4B 15 -1.041923785 3.58E-05 ENSG00000110455 ACCS 11 -1.041940954 4.71E-05 ENSG00000010278 CD9 12 -1.044077984 0.000215569 ENSG00000268156 FKSG61 14 -1.044627875 0.000174675 ENSG00000186815 TPCN1 12 -1.049408198 6.74E-06 ENSG00000121769 FABP3 1 -1.055140618 0.001679412 ENSG00000111110 PPM1H 12 -1.058317781 0.0004554 ENSG00000114770 ABCC5 3 -1.059484962 1.42E-05 ENSG00000116014 KISS1R 19 -1.060759396 0.002142983 ENSG00000118520 ARG1 6 -1.06492189 0.00145524 ENSG00000171659 GPR34 X -1.070787008 0.011618135 ENSG00000180539 C9orf139 9 -1.072580941 0.000327015 ENSG00000130304 SLC27A1 19 -1.08677508 0.000205809 ENSG00000173068 BNC2 9 -1.098253827 0.000205023 ENSG00000105612 DNASE2 19 -1.106788215 3.49E-07 ENSG00000198910 L1CAM X -1.114352948 7.06E-06 ENSG00000185052 SLC24A3 20 -1.125921527 0.000152147 ENSG00000233901 RP11-65J3.1 9 -1.126444285 0.000335429

226

log fold Chromo- EnsemblID Symbol change over p value some unstimulated

ENSG00000109667 SLC2A9 4 -1.128640485 0.004966866 ENSG00000135116 HRK 12 -1.131898488 2.17E-06 ENSG00000140465 CYP1A1 15 -1.137678724 0.007136538 ENSG00000007237 GAS7 17 -1.141180937 1.77E-05 ENSG00000136634 IL10 1 -1.147678785 0.002216828 ENSG00000080854 IGSF9B 11 -1.149122706 0.000763869 ENSG00000020577 SAMD4A 14 -1.163489666 0.000177341 ENSG00000122642 FKBP9 7 -1.172225739 0.012272813 ENSG00000105697 HAMP 19 -1.178594739 0.007634639 ENSG00000092200 RPGRIP1 14 -1.183398801 5.66E-05 ENSG00000141401 IMPA2 18 -1.214291555 0.000107432 ENSG00000171766 GATM 15 -1.220936604 3.08E-08 ENSG00000110799 VWF 12 -1.22270802 0.008030771 ENSG00000169413 RNASE6 14 -1.246442194 9.61E-07 ENSG00000178538 CA8 8 -1.250102884 1.94E-05 ENSG00000142621 FHAD1 1 -1.260129358 0.001915035 ENSG00000170486 KRT72 12 -1.261051679 0.001518274 ENSG00000128274 A4GALT 22 -1.268531774 0.002774291 ENSG00000213876 RPL7AP64 17 -1.283387166 0.008486858 ENSG00000170448 NFXL1 4 -1.29211184 3.50E-07 ENSG00000185339 TCN2 22 -1.293403298 0.00942196 RP11- ENSG00000257838 368J21.3 16 -1.296241829 0.025310663 ENSG00000073060 SCARB1 12 -1.309113156 4.59E-05 ENSG00000111261 MANSC1 12 -1.332438503 6.05E-09 ENSG00000171227 TMEM37 2 -1.336703424 0.009972707 ENSG00000178789 CD300LB 17 -1.352569647 1.84E-06 RP11- ENSG00000262019 473M20.8 16 -1.354507905 0.001304382 ENSG00000146409 SLC18B1 6 -1.357491757 6.03E-06 ENSG00000088827 SIGLEC1 20 -1.366473386 0.04175971 ENSG00000136999 NOV 8 -1.384194978 6.52E-05 ENSG00000133116 KL 13 -1.393299272 0.000340342 ENSG00000197766 CFD 19 -1.394601722 1.07E-07 ENSG00000079393 DUSP13 10 -1.396567409 0.001671783 ENSG00000172037 LAMB2 3 -1.41805413 0.000638968 ENSG00000172594 SMPDL3A 6 -1.433704124 0.000350489 ENSG00000133121 STARD13 13 -1.435786746 0.001154713 ENSG00000111247 RAD51AP1 12 -1.443630464 2.70E-05

227

log fold Chromo- EnsemblID Symbol change over p value some unstimulated

ENSG00000130208 APOC1 19 -1.45941525 0.01267028 ENSG00000196664 TLR7 X -1.463079518 1.60E-06 ENSG00000157445 CACNA2D3 3 -1.483217867 0.007488766 ENSG00000100600 LGMN 14 -1.510309078 0.000347844 ENSG00000197256 KANK2 19 -1.520760684 1.47E-06 ENSG00000162337 LRP5 11 -1.542143658 4.37E-08 ENSG00000157404 KIT 4 -1.551689446 1.35E-08 ENSG00000134247 PTGFRN 1 -1.555904217 9.53E-06 ENSG00000071967 CYBRD1 2 -1.580307475 1.24E-05 ENSG00000188158 NHS X -1.585790407 2.39E-06 ENSG00000246100 CTC-774J1.2 11 -1.601903653 0.000128702 ENSG00000179163 FUCA1 1 -1.61349237 0.000944629 ENSG00000017427 IGF1 12 -1.645084652 7.56E-06 ENSG00000137801 THBS1 15 -1.66888803 0.000608136 ENSG00000214063 TSPAN4 11 -1.716713986 6.53E-05 ENSG00000106066 CPVL 7 -1.729198142 1.01E-06 ENSG00000182985 CADM1 11 -1.731374794 6.75E-06 ENSG00000050767 COL23A1 5 -1.731901739 1.71E-05 ENSG00000170271 C5orf4 5 -1.735492847 2.02E-06 ENSG00000152767 FARP1 13 -1.736730122 3.16E-05 ENSG00000134508 CABLES1 18 -1.749308767 1.35E-07 ENSG00000010327 STAB1 3 -1.76868965 1.15E-05 ENSG00000166578 IQCD 12 -1.806979448 0.000604431 ENSG00000224307 RP11-344B5.2 9 -1.813307642 1.56E-06 ENSG00000155792 DEPTOR 8 -1.843286851 4.89E-06 ENSG00000162174 ASRGL1 11 -1.853368625 2.30E-07 ENSG00000151790 TDO2 4 -1.885155987 0.004966727 ENSG00000164684 ZNF704 8 -1.887221432 1.14E-08 ENSG00000134028 ADAMDEC1 8 -1.919447384 6.06E-07 ENSG00000116962 NID1 1 -1.947348878 2.84E-06 ENSG00000082781 ITGB5 3 -1.955288316 3.09E-05 ENSG00000156535 CD109 6 -2.053264711 3.21E-06 ENSG00000130203 APOE 19 -2.079168938 4.17E-06 ENSG00000004799 PDK4 7 -2.141110453 4.24E-06 ENSG00000136235 GPNMB 7 -2.186671095 1.48E-05 ENSG00000129538 RNASE1 14 -2.188539568 5.93E-06 ENSG00000143387 CTSK 1 -2.222534129 9.10E-06

228

log fold Chromo- EnsemblID Symbol change over p value some unstimulated

ENSG00000019991 HGF 7 -2.373773749 2.52E-06 ENSG00000115414 FN1 2 -2.412020823 0.000298221 ENSG00000147614 ATP6V0D2 8 -2.495651849 2.94E-05 ENSG00000104055 TGM5 15 -2.519485591 9.95E-06 ENSG00000214274 ANG 14 -2.540930014 5.72E-07 ENSG00000122254 HS3ST2 16 -2.590904173 1.60E-05 ENSG00000135094 SDS 12 -3.049924374 1.97E-07 ENSG00000135931 ARMC9 2 -3.394147136 1.80E-09 ENSG00000006747 SCIN 7 -3.692346327 9.45E-06 ENSG00000250722 SEPP1 5 -4.286439156 5.06E-09 ENSG00000120457 KCNJ5 11 -4.856247879 3.02E-09

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Appendix 8

Differentially expressed genes between IL-3 stimulated, and unstimulated lysed whole blood from n = 7 healthy donors, at 6 hours

log fold Chromo- EnsemblID Symbol change over p value some unstimulated

ENSG00000114115 RBP1 3 3.354903699 0.009235482 ENSG00000072694 FCGR2B 1 2.746550406 4.52E-08 ENSG00000162746 FCRLB 1 2.146325982 3.63E-06 ENSG00000179348 GATA2 3 1.913740845 1.75E-08 ENSG00000092969 TGFB2 1 1.763959913 0.042174773 ENSG00000152128 TMEM163 2 1.694777758 0.000185511 ENSG00000134061 CD180 5 1.693616479 1.12E-06 ENSG00000119121 TRPM6 9 1.683399444 2.96E-05 ENSG00000157168 NRG1 8 1.633381932 0.003582262 ENSG00000176834 VSIG10 12 1.577701874 0.018805375 ENSG00000153071 DAB2 5 1.470254854 8.83E-05 ENSG00000123342 MMP19 12 1.386925481 0.006826742 ENSG00000112759 SLC29A1 6 1.373479119 1.89E-06 ENSG00000139354 GAS2L3 12 1.353643775 0.000424424 ENSG00000113749 HRH2 5 1.335845251 0.002630204 ENSG00000171631 P2RY6 11 1.319709645 0.000362575 ENSG00000141527 CARD14 17 1.299028958 0.047038991 ENSG00000225217 HSPA7 1 1.245197396 0.002847234 ENSG00000225331 AP001055.6 21 1.220097724 4.72E-05 ENSG00000171657 GPR82 X 1.214753338 0.031826555 ENSG00000054277 OPN3 1 1.181982375 4.52E-08 ENSG00000244682 FCGR2C 1 1.18052543 0.002959521 ENSG00000135605 TEC 4 1.180026323 0.000264296 ENSG00000242265 PEG10 7 1.130896508 4.85E-05 ENSG00000079215 SLC1A3 5 1.110268818 0.028486146 ENSG00000105976 MET 7 1.103679874 0.014541233 ENSG00000187098 MITF 3 1.100126625 0.012666964 ENSG00000180509 KCNE1 21 1.090761565 0.047038991

230

log fold Chromo- EnsemblID Symbol change over p value some unstimulated

ENSG00000131016 AKAP12 6 1.085542431 0.001964665 ENSG00000237604 AP001056.1 21 1.081124702 0.001494007 ENSG00000144843 ADPRH 3 1.076683675 5.46E-06 ENSG00000182240 BACE2 21 1.072754206 0.000355917 ENSG00000186522 SEPT10 2 1.069406173 8.87E-06 ENSG00000115602 IL1RL1 2 1.061679132 0.025617065 ENSG00000169224 GCSAML 1 1.056919669 0.000682316 ENSG00000222047 C10orf55 10 1.017261447 0.048621227 ENSG00000167414 GNG8 19 1.004201391 0.000707894 ENSG00000240350 AC017002.1 2 1.002050166 0.000144749 ENSG00000196935 SRGAP1 12 -1.036079116 0.026912153 ENSG00000125266 EFNB2 13 -1.076998719 0.010364778 ENSG00000008056 SYN1 X -1.104684036 0.001310723 ENSG00000133317 LGALS12 11 -1.109452972 0.000734052 ENSG00000011028 MRC2 17 -1.12314572 0.000173512 ENSG00000122025 FLT3 13 -1.133100345 0.000405509 ENSG00000112299 VNN1 6 -1.16714447 0.000460757 ENSG00000163053 SLC16A14 2 -1.167513217 0.002311526 ENSG00000128805 ARHGAP22 10 -1.23294503 0.012396204 ENSG00000185070 FLRT2 14 -1.495634968 0.033039239 ENSG00000163751 CPA3 3 -1.522709278 1.63E-05 ENSG00000198795 ZNF521 18 -1.558042293 0.000128538

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Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Oon, Shereen

Title: IL-3R as a novel therapeutic target in systemic lupus erythematosus

Date: 2017

Persistent Link: http://hdl.handle.net/11343/168290

File Description: IL-3R as a novel therapeutic target in Systemic Lupus Erythematosus

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