Gut-selective -targeted therapies for inflammatory bowel disease

Christopher A. Lamb1,2, Sharon O’Byrne3, Mary E. Keir4, Eugene C. Butcher5,6

Short title: Gut-selective integrin-targeted therapies for IBD

Correspondence: Dr. Chris Lamb ([email protected])

Institute of Cellular Medicine The Medical School William Leech Building M3089 Newcastle University Framlington Place Newcastle upon Tyne NE2 4HH United Kingdom

Tel: +44 191 208 8578 Fax: +44 191 222 5851

Institutions:

1. Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, NE2 4HH. United Kingdom. 2. Department of Gastroenterology, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, NE1 4LP. United Kingdom. 3. Global Medical Affairs, Takeda Pharmaceuticals International AG, Thurgauerstrasse 130, 8152 Glattpark-Opfikon, Zurich, Switzerland. 4. Genentech Research & Early Development, South San Francisco, California 94080. United States of America. 5. Laboratory of and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, United States of America. 6. Veterans Affairs Palo Alto Health Care System and The Palo Alto Veterans Institute for Research, Palo Alto, California 94304, United States of America.

Keywords:

Vedolizumab, , , abrilumab, α4β7, αE, αEβ7, CD103, CD49d, αLβ2, LFA-1, integrin, lymphocyte, leukocyte, trafficking, inflammatory bowel disease, IBD, , UC, Crohn’s disease, CD, MAdCAM-1, VCAM-1, ICAM-1, AJM300, carotegrast methyl, PTG-100, HIV, human immunodeficiency virus

1

Abstract

Integrins are cell surface receptors with bi-directional signaling capabilities that can bind to adhesion molecules in order to mediate homing of leukocytes to peripheral tissues. Gut- selective leukocyte homing is facilitated by interactions between α4β7 and its ligand

MAdCAM-1, while retention of lymphocytes in mucosal tissues is mediated by αEβ7 binding to its ligand E-. Therapies targeting gut-selective trafficking have shown efficacy in inflammatory bowel disease (IBD), confirming the importance of leukocyte trafficking in disease pathobiology. This review will provide an overview of integrin structure, function and signaling, and highlight the role that these molecules play in leukocyte homing and retention.

Anti-integrin therapeutics, including gut-selective antibodies against the β7 integrin subunit

(etrolizumab) and the α4β7 integrin heterodimer ( and abrilumab), as well as the non-gut selective anti-α4 integrin (natalizumab), will be discussed as well as novel targeting approaches using small molecules.

Word count of abstract: 140

Word count of manuscript: 9347

Statement of Originality

The authors confirm this manuscript has not been previously published and is not under consideration for publication elsewhere.

2 Introduction

Inflammatory bowel disease (IBD), which includes patients diagnosed with Crohn’s disease

(CD) and ulcerative colitis (UC), is characterised by a dysregulated mucosal immune response to gut microbiota. Increased activation and recruitment of leukocytes to the gut mucosa is observed in patients with IBD. Leukocyte migration from the peripheral circulation into tissues is tightly regulated by a combination of soluble and cell-surface molecules, and blockade of gut-selective homing mechanisms is an effective approach for the treatment of

IBD. In this review, we examine the role of in gut-selective homing and evaluate current and emerging integrin-targeting therapies.

Integrin structure, activation and signaling

Integrins are broadly expressed transmembrane glycoproteins of >1500 amino acids that mediate cell-cell and cell-extracellular matrix interactions.1 Twenty-four known integrin heterodimers have been described, made up of 18 α subunits and 8 β subunits. Dimers are grouped according to cell type or ligand specificity: leukocyte receptors, laminin receptors, collagen receptors and arginine-glycine-aspartic acid (RGD) receptors (Figure 1). RGD receptors are responsible for integrin-binding to extracellular matrix (ECM) such as fibronectin or fibrinogen. The RGD sequence may be exposed in ECM upon proteolytic cleavage or denaturation.2

Integrin heterodimers on the cell surface are normally found in a bent ‘closed’ resting state

(Figure 2A) during which time they are functionally inactive.3 The cytoplasmic domain of all β integrins are bound to cytoskeleton proteins including α-actin, talin, vinculin, , filamin and tensin in the closed state. Integrins assume an ‘open’ configuration following activation of cell-surface receptors such as T cell receptors or chemokine receptors; this process is

3 termed ‘inside-out signaling’, as rapid intracellular signaling leads to changes in integrin conformation. As one of the best studied leukocyte-expressed integrins, Figure 2B summarises the chemokine-induced signaling cascade associated with inside-out signaling in

αLβ2 (lymphocyte function-associated antigen 1; LFA-1) activation.4 Inside-out signaling leads to extension of the integrin structure and separation of the cytoplasmic tails. This straightening process has been termed the ‘flick knife’ or ‘switch-blade’ model,5 and improves integrin ‘affinity’ (ability to bind ligand).

‘Outside-in signaling’ occurs when an integrin heterodimer binds its ligand, leading to activation and conformational change. Outside-in signaling occurs via integrin associated signaling proteins including FAK, Src and PI3K.6 Clustering of integrins on the leukocyte plasma membrane at the site of contact with endothelial molecules (CAMs) improves

‘valency’, the number of receptor-ligand bonds, and ‘avidity’, or strength of cellular adhesiveness, resulting in stronger association. Mobility of integrins in the plasma membrane is also regulated by chemokines and contributes to cell-cell interactions.7

Role of β7 integrins in lymphocyte migration to the gut and retention in the epithelium

β7 integrin-containing heterodimers play a central role in leukocyte homing to the gut and retention at epithelial surfaces. The β7 integrin subunit can form a dimer with either the α4

(CD49d) or αE (CD103, αIEL, αM290, OX-62) integrin subunit to form α4β7 (lymphocyte

Peyer’s patch adhesion molecule-1; LPAM-1) or αEβ7 integrin respectively. The α4β7 integrin mediates lymphocyte migration from the circulation across the vascular endothelial barrier into gut-associated secondary lymphoid tissue or the intestinal lamina propria (LP) through interaction with its counter-receptor mucosal addressin cellular adhesion molecule-1

4 (MAdCAM-1), which is selectively expressed on the vascular endothelium within the intestine.8-10 Once within the intestinal mucosa, a subset of T cells then upregulate αEβ7 which forms retentive interactions with E-cadherin expressed on intestinal epithelial cells

(Figure 3).11

α4β7 integrin

Circulating T lymphocytes enter the gut either as naïve cells moving to gut-associated lymphoid tissue (GALT), or as circulating memory/effector cells that have previously left the

GALT and that are returning to GALT or the LP via the systemic circulation. In either circumstance, lymphocytes must cross the endothelial barrier. This occurs in post capillary venules (PCVs) or high endothelial venules (HEV), a specialised PCV found in mesenteric lymph nodes (MLN) and Peyer’s patches (PP) in GALT.12,13 Although cells travel at relatively high velocity through these vessels, adhesion receptors on microvilli enable tethering to the vessel wall under physiologic shear stress (Figure 3).14,15 Unique structural and biophysical features of and α4β7 integrin facilitate tethering and rolling.16 Selectins are a family of single- chain transmembrane cell adhesion molecules containing an N-terminal extracellular lectin domain, an epidermal growth factor-like domain and a series of 2-9 consensus repeats with homology to sequences found in complement proteins.17 Naïve T lymphocytes expressing

CCR7 and low levels of α4β718 initially contact endothelium in HEVs through L- interactions, then become activated by CCR7 inside-out signaling via CCL19 and CCL21, thus triggering α4β7:MAdCAM-1 and αLβ2:(intercellular adhesion molecule-1)ICAM-1 interactions and cellular arrest.19,20 Mice lacking functional L-selectin have severely reduced numbers of lymphocytes in both peripheral and mucosal lymph nodes and PP, and display reduced leukocyte endothelial rolling during inflammation.21

5 Naïve and memory B cells also employ α4β7:MAdCAM-1 and LFA-1:ICAM-1 interactions, but in addition express the immunoglobulin family lectin CD22, which interacts with St6gal1- dependent carbohydrate ligands on venules in GALT, thus conferring preferential B cell localisation to GALT in support of the intestinal secretory immune response.13,22 MAdCAM-1- deificient mice have a reduced number of IgA secreting plasma cells in the small intestine lamina propria, and fail to mount a normal intestinal IgA response following mucosal immunisation.23

MAdCAM-1 is a type 1 transmembrane adhesion molecule containing two distal immunoglobulin-like domains, and a mucin-like domain that can be modified by carbohydrate ligands for L-selectin.24,25 Murine MAdCAM-1 includes an additional, membrane-proximal immunoglobulin domain homologous to IgA.26,27 The activity of MAdCAM-1 as a ligand for L- selectin is regulated by post-translational carbohydrate sulphation.24 MAdCAM-1 expression is constitutive in LP venules and GALT HEV, but can be further induced by pro-inflammatory factors such as TNFα, IL-1β and LPS during intestinal inflammation.9,28,29

Once naïve lymphocytes enter GALT, they encounter specialised stromal cells30 and CD103+ migratory dendritic cells (DCs) from the gut that express retinal dehydrogenase, which converts vitamin A to retinoic acid (RA).13,31 CD103+ DCs can elicit a gut-homing phenotype in

T cells32 and activation by antigen in the presence of RA induces up-regulation of α4β7 and

CCR9 on lymphocytes.31,33 The CCR9 ligand chemokine CCL25, together with α4β7 mediates lymphocyte homing to the small intestine. By this mechanism, lymphocytes encountering

CD103+ DCs become enterotropic, upregulating α4β7 integrin to enable binding to MAdCAM-

1 and homing to the gut mucosa.

After differentiated lymphocytes return to the circulation, homing to the intestinal LP is facilitated by α4β7 integrin. CCL25 secreted by small intestinal endothelium is responsible for

6 recruitment of CCR9-expressing lymphocytes.34 Other chemokine receptors also assist gut homing including the Th17-associated receptor CCR6 and its ligand CCL20,35-37 as well as

GPR15 and its colon-expressed chemokine-like ligand GPR15L which are of particular importance in colonic T cell trafficking.38-42 Gut homing memory and effector cells express higher levels of α4β7 than naïve T cells, enhancing their interactions and homing via

MAdCAM-1.43 Chemokines trigger integrin activation and arrest, and cell movement across the endothelial barrier is then facilitated by a chemokine gradient (Figure 3).

Regulatory T cells (TReg), which mediate a suppressive function, can be generated in gut- draining lymph nodes and proliferate after homing to the intestine mediated by CXC3R1 and

α4β7/MAdCAM-1 interactions.44 In β7-/- or MAdCAM-1-/- mouse models of oral tolerance, gut homing and intestinal expansion of TReg is impaired leading to an inability to suppress delayed type hypersensitivity. Similar effects on migration to the intestine have been observed in β7-/- T cell transfer colitis models with no subsequent effect on disease.45

Whilst the principal integrin responsible for migration of lymphocytes to the intestine is α4β7 interacting with its ligand MAdCAM-1,9,13,43,46 α4β7 integrin can also bind to vascular -1 (VCAM-1), the expression of which may be increased in active IBD under the influence of pro-inflammatory cytokines such as TNFα.47,48 This could theoretically provide an alternative route by which α4β7+ cells can migrate to the gut, though in vivo homing via α4β7:VCAM-1 interactions have not been shown to date. On the other hand,

α4β7+ lymphocytes also express α4β1, albeit at lower levels than expressed by α4β7- β1+ effector and memory cells.43,49 Indeed, recent evidence suggest that α4β1 can mediate T cell homing to the gut, contributing to α4β7+ T cell localization but also allowing α4β7- T cell recruitment to the ileum and/or colon, particularly in CD.50 The role of α4β1 may be particularly significant in the inflamed ileum, the clinical significance of which will need to be

7 elucidated using objective measures (ileocolonoscopy or MRE) of inflammation in this part of the bowel. αLβ2 (lymphocyte function-associated antigen; LFA-1) interacting with ICAM-1 also contributes to gut homing.19,51,52 Confirming the relative importance of α4β7 integrin for the lymphocyte populations within the gut, lymphocyte migration to gut tissue in β7-/- mice is severely impaired, leading to poorly developed, small PP with diminished cellularity of the lamina propria and intraepithelial compartments.53,54 Similarly, α4β7 inhibition in mice leads to a reduction in B lymphocyte infiltration in the LP, as well as a reduction in lymphoid follicle formation in both uninflamed intestinal tissue and in acute DSS colitis.55

Under steady state conditions and in both CD and UC, α4β7 is expressed on approximately half of circulating memory/effector phenotype CD4+ lymphocytes.43,56 The integrin is expressed by all naïve CD4+ cells at low levels. MAdCAM-1 is up-regulated in active CD and

UC,9,57,58 and may be expressed in other organs during IBD leading to aberrant homing of intestinal lymphocytes and therefore extra-intestinal manifestations of the disease, for example in liver portal tracts during primary sclerosing cholangitis (PSC).59,60 However further study is required to determine the relative importance of α4β7+ leukocytes in PSC aetiology and whether integrin targeted therapy in this setting may be beneficial.

αEβ7 integrin

The αEβ7 integrin is expressed predominantly by mucosal lymphocytes and dendritic cells, generally referred to as CD103+ DCs, where it plays a role in adhesion or retention of these cells due to interactions with E-cadherin, the only known ligand for αEβ7.11,61,62

Less than 3% of peripheral circulating T lymphocytes express the αEβ7 integrin, yet αEβ7 is readily observed on lymphocytes in the gut. The majority of αEβ7+ cells in the intestine are

CD3+ T lymphocytes,63 and neither B lymphocytes nor macrophages in the gut express the

8 αEβ7 integrin.64 αEβ7 is expressed on approximately 90% of intraepithelial lymphocytes (IELs) and approximately 40% of T cells in the LP.63,65 The αE knockout mouse has reduced epithelial and LP lymphocyte numbers, however PP and splenic populations remain intact.66 E-cadherin, expressed on basolateral epithelial surfaces, is a member of the cadherin family of glycoproteins responsible for Ca2+ dependent cell-cell adhesion and cellular signaling via a histidine-alanine-valine sequence in the first extracellular domain.67,68 αEβ7 binds to E- cadherin at a separate site from regions involved in maintaining epithelial polarity and para- cellular junctions.69-71 Inside-out signaling through TCR stimulation can improve the avidity of

αEβ7 for E-cadherin and may be a mechanism for retention of lymphocytes at the site of epithelial injury during inflammation.72 Outside-in signaling from binding of the αEβ7 integrin to E-cadherin enhances T cell proliferation.73,74

Expression of αEβ7 integrin is regulated by transforming growth factor beta (TGFβ). 75,76

Genes encoding both αE and β7 integrin contain the TGFβ response element.77,78 Conversely transcription of the α4 integrin subunit was shown to be reduced in the presence of TGFβ.78

Indeed up to 40% of αEβ7+ T cells in the gut do not express α4β7,63 and an additive effect on migration to the intestinal mucosa has been shown for blocking both α4β7 and αEβ7 in comparison to α4β7 alone.79 Activated T cells cultured in the presence of TGFβ1 develop an

IEL-like phenotype with increased surface expression of αEβ7.62,75,80 This suggests that αE is upregulated on populations of T cells after homing to the intestine and that are exposed to active TGFβ.

There are key differences between αEβ7+ T cells in humans and mice. The majority of IELs

(and hence αEβ7 expressing lymphocytes) are CD8+ in both mice and humans.81,82 Human

αEβ7+ T cells have been demonstrated to have an inflammatory phenotype. αEβ7 expression is more common on effector memory (CD45RO+) than naïve (CD45RA+) T cells.83 Studies in

9 peripheral blood T cells showed increased αEβ7 expression on Th9 and Th17 cells in comparison to TReg cells.79 In the gut, very few αEβ7 cells were observed to co-express FOXP3 ,63,79 and sorted αEβ7+ T cells had higher expression of inflammatory cytokines such as IFN, IL17, and TNFα as well as effector molecules including granzyme A.63,84 These data support an inflammatory role for αEβ7+ T cells in humans.

In mice, αEβ7 integrin expression on CD4+ T cells marks a subset of potent TReg preferentially located in GALT and MLN, that are capable of expressing high levels of IL-10 and TGFβ1.85

CD4+αE+ activated TReg were found to reduce the severity and increase the survival of recipients in a chronic graft-versus-host disease (GvHD) rodent model, and adoptive transfer of CD4+αE+ TReg can ameliorate disease in recipient animals.86 Murine αEβ7+ TReg subsets are able to suppress colitis in the adoptive T cell transfer model, express CTLA-4, have increased IL-10 and FOXP3, and migrate in response to CXCL9, CCL17 and CCL20 chemokines.87,88 This suggests that in mice αEβ7+ TReg have a greater potential to migrate to the site of inflammation than αEβ7- cells.

However, published data also support a pro-inflammatory role for αEβ7+ cells in mice. In the

IL2-TNP-OVA immunisation model of colitis, inflammation is driven by a Th1 response.89

Administration of an anti-αE mAb led to a reduction in colitis severity, accompanied by a reduction in LP CD4+ cells, and reduced release of IFNγ from these cells.90 In experimental

GvHD, host-specific pathogenic CD8+ effector T cells required TGFβ for αEβ7 integrin expression and migration to the intestinal epithelium.91 Consistent with active TGFβ being expressed by intestinal epithelium, the same group demonstrated that in an allogeneic stem cell transplantation sarcoma mouse model, αE was preferentially expressed by intestinal intraepithelial CD8+ T cells and not tumour cells.92 Thus mice receiving αE deficient alloreactive CD8+ T cells displayed reduced severity GvHD within the intestine, whilst the

10 therapeutic graft versus tumour immune response was retained. Overall, these data demonstrate a more mixed role for αEβ7+ T cells in mice in comparison to humans.

Whilst αEβ7+ T cells can be detected in interstitial lung disease, dermatitis, and during renal transplant rejection,75,83,93,94 αEβ7 deficient mice demonstrate a dramatic reduction in intestinal T lymphocytes (both LPL and IEL) with overall maintenance of mucosal T lymphocyte cell numbers elsewhere specifically in the bronchopulmonary, and splenic regions.66 This suggests therapeutic mechanisms targeting αEβ7 expressing cells in gastrointestinal disease should be gut-selective.

Integrins as therapeutic targets in inflammatory bowel disease

Early studies demonstrated that blockade with either anti-β7 or anti-MAdCAM1 monoclonal antibodies significantly reduced the severity of colitis in SCID mice reconstituted with CD4+ T cells enriched for the CD45RBhigh subpopulation.95 Emerging data suggests that trafficking to the inflamed ileum may be α4β7-independent in a murine adoptive transfer model.50 In captivity, the cotton-top tamarin monkey (Saguinus oedipus) has a well characterized susceptibility to the development of colonic inflammation, with histological similarities to UC, and responsiveness to sulphasalzine.96 Binding of α4-integrin (and hence dual α4β1 and α4β7 inhibition) or specific α4β7 dimer blockade was shown to ameliorate colitis in this model.97,98

α4β7 blockade was associated with a reduction in β7+ T cells in the colonic mucosa with no overall reduction in the peripheral circulation.98 α4 integrin blockade was also associated with stable peripheral lymphocyte counts with improvement in histological inflammation scores.97

Integrin-targeted therapeutics approved or currently under investigation for IBD and their proposed mechanisms of action are summarised in Table 1. As cell adhesion mediators with bi-directional signaling capabilities, integrins play a crucial role in leukocyte homing and cell

11 differentiation in homeostasis, inflammation and cancer.99 This therefore makes integrins intriguing targets for therapeutic intervention, but raises the concern of undesirable off- target effects.

Although integrins have the potential to be gut-selective, they also are involved in leukocyte migration into other tissues. Of particular interest is their role in homing to the central nervous system (CNS). blockade of either α4 integrin or α4β7 have consistently been shown to inhibit the development of experimental autoimmune encephalitis (EAE) in murine models, but mixed findings have been reported using β7 integrin blockade.100,101 More recent studies have found that β7 blockade has no effect on EAE,102 and are consistent with findings using anti-α4β7103 and genetic data showing that β1 is required for T cell migration into the CNS and subsequent EAE induction.104 Consistent with the requirement for β1 integrin for T cell migration, VCAM-1 but not MAdCAM-1 is found in the

CNS,105 with maximal VCAM-1 staining in acute active plaques in multiple sclerosis (MS) patients in comparison to controls. Choroid plexus may also allow entry of immune cells into the CNS,106 and MAdCAM-1, along with ICAM-1 and VCAM-1, is upregulated on choroid plexus epithelium in EAE.107 The co-expression of VCAM-1 and ICAM-1 by the choroid epithelium suggests redundancy in entry mechanisms at this site. Taken together, these data are consistent with the importance of α4β1 integrin, but not β7 subunit containing integrin pairs, in cell migration to the CNS.

Gut selective therapeutics that target leukocyte trafficking pathways are attractive for the potential to limit effects to the site of inflammation, in this case the gastrointestinal tract, whilst preserving immune cell trafficking to other organs.

12 Natalizumab (anti-α4 integrin monoclonal antibody)

Natalizumab is a humanised monoclonal IgG4 antibody that selectively binds the α4 integrin subunit, blocking both α4β7:MAdCAM-1 as well as α4β1:VCAM-1 interactions. α4β1 is an integrin expressed by most leukocytes, including lymphocytes, and can bind to VCAM-1, fibronectin and thrombospondin.108 Early studies in CD demonstrated a significant increase in the number of circulating TCRαβ+ T lymphocytes, and CD19+ B lymphocytes in patients receiving the drug, suggesting blockade of lymphocyte transmigration to the intestine.109 In the ENACT-1 study, 905 CD patients were randomised to receive natalizumab 300mg or placebo i.v. at week 0, 4 and 8. At week 10, response rates to natalizumab vs. placebo were

56% vs. 49% respectively (p=0.05), with a remission rate of 37% vs. 30% (p=0.12).110 In ENACT-

2, 339 responders from ENACT-1 were re-randomised to natalizumab 300mg or placebo i.v. every month . Response rates at 36 weeks were 61% vs. 28% (p<0.001) and remission 44% vs.

26% (p=0.003). In ENACT-1 and ENACT-2, 8% and 9% of patients respectively tested positive for anti-natalizumab antibodies. In ENCORE, 509 CD patients were randomised to receive either placebo, or natalizumab 300mg IV at week 0, 4 and 8 with sustained response and remission measured over 12 weeks.111 Natalizumab treated patients had 48% response and

26% sustained remission in comparison to 32% response and 16% sustained remission in patients receiving placebo.

Natalizumab is also licensed for the treatment of relapsing-remitting MS, where α4β1 blockade reduces the migration of T, B and plasma cells across the blood-brain barrier.112,113

The drug was withdrawn from the market temporarily in 2005 due to an association with three cases of progressive multifocal leukoencephalopathy (PML), 1 in CD and 2 in MS.114-116

α4β1 integrin binds to VCAM-1 to mediate trafficking of lymphocytes into the CNS. PML is a potentially fatal opportunistic brain infection caused by the John Cunningham (JC) virus,

13 leading to destruction of CNS oligodendrocytes.117 Subsequently there have been over 600 cases of PML reported in association with natalizumab therapy in MS, with an incidence of

4.22 per 1,000 treated patients.118 The risk of PML development is higher in those who are positive for JC virus antibodies and is positively correlated with prior use of immunosuppressants and duration of natalizumab therapy. Owing to the risk of PML, natalizumab is only licensed in the USA for CD under a tightly controlled risk management program called the TOUCH Prescribing Program.

Another anti-integrin, ,119 a humanized monoclonal IgG1 antibody that targets αL integrin and blocks αLβ2 interactions with ICAM, was originally approved for moderate to severe psoriasis.119 When tested in an open-label study of 15 CD patients 67% showed response and 40% remission at 8 weeks.120 However, efalizumab was voluntarily removed from the market in 2009 following reports of 3 PML cases among psoriasis patients, precluding further study.121

Vedolizumab (anti-α4β7 integrin)

Vedolizumab, approved for both CD and UC is a humanised monoclonal IgG1 antibody specific for the α4β7 heterodimer, blocking only α4β7:MAdCAM-1 interactions and not α4β1:VCAM-

1 interactions.122,123 The gut-selective activity of vedolizumab has been confirmed by several experiments. Vedolizumab did not prevent CNS inflammation in a non-human primate model of EAE.103 Flow cytometric analysis of cerebrospinal fluid (CSF) in 14 healthy subjects demonstrated no significant difference in T cell numbers or phenotype after single-dose treatment with vedolizumab, confirming that vedolizumab does not interfere with lymphocyte migration to the CNS.124 In 64 healthy subjects treated with single-dose vedolizumab, seroconversion on parenteral administration of hepatitis B vaccine was not

14 altered as opposed to a significantly attenuated seroconversion on enteral administration of cholera vaccine, demonstrating the gut selective mechanism of action of vedolizumab.125

A phase 2 placebo-controlled trial of an earlier version of vedolizumab (MLN02/MLN0002/

LDP-02) randomised 181 patients with active UC to placebo, or 0.5mg/kg or 2mg/kg of active drug intravenously on day 1 and day 29.126 Clinical and endoscopic assessment was made at

6 weeks. Clinical remission was noted in 33% and 32% in those receiving 0.5mg/kg and 2mg/kg of MLN02 respectively, compared to 14% who received placebo (p=0.03). Endoscopic remission was seen, respectively in 28% and 12% was in those receiving 0.5mg/kg and 2mg/kg of MLN02, and in 8% of those receiving placebo (p=0.007). Anti-drug antibodies (ADAs) to

MLN02 were identified in 44% of enrolled subjects. To reduce immunogenicity, vedolizumab was produced in Chinese Hamster Ovary (CHO) cells and tested in a small additional phase 2 study of the reformulated form of vedolizumab.123,127 Importantly CHO-produced vedolizumab did not alter the epitope binding for α4β7,123 and this formulation was used in the phase 3 GEMINI studies in both UC and CD.

Pivotal phase 3 clinical trials

GEMINI 1 was an integrated trial of vedolizumab to induce and maintain remission in 895 patients with moderate to severely active UC.128 40% of patients had previously failed anti-

TNF therapy. Clinical response was defined as a reduction in Mayo Clinic Score129 of ≥3 points and a decrease of ≥30% from baseline. Clinical remission was defined as a total Mayo score of ≤2, with no sub-score >1. Patients received a 300mg loading dose of vedolizumab or placebo IV at weeks 0 and 2 and clinical assessment was performed at week 6. Response rates for vedolizumab vs. placebo were 47% vs. 26% (p<0.001) at week 6. Patients who had responded to vedolizumab by week 6 were then re-randomised to vedolizumab or placebo every 4 or 8 weeks for maintenance therapy from week 6. At 52 weeks, 45% and 42% of those

15 receiving vedolizumab every 4 and 8 weeks, respectively, were in clinical remission in comparison to 16% of patients who were randomised-withdrawn to placebo from week 6.

The safety profile of vedolizumab was comparable to placebo.

GEMINI 2 evaluated the efficacy of vedolizumab compared with placebo in the induction and maintenance of clinical remission in 1115 patients with moderate to severely active CD as assessed using the CDAI (CDAI 220-450).130 The study design was similar to GEMINI 1, with clinical response defined as a reduction in CDAI of ≥100 points from baseline and clinical remission defined as a CDAI of ≤150 points, with assessment at week 6 after 2 doses of vedolizumab 300mg or placebo IV at weeks 0 and 2. 50% of patients in GEMINI 2 had previously been exposed to anti-TNF therapy. The week 6 clinical response rates for vedolizumab vs. placebo were not significantly different at 31% vs. 26% (p=0.23). However, clinical remission rates at 6 weeks favoured vedolizumab at 15% vs. 7% (p=0.02). At 52 weeks of maintenance therapy 36% of patients receiving vedolizumab every 4 weeks and 39% of patients receiving vedolizumab every 8 weeks were in clinical remission, compared to 22% of placebo patients (p=0.004 and p<0.001 respectively). In this study, only 4% of patients developed ADAs, demonstrating that the earlier immunogenicity issues with the monoclonal antibody were resolved. Nasopharyngitis was seen more commonly in vedolizumab treated patients, with headache and abdominal pain more common in those receiving placebo.

Overall when compared to placebo vedolizumab treatment was associated with a higher rate of serious adverse events (24.4% vs. 15.3%), infections (44.1% vs. 40.2%), and serious infections (5.5% vs. 3.0%).

GEMINI 3 was an additional induction study assessing the efficacy, safety and tolerability of vedolizumab compared with placebo in moderate to severely active CD, where a high

16 proportion of patients had prior exposure to TNF antagonists.131 Of 416 randomised patients,

76% had previously failed TNF antagonist therapy. Patients received a full induction course of

300mg vedolizumab or placebo i.v. at weeks 0, 2 and 6, with response and remission rates analysed at 6 and 10 weeks. At week 6, anti-TNF failure patients experienced no difference in remission between vedolizumab and placebo (15% vs. 12% respectively; p=0.433). However, by week 10 after 3 doses of vedolizumab or placebo a higher proportion of the vedolizumab treated patients were in remission in comparison to placebo (27% vs. 12%; p=0.001). Anti-

TNF-naïve patients had higher remission rates of 31% at week 6 and 35% at week 10 in comparison to 12% and 16% of placebo patients, respectively. These data suggest that CD patients previously treated with anti-TNF therapy and whose disease may be considered refractory, may require a longer induction treatment period than patients who are anti-TNF naïve.

Exposure-efficacy relationship

The impact of prior anti-TNF antagonist treatment and of serum vedolizumab trough concentrations on treatment efficacy has recently been examined in a post hoc analysis of

GEMINI clinical trial data.132 An exposure-efficacy relationship was most apparent in UC patients within GEMINI 1 induction, where higher trough concentrations of drug were associated with greater efficacy at week 6. Patients in the lowest quartile of serum trough drug levels had remission rates similar to placebo, while patients in the highest quartile had a 30% improvement over placebo remissions rates. A less steep exposure-efficacy relationship was observed for induction in CD patients, again with similar remission rates to placebo in the lowest quartile and a 14% improvement over placebo in patients that fell into the highest quartile of serum drug levels. These findings were not reproduced in CD studies, with a less clear relationship observed In GEMINI 3. Overall those with no prior anti-TNF

17 antagonist exposure, and those with higher serum albumin concentrations consistently had a higher probability of clinical remission.

Based on the GEMINI clinical trial data, an exposure-response relationship appears to be more definitive for UC than for CD. The data for CD in the GEMINI trials is based on the use of CDAI as an assessment of disease activity, however, with no objective ileo-colonoscopy data to confirm active mucosal inflammation in the bowel. A further assessment of the exposure- response relationship in CD will be needed when endoscopy and serum trough concentration data become available.

Long term safety and efficacy data from clinical trials

Following the pivotal GEMINI studies, further data on the long-term efficacy and safety of vedolizumab have emerged from an open label extension study to the Phase 2, and Phase 3

GEMINI trials.

Combined long term safety data integrated from the six Phase 2 and 3 trials of vedolizumab, representing 2830 vedolizumab-treated patients for up to 5 years duration, and a total of

4811 person years of drug exposure to vedolizumab, showed no increased risk of infections, including serious opportunistic infections (e.g. disseminated TB, systemic candidiasis, disseminated herpes zoster, extra-intestinal CMV or pneumocystis pneumonia) nor malignancy, nor any cases of PML.133 The exposure-adjusted incidence of lower respiratory tract infections was comparable between placebo and vedolizumab groups at 7.7 (3.9-11.5) and 6.1 (5.3-6.8)/100 person years (PY) respectively. The GEMINI long term safety (LTS) study is an ongoing single arm, open label continuation of the Phase 2 and Phase 3 GEMINI UC and

CD trials, designed to test long term effectiveness and safety of treatment with vedolizumab

300mg i.v. dosed every 4 weeks. In addition to the patients from the GEMINI trials rolling over to the LTS study, a cohort of IBD patients naïve to vedolizumab who received drug open-label

18 are included. An interim analysis of the study data for both UC and CD patients enrolled from

May 2009 up to a data cut-point of June 2013 (152 weeks – 3 years) was recently published.134,135

In UC, health related quality of life (HRQL) and clinical improvements, as measured by the partial Mayo clinic score (pMCS; total MCS without the endoscopic subscore136) were observed in patients treated with vedolizumab.134 In GEMINI 1, of the 247 UC patients who responded to vedolizumab by week 6 and received vedolizumab in the maintenance phase,

154 (82%) completed maintenance and were enrolled in the LTS study. Of the 154 patients who rolled over into the LTS study, 42 had failed anti-TNF therapy, a further 107 were TNF- naïve and 5 patients had either been exposed to anti-TNF previously or their TNF-responder status was unknown. Of those patients who had data available at the time of the interim analysis (as observed analysis), 88% (n=120/136) remained in remission at 104 weeks and

96% (n=70/73) were in remission at 152 weeks. The clinical remission calculations to 152 weeks did not take into account patients who may have discontinued the LTS study due to lack of efficacy. Adopting a conservative approach where missing data at each study visit was assumed to indicate treatment failure (non-responder imputation analysis), clinical remission was calculated as 74%, 78% and 46% of patients after 52 weeks, 104 weeks and 152 weeks, respectively. However, a limitation of this approach is that patients who remained in the study but had not yet reached the 152-week time-point at the time of the interim analysis, were deemed treatment failures thus providing a more stringent assessment of drug efficacy.

Similar long term clinical remission rates were observed whether patients had failed, or were naïve to anti-TNF agents.

32 patients on every 8-week dosing of vedolizumab withdrew from GEMINI 1 before week 52 due to lack of efficacy or worsening of symptoms, and enrolled in the LTS study where they

19 received vedolizumab infusions every 4 weeks. After 52 weeks, clinical response and remission were 41% and 28% respectively, an increase from 19% and 6% respectively from before dose increase, suggesting patients not responding to 8-weekly dosing may receive clinical benefit from shortening of dosing intervals to every 4 weeks.134

The most common adverse events in the GEMINI LTS UC cohort were worsening of UC (24%), nasopharyngitis (23%), headache (16%), upper respiratory tract infection (15%) and arthralgia

14%). The incidence of malignancy was <1%. Overall, 3% of patients experienced infusion reactions. No cases of PML were observed.

For CD, clinical effectiveness in the LTS study was assessed using the Harvey Bradshaw Index

(HBI).135 Clinical response was defined as a reduction in HBI of ≥3 points from baseline and clinical remission was defined as an HBI of ≤4. In GEMINI 2, of the 308 patients who responded to vedolizumab by week 6 and received vedolizumab in the maintenance phase, 146 (47%) completed maintenance and were enrolled in the LTS study. Of the 146 CD patients who rolled over into the LTS study, 57 had failed anti-TNF therapy, a further 81 were anti-TNF- naïve and the remaining 8 patients had either been exposed to anti-TNF previously or their anti-TNF responder status was unknown. Of those patients who had available data at the time of the interim analysis, 83% (n=97/120) remained in clinical remission at 104 weeks and 89%

(n=62/70) were in remission at 152 weeks. Similar to the analysis for UC, patients who had discontinued the LTS study for various reasons, including due to lack of efficacy, were not included in this analysis. Again, correcting for this by assuming all missing data was due to lack of efficacy, clinical remission was calculated in 71%, 69% and 43% of patients after weeks

52, 104 and 152 respectively. As in the UC analysis, this approach is limited by the fact that some patients remain in the LTS study but had not reached the 152-week time-point at the

20 time of the interim analysis, thus providing a more stringent assessment of efficacy at the later time points. Similar long term clinical remission rates were observed whether patients had failed, or were naïve to anti-TNF agents.135

57 patients on every 8-week dosing of vedolizumab withdrew from GEMINI 2 before week 52 due to lack of efficacy or worsening of symptoms and enrolled in the LTS study where they received vedolizumab infusions every 4 weeks. After 52 weeks, clinical response and remission were achieved in 47% and 32% respectively, an increase from 39% and 4% respectively, before the dose increase, suggesting, as observed for UC, patients who lose response to vedolizumab dosing every 8 weeks may experience an improved clinical outcome by increasing the frequency of infusions.

The most common adverse events in the GEMINI LTS CD cohort were worsening of CD (26%), nasopharyngitis (21%), arthralgia (20%), headache (19%), abdominal pain (18%) and nausea

(14%). Malignancy was observed in <1% of patients and infusion reactions in 4%. No cases of

PML were observed.

HRQL were also assessed for up to 152 weeks of cumulative treatment in both UC and CD patients and showed long-term benefits.134,135

A further 248 week (5 year) interim analysis of the LTS study has recently been reported in abstract form. These data demonstrate as-observed clinical remission in 90% (57/63) of UC137 and 89% (54/61) of CD138 patients respectively on cumulative treatment who initially responded to vedolizumab, the majority of whom were corticosteroid free at week 248. HRQL improvements continued to be observed. The long-term safety of vedolizumab was consistent with that previously reported with 9/61 CD and 10/63 UC patients experiencing a serious infection. There were no cases of PML.

21 Immunogenicity

In the GEMINI 1 and 2 studies, 56 of 1434 patients (4%) who received continuous vedolizumab for 52 weeks developed ADAs.133 Concomitant administration of an immunomodulator at baseline reduced the ADA incidence from 4% to 3%. In patients receiving vedolizumab induction at weeks 0 and 2, followed by placebo maintenance therapy, 18% of those not receiving an immunomodulator at baseline developed ADAs compared to only 3% of subjects who were receiving an immunomodulator at baseline. Available evidence from the GEMINI

LTS suggests that ADA incidence is not dependent on length of drug holiday.134,135 In the

GEMINI 1 and 2 studies, 6 UC patients and 3 CD patients had persistent ADAs, defined as two consecutive ADA positive results during treatment, as quantified by ELISA. Persistently positive ADA titres were associated with decreases in serum trough concentration of vedolizumab in 3 out of the 6 UC patients and 2 out of the 3 CD patients with trough concentrations below the limit of detection (BLQ < 0.125 µg/ml).133,139

The assay used to detect ADAs in the GEMINI studies was drug sensitive and so potentially may underestimate detection of these antibodies in the presence of vedolizumab.

Accordingly, a drug-resistant assay has been developed in Leuven, Belgium and validated outside of the GEMINI programme to detect ADAs in the presence of high vedolizumab concentrations and compared to the drug sensitive assay.140 In an induction and maintenance study of 179 vedolizumab patients, the drug-sensitive assay identified one patient (0.6%) as having developed ADAs whereas the drug resistant assay identified 4 patients respectively as having developed ADAs (2.2%). In 3 of the 4 patients with ADAs, antibodies developed during induction dosing. ADAs were transient and disappeared on serial measurement in all patients and no correlation with vedolizumab trough concentrations was observed. The transient nature of ADAs to vedolizumab and the lack of correlation with serum trough levels in this

22 small number of patients may suggest that immunogenicity does not influence treatment response, however further study is needed to determine the influence of ADAs on long term treatment efficacy and the impact of co-administration of immunomodulators.

In the context of anti-TNF therapy the presence of ADAs is recognised as a cause of secondary loss of response,141 and co-administration of an immunodulator such as methotrexate or azathioprine has been shown to reduce ADA formation.142 However, immunomodulators are associated with serious adverse effects including skin and haematological malignancies.44 As avoidance of systemic immunosuppression via gut selectivity is a major advantage of vedolizumab therapy, it remains to be determined whether systemic immunosuppression is required in the long-term use of vedolizumab.

Real World Clinical Experience with vedolizumab

Clinical effectiveness and safety data from several multicenter retrospective and prospective cohort studies reflecting real-world clinical experience (RWE) with vedolizumab have been published. A recent systematic review of 6 ‘real-world’ cohort studies followed 1049 patients.143 In this review, the most common non-infectious side effects were arthralgia (3%), arthritis (1%) and headache (2%). The risk of any infection was 7.8% overall. Specifically, the risk of gastroenteritis was 0.2%, and Clostridium difficile 1.2%. No cases of PML were identified in any of these studies.

A further systematic review and meta-analysis of real world effectiveness and safety of vedolizumab has been completed.144 Despite clinical observations in a predominantly treatment-refractory IBD population over the first 3 years post approval of vedolizumab, RWE confirms the long-term benefit and safety of vedolizumab as demonstrated in the clinical trial patient population.

23 A potential role for vedolizumab in human immunodeficiency virus (HIV) infection?

Advances in the development of anti-retroviral therapy (ART) for HIV have led to substantial reductions in mortality and longer life expectancy for HIV+ patients.145 However, chronic ART is required in order to suppress viral replication and to prevent rebound of viraemia.146 ART is associated with significant effects on patient health, including myelosuppression, lactic acidosis, hepatic steatosis, neuro-psychiatric complications, rash, osteonecrosis, and diabetes mellitus.147-149 Therefore, cost-effective alternatives with good tolerability and safety profiles are desirable. Emerging research highlights intriguing evidence of the importance of the gut and the α4β7 integrin in the pathogenesis of HIV-1.

In HIV-1 infection, rapid viral replication occurs in the gastrointestinal tract, causing depletion of CD4+ T cells in the gut and formation of viral reservoirs.150 HIV-1 viral entry is mediated through binding of the HIV-1 envelope protein gp120 to CD4. Both CXCR4 and CCR5 and other

G protein coupled receptors can serve as co-receptors for this viral entry. Intriguingly, gp120 can also bind and activate α4β7,151 and in acute SIV or HIV infection the virus preferentially infects cells expressing high levels of α4β7.152-155 Additionally recent human data demonstrates that high pre-infection numbers of CD4+α4β7+ T cells is associated with increased rates of HIV acquisition, independent of other T cell subsets.156 α4β7 integrin is preferentially incorporated into HIV-1 and SIV virions during budding,157 with the α4β7 integrin-containing virions are highest at early time points post-infection. Administration of anti-α4β7 antibodies before and during intravaginal SIV infection in rhesus macaques conferred protection from infection.158

24 A recent study has found that anti-α4β7 therapy also has a profound impact on viral remission.159 SIV-infected macaques received 90 days of ART initiated five weeks after primary intravenous SIV infection. From weeks 9 to 18 a cohort of these animals received 8 infusions of anti-α4β7 at 3-weekly intervals and were compared to a cohort receiving ART and non-specific IgG infusions, dosed at the same frequency and for the same duration. Anti-α4β7 and IgG treatment was continued following ART withdrawal at week 18, with all treatment being discontinued by week 32. Viraemia rebounded within a fortnight to high levels in IgG- treated animals following ART cessation, whereas in the anti-α4β7 treated animals, viral suppression or transient viraemia was observed. Virologic control without ART was maintained for nearly 1 year following cessation of anti-α4β7 therapy, while IgG-treated animals had persistently high viral titers throughout the remainder of the study. Normal CD4+

T cell numbers were observed in the intestine and peripheral circulation of anti-α4β7 treated animals.

Currently an exploratory, open label study is now recruiting HIV-1+ patients to investigate the safety and efficacy of vedolizumab in controlling HIV-1 infection upon discontinuation of

ART.160

Etrolizumab (anti-β7 integrin)

Etrolizumab (rhuMAbBeta7) is a humanised IgG1 monoclonal antibody specific for the β7 integrin subunit. Through binding to β7 integrin, this drug has the dual effect of blocking gut homing α4β7:MAdCAM-1 interactions, and also antagonises lymphocyte retaining αEβ7:E- cadherin interactions. It is a humanised version of the rat monoclonal antibody FIB504, which was unique among a panel of anti-β7 integrin antibodies in blocking all known α4β7-

25 dependent interactions, including T cell aggregation, and T cell binding to fibronectin and

VCAM-1, in addition to MAdCAM-1.161

To date, published phase 2 etrolizumab studies have only been performed in UC patients,162 although recent phase 3 data has been published for both UC and CD in abstract form.163,164

A combined single ascending dose, and multiple dosing Phase 1 placebo-controlled study demonstrated no dose limiting toxicity, and no infusion or injection site reactions with etrolizumab.165 Anti-etrolizumab antibodies were detected in 2 of 38 drug-treated patients.

In the EUCALYPTUS Phase 2 study, 124 patients with moderately to severely active UC were randomised 1:1:1 to subcutaneous etrolizumab 100mg at week 0, 4 and 8 (with placebo at week 2) or a loading dose of 420mg at week 0, followed by 300mg at week 2, 4 and 8, or placebo.162 The primary end-point was remission at week 10 defined as a Mayo Clinic Score of ≤2 with no individual subscore >1. At week 10, 0% of the placebo-controlled group were in remission, compared to 21% of the 100mg etrolizumab group and 10% in the 300mg plus loading dose group. In those patients who were anti-TNF-naïve the remission rates were 0% for placebo, 44% for the 100mg etrolizumab group and 25% for 300mg plus loading dose group. This was in comparison to poor remission rates in anti-TNF-experienced patients with remission rates of 0% for placebo, 5% for the 100mg etrolizumab group and 4% for the 300mg plus loading dose group. Post-hoc analysis presented in abstract form showed no difference between dose groups when the Physician’s Global Assessment component was removed from the calculation of efficacy by Mayo Clinic Score.166 Phase 3 studies in UC and CD are currently recruiting, with interim data from an open label induction cohort in UC showing a reduction in stool frequency and rectal bleeding in anti-TNF-refractory patients receiving etrolizumab as early as 4 weeks post-treatment.167

26 Further post-hoc analysis of colonic biopsies stratified patients by baseline whole-biopsy gene expression or IHC expression of the αE integrin subunit. High or low expression in each case was defined as above or below, respectively, a median cut-off for the cohort. In anti-TNF- naïve patients with high gene or IHC expression of αE, remission was further enriched at 67% in the 100mg etrolizumab group and 50% in the 300mg plus loading dose group. Sequencing studies showed similar predictive potential for αE and granzyme A, an effector molecule expressed by a subset of αE+ T cells.84

More recently, data regarding etrolizumab therapy in anti-TNF refractory UC has been presented in abstract form.164 The ongoing, placebo-controlled Phase 3 HICKORY study is evaluating etrolizumab for the induction (week 14) and maintenance of remission (week 66) in patients with moderate to severe UC who have previously failed an anti-TNF therapy. Initial results from the open label induction cohort (n=130 patients) showed that treatment with etrolizumab (105mg q4w) resulted in 50.8% of patients achieving a clinical response (≥3-point decrease and 30% reduction of MCS with a ≥1-point decrease in rectal bleeding score or rectal bleeding score ≤1) and 12.3% of patients achieving clinical remission (MCS ≤2, with individual subscores ≤1 and rectal bleeding score of 0) at week 14. Additionally, 43.9% of patients experienced a ≥1-point improvement from baseline endoscopic score, 52.3% were in rectal bleeding remission (rectal bleeding score of 0) and 35.4% were in stool frequency remission

(stool frequency score ≤1 with ≥1-point reduction from baseline) at week 14; endoscopic improvement was associated with lower stool frequency scores and rectal bleeding remission.

Emerging data, also presented in abstract form suggests that etrolizumab is effective in CD.163

The ongoing, placebo-controlled Phase 3 study BERGAMOT is evaluating patients with moderate to severe CD who have been previously treated with immunosuppressants,

27 corticosteroids, and/or anti-TNFs. In the exploratory induction cohort, 300 patients were randomized 2:2:1 to receive subcutaneously etrolizumab 105mg every 4 weeks, etrolizumab

210 mg on weeks 0, 2, 4, 8, and 12, or placebo during a 14-week period. Endpoints assessed included CDAI remission (CDAI <150), endoscopic improvement (≥50% reduction in Simple

Endoscopic Score for Crohn’s Disease [SES-CD]) and symptomatic remission (abdominal pain

<1 and loose/very soft stool frequency <3). CDAI remission was achieved at week 14 by 23.3% and 28.9% of patients receiving etrolizumab 105mg and 210mg, respectively, compared with

16.9% of those receiving placebo. At week 14, treatment with both etrolizumab 105mg and

210mg resulted in higher rates of endoscopic improvement compared with placebo (21.0% and 17.4% vs 3.4%, respectively). Symptomatic remission was achieved with both doses of etrolizumab as early as week 6 and observed consistently through week 14; 20.8% of etrolizumab 105mg and 24.8% of etrolizumab 210mg patients achieved symptomatic remission at week 14 compared with 11.9% of patients receiving placebo.

Etrolizumab has been well tolerated in clinical studies reported to date, with no reported cases of PML, consistent with murine studies showing no role for β7 subunit containing integrin pairs in cell migration to the CNS.162,165 Adverse events, including infections, in the etrolizumab Phase 2 study in UC were similar between the etrolizumab and placebo arms.162

Phase 3 trials in UC and CD are ongoing.

Abrilumab (anti-α4β7 integrin)

Abrilumab (AMG 181) is an IgG2 monoclonal antibody that, like vedolizumab, selectively binds the α4β7 integrin heterodimer.168 A first-in-human single ascending dose study confirmed

AMG 181 to be safe and well tolerated with suitable pharmacokinetic and pharmacodynamic

28 profiles by subcutaneous injection.169 Phase 2 data in both moderate to severe CD and UC have recently been presented in abstract form.

The Phase 2b CD trial included 249 adult patients who had failed corticosteroid, immunosuppressive or anti-TNF therapy.170 Participants had moderate to severe disease

(CDAI 220-450) and evidence of active inflammation by endoscopy, or CRP or raised faecal calprotectin. Patients were randomised to placebo, a single subcutaneous 210mg dose of abrilumab, or repeated 21mg or 71mg doses of abrilumab by subcutaneous injection on day

1 and at weeks 2 and 4, then every 4 weeks (Q4W) for up to 24 weeks. The primary endpoint was a CDAI remission score of <150 at week 8, and was not achieved in any treatment group.

Secondary endpoints included remission at week 12 and a CDAI response at week 8 or 12

(defined as a reduction of 100 points or more). CDAI response was observed in the 70mg Q4W group at week 8 and 12 (43.2% and 48.9% respectively) and the 210mg single dose group at week 12 (46.6%) with comparison to placebo response rates of 27% and 29.6% respectively at week 8 and 12. Higher remission was also observed in the 21mg Q4W group at week 12

(41.7% compared to 17.6% in placebo). Stratifying according to prior anti-TNF use demonstrated greater efficacy in those who had failed anti-TNF therapy, where higher rates of remission were observed at week 12 in all treatment groups with remission also seen at week 8 in the 210mg single dose arm. Wide variation in placebo response rates were observed between anti-TNF-failure and anti-TNF-naïve patients which may have contributed to the lack of efficacy observed in this study.

The Phase 2b UC study enrolled 354 patients with moderate to severe UC defined as a Mayo

Score of 6-12 with an endoscopic score of ≥2.171 Subjects received placebo, a single 210mg subcutaneous dose of abrilumab, or repeated abrilumab dosing at 7mg, 21mg or 70mg on day 1, followed by week 2, week 4 and then every 4 weeks for 24 weeks. The primary endpoint

29 was remission at week 8 (defined as a total Mayo score of ≤2 with no individual subscore >1).

Endoscopies were centrally read. Higher rates of remission were observed in the 70mg Q4W and 210mg single dose group compared to placebo (respectively, 13.5%, 13.4% and 4.4%).

Response rates and mucosal healing rates were also higher in these treatment groups.

In both the UC and CD studies no cases of PML were observed, adverse events were balanced between groups and no patients developed neutralising antibodies to the drug.170,171 Maximal receptor occupancy was observed in all treatment groups. Pharmacokinetic studies in the UC cohort indicated that maximal remission rates were observed in patients with drug serum trough-levels of >10µg/ml.171

PF-00547659 (anti-MAdCAM-1)

PF-00547659 is a subcutaneously delivered, fully humanized IgG2 anti-MAdCAM-1 monoclonal antibody that blocks binding of α4β7 integrin to MAdCAM-1, thus is intended to reduce trafficking of α4β7+ leukocytes to the intestine.172 To demonstrate that this mechanism of action should not increase risk of PML, the TOSCA study enrolled 49 patients with moderate to severely active CD to undergo lumbar puncture and CSF analysis of lymphocyte populations.173 10 patients underwent two lumbar punctures 14 days apart to determine feasibility of identifying lymphocytes in CSF of patients with CD who had received prior immunosuppressants. Patients then received induction therapy with three doses of

225mg PF-00547659 administered 4 weeks apart. The remaining 39 patients underwent CSF analysis before and after 12 weeks induction therapy. Consistent with blockade of

α4β7:MAdCAM-1 interactions and thus impairment of lymphocyte trafficking to the gut, higher numbers of circulating β7+ memory T cells were observed in blood. In keeping with gut selectivity in mechanism of action, CSF analysis revealed no reduction in CSF T cell subsets

30 or CD4:CD8 ratio. Whilst 74% of patients at baseline had positive anti-JCV serology, no JCV virus DNA was detected in CSF from 19 patients tested following therapy. Open label analysis of efficacy using the HBI demonstrated clinical response (reduction in HBI of ≥3 points) in 80% and clinical remission (defined as an HBI of <5 points) in 79% of patients. The most common adverse events considered related to therapy included nasopharyngitis (16.3%), arthralgia

(18.4%) and headache (12.2%).

More recently double blind placebo controlled phase 2 trial data has been published from the

TURANDOT study in UC,174 and the OPERA study in CD.175 In the TURANDOT trial, 357 patients with moderate to severely active UC (Mayo score ≥6 and endoscopic sub score ≥2) were randomised to placebo or 7.5mg, 22.5mg, 75mg or 225mg of PF-00547659 every 4 weeks.

Remission rates at week 12 (defined as Mayo score ≤2with no subscore >1 and rectal bleeding subscore ≤1) were 2.7% in the placebo group. In the active treatment groups remission rates were 11.3% (7.5mg dose), 16.7% (22.5mg dose), 15.5% (75mg dose) and 5.7% (225mg). With comparison to placebo all doses except 225mg reached significance. In this induction study, adverse events were similar between placebo and PF-00547659 treated patients.

In the OPERA trial, 265 patients with moderate to severe CD (defined as CDAI 220-450, CRP

>3mg/L and ulcers on colonoscopy) who had failed treatment with anti-TNF therapy or immunosuppressants were randomised to placebo, 22.5mg, 75mg or 225mg of PF-00547659 every 4 weeks.175 The primary end point of a decrease in CDAI of 70 points or more from baseline at week 8 or 12 was not reached, though outcomes may have been confounded by high CDAI placebo response rates. Pharmacological activity of the drug was evidenced by higher circulating β7+ memory T cells post therapy and a reduction in soluble MAdCAM-1. No adverse safety signal was identified across treatment groups or in comparison to placebo.

31 Oral integrin-targeted therapy in IBD

The licensed and late-phase clinical trial anti-trafficking agents discussed so far are monoclonal antibodies that require intravenous or subcutaneous dosing. Successful development of oral therapeutics may potentially offer greater convenience to patients, and future development of small molecule therapies may allow targeting, not only of ligand binding, but alternate integrin-focused mechanisms of action such as conformational stabilisation.176 To date, two oral integrin-targeted compounds, AJM300 (carotegrast methyl) and PTG-100 have been studied for IBD.

AJM300 is an oral small molecule antagonist of α4 integrin that targets both α4β7 and α4β1 integrin. Experimental colitis induced by adoptive transfer of CD4+ T cells from IL-10-/- mice into SCID mice confirmed that oral treatment with AJM300 led to decrease of T lymphocytes within the LP, and reduced histopathological inflammatory score.177 A multiple-dose, randomised, double-blind clinical trial of 71 patients with active CD (defined as CDAI of 150 or higher) has been presented in abstract form only.178 In this study AJM300 at doses of 40mg,

120mg or 240mg three times daily for 8 weeks was compared to placebo. The primary end point was a decrease in CDAI from baseline to week 4 or later. Reductions in CDAI for those receiving AJM300 were not significantly different to placebo.

More recently a double-blind, placebo controlled Phase 2a study of 102 patients with moderately active UC (Mayo Score of 6-10 with an endoscopic subscore of ≥2, and a rectal bleeding subscore of ≥1) has been reported.179 In this study patients were assigned to 960mg of AJM300 three times daily for 8 weeks or placebo. The primary end point was clinical response at week 8, defined as a decrease in Mayo Clinic Score of at least 3 points, with a decrease of at least 30% from baseline, and a decrease in the rectal bleeding subscore of 1 or more, or an absolute rectal bleeding subscore of 0 or 1. Clinical response at week 8 was

32 significantly greater in those receiving AJM300 at 62.7%, compared with 25.5% in the placebo group (p=0.0002). Higher rates of clinical remission and mucosal healing were also noted in those receiving AJM300. The proportion of patients previously treated with anti-TNF therapy and the relative outcome for this group were not reported. In common with other integrin- targeted therapies an increase in peripheral circulating lymphocytes was observed. No serious adverse events, including no episodes of PML were reported. A Phase 3 programme studying AJM300 in UC is currently underway in Japan.180

PTG-100 is an orally available α4β7 integrin-specific cysteine knot peptide under development by Protagonist Therapeutics. Animal studies presented in abstract form report a dose-dependent exposure of the peptide in GI tissue with a much lower exposure of the drug in blood.181 A Phase 2b multicenter, placebo controlled trial of this drug is currently recruiting, and will evaluate safety and efficacy of once daily doses of PTG-100 in moderate to severe UC.182

Concluding remarks and positioning of anti-integrin therapy in IBD management

Our understanding of integrin biology has led to the development of a new class of effective biological therapy in IBD. Vedolizumab is established in clinical practice, and several compounds that are in advanced clinical development may contribute additional effective treatment options for UC and CD. Integrin-targeted therapy can offer the potential of being gut-selective, which may be attractive in comparison to the systemic mechanism of action of anti-cytokine therapies including anti-TNF drugs and the recently licensed anti-p40 therapy . Anti-TNF therapy can induce rapid clinical response, such that this class of drug is now used in the setting of acute severe colitis.183 Ongoing head-to-head comparison trials

33 between anti-integrin therapies and anti-TNFs will help to better understand the relative efficacy and speed of impact of these classes of drugs.184-187 Once a patient achieves clinical remission, the durability of clinical remission is impressive with anti-trafficking agents.110,126,130,134,135 Long clinical experience with anti-TNFs have identified therapeutic drug levels as of importance for efficacy. At present the role of therapeutic drug monitoring for anti-integrin drugs is not firmly established, but with emerging evidence of serum trough level-efficacy relationships, particularly in UC, this is likely to be an important area of future study.

Due to the excellent safety profile and good first-line efficacy of gut-selective anti-integrin therapy, in UC, European and Canadian guidance now recommends consideration of vedolizumab as a first-line biological therapy after incomplete response or failure to 5-

ASA.188,189 Given the known association of infections as an adverse effect in anti-TNF therapy, future algorithms may place vedolizumab as a first line therapy in higher risk, for example, older patients more at risk of respiratory disease. Further efficacy and safety data, including regarding treatment impact on extra-intestinal manifestations of IBD, and an improved understanding of discriminating mechanisms of action between anti-integrin therapies, as well as further exploration of therapeutic biomarkers for stratification or response to therapy may further alter treatment paradigms in the future.

34 Acknowledgements

Funding

CAL is a Clinical Lecturer supported by the National Institute for Health Research (NIHR).

Conflicts of Interest

CAL has received research support from, and consulted for Genentech, and has spoken at

Genentech and Takeda educational events. SOB was an employee of Takeda during the development of this manuscript and was a previous employee of Genentech. MEK is a current employee of Genentech. ECB was a founder of Leukosite Inc. and advisor to Millennium

Pharmaceuticals, developers of vedolizumab, and has consulted for Genentech.

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44 154. Lu X, Li Z, Li Q, et al. Preferential loss of gut-homing alpha4beta7 CD4+ T cells and their circulating functional subsets in acute HIV-1 infection. Cell Mol Immunol. 2016;13(6):776-784. 155. Martinelli E, Veglia F, Goode D, et al. The frequency of alpha(4)beta(7)(high) memory CD4(+) T cells correlates with susceptibility to rectal simian immunodeficiency virus infection. J Acquir Immune Defic Syndr. 2013;64(4):325-331. 156. Sivro A, Schuetz A, Sheward D, et al. Integrin alpha4beta7 expression on peripheral blood CD4(+) T cells predicts HIV acquisition and disease progression outcomes. Sci Transl Med. 2018;10(425). 157. Guzzo C, Ichikawa D, Park C, et al. Virion incorporation of integrin α4β7 facilitates HIV-1 infection and intestinal homing. Science Immunology. 2017;2(11):eaam7341. 158. Byrareddy SN, Kallam B, Arthos J, et al. Targeting alpha4beta7 integrin reduces mucosal transmission of simian immunodeficiency virus and protects gut-associated lymphoid tissue from infection. Nat Med. 2014;20(12):1397-1400. 159. Byrareddy SN, Arthos J, Cicala C, et al. Sustained virologic control in SIV+ macaques after antiretroviral and alpha4beta7 antibody therapy. Science. 2016;354(6309):197- 202. 160. Vedolizumab (Anti-alpha4beta7) in Subjects With HIV Infection Undergoing Analytical Treatment Interruption. https://clinicaltrials.gov/ct2/show/NCT02788175. Accessed 1st March 2018. 161. Andrew DP, Berlin C, Honda S, et al. Distinct but overlapping epitopes are involved in alpha 4 beta 7-mediated adhesion to vascular cell adhesion molecule-1, mucosal addressin-1, fibronectin, and lymphocyte aggregation. J Immunol. 1994;153(9):3847- 3861. 162. Vermeire S, O'Byrne S, Keir M, et al. Etrolizumab as induction therapy for ulcerative colitis: a randomised, controlled, phase 2 trial. Lancet. 2014;384(9940):309-318. 163. Sandborn WJ, Panes J, Jones J, et al. Etrolizumab as induction therapy in moderate to severe Crohn's disease: Results from BERGAMOT cohort 1. United European Gastroenterology Journal. 2017;5(8):1139. 164. Peyrin-Biroulet L, Rubin DT, Feagan BG, et al. Etrolizumab Induction Therapy Improved Endoscopic Score, Patient-Reported Outcomes, and Inflammatory Biomarkers in Patients With Moderate to Severe UC Who Had Failed TNF Antagonist Therapy: Results from the HICKORY Open-Label Induction (OLI) trial. United European Gastroenterology Journal. 2017;5(8):1138-1139. 165. Rutgeerts PJ, Fedorak RN, Hommes DW, et al. A randomised phase I study of etrolizumab (rhuMAb beta7) in moderate to severe ulcerative colitis. Gut. 2013;62(8):1122-1130. 166. Sandborn WJ, Schreiber S, Tan MT, Tatro AR, Oh YS, Maciuca R. Etrolizumab demonstrated no difference among doses in symptomatic and endoscopic-based evaluation of remission in anti-TNF-α-naïve patients in a post-hoc analysis of the phase 2 ulcerative colitis trial (EUCALYPTUS). J Crohns Colitis. 2017;11(Suppl 1):S278- S279. 167. Peyrin-Biroulet L, Feagan BG, Mansfield J, et al. Etrolizumab Treatment Leads to Early Improvement in Symptoms and Inflammatory Biomarkers in Anti-Tnf refractory Patients in the Open-Label Induction Cohort of the Phase 3 Hickory Study. Gastroenterology. 2017;152(5 Suppl 1):S603.

45 168. Pan WJ, Hsu H, Rees WA, et al. Pharmacology of AMG 181, a human anti-alpha4 beta7 antibody that specifically alters trafficking of gut-homing T cells. Br J Pharmacol. 2013;169(1):51-68. 169. Pan WJ, Kock K, Rees WA, et al. Clinical Pharmacology of AMG 181, a Gut-Specific Human Anti-alpha beta Monoclonal Antibody, for Treating Inflammatory Bowel Diseases. Br J Clin Pharmacol. 2014. 170. Sandborn WJ, Cyrille M, Berner Hansen M, et al. Efficacy and safety of abrilumab (AMG1181/MEDI 7183) therapy for moderate to severe Crohn's disease. Gastroenterology. 2017;152(5, Suppl 1):S598. 171. Sandborn WJ, Cyrille M, Berner Hansen M, et al. Efficacy and safety of abrilumab in subjects with moderate to severe ulcerative colitis: Results of a Phase 2B, randomized, double-blind, multiple-dose, placebo controlled study. Gastroenterology. 2017;152(5, Suppl 1):S198. 172. Pullen N, Molloy E, Carter D, et al. Pharmacological characterization of PF-00547659, an anti-human MAdCAM monoclonal antibody. Br J Pharmacol. 2009;157(2):281- 293. 173. D'Haens G, Vermeire S, Vogelsang H, et al. Effect of PF-00547659 on Central Nervous System Immune Surveillance and Circulating beta7+ T Cells in Crohn's Disease: Report of the TOSCA Study. J Crohns Colitis. 2018;12(2):188-196. 174. Vermeire S, Sandborn WJ, Danese S, et al. Anti-MAdCAM antibody (PF-00547659) for ulcerative colitis (TURANDOT): a phase 2, randomised, double-blind, placebo- controlled trial. Lancet. 2017;390(10090):135-144. 175. Sandborn WJ, Lee SD, Tarabar D, et al. Phase II evaluation of anti-MAdCAM antibody PF-00547659 in the treatment of Crohn's disease: report of the OPERA study. Gut. 2017. 176. Shimaoka M, Springer TA. Therapeutic antagonists and conformational regulation of integrin function. Nat Rev Drug Discov. 2003;2(9):703-716. 177. Sugiura T, Kageyama S, Andou A, et al. Oral treatment with a novel small molecule alpha 4 integrin antagonist, AJM300, prevents the development of experimental colitis in mice. J Crohns Colitis. 2013;7(11):e533-542. 178. Takazoe M, Watanabe M, Kawaguchi T, et al. Oral Alpha-4 Integrin Inhibitor (AJM300) in Patients with Active Crohn's Disease — A Randomized, Double-Blind, Placebo-Controlled Trial. Gastroenterology. 2009;136(5 Suppl 1):A181. 179. Yoshimura N, Watanabe M, Motoya S, et al. Safety and Efficacy of AJM300, an Oral Antagonist of alpha4 Integrin, in Induction Therapy for Patients With Active Ulcerative Colitis. Gastroenterology. 2015;149(7):1775-1783 e1772. 180. AJM300の活動期潰瘍性大腸炎患者を対象とした第3相臨床試験. http://www.clinicaltrials.jp/user/search/directCteDetail.jsp?clinicalTrialId=13191. Accessed 1st March 2018. 181. Mattheakis L, Bhandari A, Bai L, et al. PTG-100, An Oral Peptide Antagonist of Integrin α4β7 that Alters Trafficking of Gut Homing T Cells in Preclinical Animal Models. Inflamm Bowel Dis. 2016;22 Suppl 1:S48. 182. Safety and Efficacy Study of PTG-100 in the Treatment of Moderate to Severe Ulcerative Colitis. https://clinicaltrials.gov/ct2/show/record/NCT02895100. Accessed 1st March 2018.

46 183. Williams JG, Alam MF, Alrubaiy L, et al. versus ciclosporin for steroid- resistant acute severe ulcerative colitis (CONSTRUCT): a mixed methods, open-label, pragmatic randomised trial. Lancet Gastroenterol Hepatol. 2016;1(1):15-24. 184. An Efficacy and Safety Study of Vedolizumab Intravenous (IV) Compared to Subcutaneous (SC) in Participants With Ulcerative Colitis. https://clinicaltrials.gov/ct2/show/NCT02497469. 185. A Study Comparing the Efficacy and Safety of Etrolizumab With Adalimumab and Placebo in Participants With Moderate to Severe Ulcerative Colitis (UC) in Participants Naive to Tumor Necrosis Factor (TNF) Inhibitors (Study #1). https://clinicaltrials.gov/ct2/show/NCT02163759. 186. A Study Comparing the Efficacy and Safety of Etrolizumab With Adalimumab and Placebo in Participants With Moderate to Severe Ulcerative Colitis (UC) in Participants Naive to Tumor Necrosis Factor (TNF) Inhibitors (Study #2). https://clinicaltrials.gov/ct2/show/NCT02171429. 187. A Study Comparing the Efficacy and Safety of Etrolizumab to Infliximab in Participants With Moderate to Severe Ulcerative Colitis Who Are Naïve to Tumor Necrosis Factor (TNF) Inhibitors. https://clinicaltrials.gov/ct2/show/NCT02136069. 188. Harbord M, Eliakim R, Bettenworth D, et al. Third European Evidence-based Consensus on Diagnosis and Management of Ulcerative Colitis. Part 2: Current Management. J Crohns Colitis. 2017;11(7):769-784. 189. Bressler B, Marshall JK, Bernstein CN, et al. Clinical practice guidelines for the medical management of nonhospitalized ulcerative colitis: the Toronto consensus. Gastroenterology. 2015;148(5):1035-1058 e1033.

47 Table 1: Integrin-targeted therapies for inflammatory bowel disease

Agent Mechanism of Class Indication Manufacturer Approval/Phase action of clinical trial

Natalizumab anti-α4 integrin subunit Humanised CD Biogen Licensed in USA mAb IgG4 mAb (α4β7 & α4β1 activity)

Vedolizumab anti-α4β7 integrin dimer Humanised CD/UC Takeda Licensed mAb IgG1 mAb

Etrolizumab anti-β7 integrin subunit Humanised UC/CD Genentech/Roche Phase 3 mAb IgG1 mAb (αEβ7 & α4β7 activity)

Abrilumab anti-α4β7 integrin dimer Humanised CD/UC Amgen /Astra Phase 2 complete mAb IgG2 mAb Zeneca/MedImmune

AJM 300 α4 integrin subunit Oral small UC EA Pharma Phase 3 (carotegrast antagonist molecule methyl)

PTG-100 α4β7 integrin-specific Orally available UC Protagonist Phase 2b peptide peptide Therapeutics

48 Figure legends

Figure 1. Mammalian integrin αβ heterodimer pairing

Twenty-four integrin heterodimer pairs have been identified in mammalian species and can be broadly grouped into functional categories, cellular expression or ligand specificity of receptors. The α4 and α9 subunits are restricted to chordates. Re-produced from Hynes99 with permission from Elsevier.

49

Figure 2: Chemo-attractant signaling triggers rapid integrin activation

A. Integrin in resting bent “closed conformation”. B. Integrins are rapidly activated by intracellular signaling (“inside-out signaling”) cascades triggered by chemokine receptor stimulation. TCR stimulation can also activate integrins, but less rapidly. The signaling cascade depicted summarises chemokine mediated signaling during activation of αLβ2, as adapted from Toffali et al.4 Here chemokine interacting with its receptor leads to tyrosine kinase or G

50 protein activation of multiple rho small GTPases. In turn activation of Rho and Rap modules leads to regulation of the activity of several actin/integrin binding proteins. Chemokines trigger both conformational change and integrin motility and repositioning, both of which are important for cellular arrest during cell trafficking. In addition to chemokine mediated signaling, TCR activation of integrins regulates cell to cell interactions. Therefore, integrin targeted therapies may elicit a biological effect within lymphoid or target tissues, independent of their impact on cell trafficking.

51

Figure 3: Integrin involvement in T cell localisation to the gut lamina propria and epithelium

T lymphocyte migration via HEVs in GALT and post-capillary venules in the lamina propria is controlled by initial microvillous presentation of receptors, initiating tethering and rolling along the endothelial surface. This process slows T lymphocytes sufficiently to allow the cells to sense chemokines presented by the endothelium. Chemokines trigger integrin activation

(Figure 2), leading to strong integrin:CAM adhesion and cell arrest. Lymphocytes then

52 undergo diapedesis through the endothelial layer in response to motility signals from outside- in integrin signaling and from chemoattractant gradients. In GALT, T cells use L-selectin for tethering and α4β7 for slow rolling on HEV. High levels of α4β7 on gut homing effector cells and blasts interacting with MAdCAM-1 can directly mediate rolling and arrest in the LP.15,19

αLβ2:ICAM-1 interactions participate in terminal arrest on the endothelium, and may also promote cellular adhesion.19,52 High expression of α4β1 on effector T cells may allow α4β7- independent interactions during inflammation in combination with L-selectin,12,57 especially in the inflamed ileum, a mechanism that may be important in CD.50 Lamina propria venules can also express P-selectin during inflammation to enhance tethering and rolling. Different lymphocyte populations can use distinct chemokine receptors for integrin activation and chemotaxis, conferring subset selectivity in recruitment (not shown). Once lymphocytes are within tissue, they may upregulate αEβ7 integrin and bind to epithelium through αEβ7:E- cadherin interactions.

53