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Review article: nonclinical and clinical pharmacology, pharmacokinetics and pharmacodynamics of etrolizumab, an anti‐β7 integrin therapy for inflammatory bowel disease

Summary Background Novel treatments with superior benefit‐risk profiles are needed to improve the long‐term prognosis of patients with inflammatory bowel disease (IBD). Etrolizumab—a monoclonal antibody that specifically targets β7 integrins—is currently under phase III clinical evaluation in IBD. Aim This review summarises the available pharmacological and pharmacokinetic/pharmacodynamic data for etrolizumab to provide a comprehensive understanding of its mechanism of action (MOA) and pharmacological effects. Methods Published and internal unpublished data from nonclinical and clinical studies with etrolizumab are reviewed. Results Etrolizumab exerts its effect via a unique dual MOA that inhibits both leucocyte trafficking to the intestinal mucosa and retention within the intestinal epithelial layer. The gut‐selectivity of etrolizumab results from its specific targeting of the β7 subunit of α4β7 and αEβ7 integrins. Etrolizumab does not bind to α4β1 integrin, which mediates lymphocyte trafficking to tissues including the central nervous system, a characteristic underlying its favourable safety with regard to progressive multifocal leucoencephalopathy. Phase I/II studies in patients with ulcerative colitis (UC) showed linear pharmacokinetics when etrolizumab was administered subcutaneously at 100 mg or higher once every 4 weeks. This dose was sufficient to enable full β7 receptor occupancy in both blood and intestinal tissues of patients with moderate to severe UC. The phase II study results also suggested that patients with elevated intestinal expression of αE integrin may have an increased likelihood of clinical remission in response to etrolizumab treatment. Conclusion Etrolizumab is a gut‐selective, anti‐β7 integrin monoclonal antibody that may have therapeutic potential for the treatment of IBD.


| INTRODUCTION
The current goals of novel therapeutics for the treatment of Crohn's disease and ulcerative colitis (UC), collectively known as inflammatory bowel disease (IBD), are to induce and maintain steroid-free remission and achieve long-term mucosal healing, so as to reduce/eliminate the need for IBD-related hospitalisation and surgery, improve patient quality of life and avoid disability. [1][2][3] The current standard of care includes 5-aminosalicylates (5-ASAs), which are used as first-line therapy for UC. However,~50% of patients do not respond to 5-ASAs alone and require more aggressive intervention with corticosteroids and/or immunosuppressant therapy (eg azathioprine, mercaptopurine and methotrexate). Corticosteroid and immunosuppressant therapies, however, are often associated with significant side effects or intolerance and may have limited efficacy; for example, corticosteroids have not shown benefit in the maintenance settings. 4,5 In patients who do not achieve or maintain remission in response to corticosteroids or immunosuppressants, biologic therapy options including monoclonal antibodies (mAbs) targeting tumour necrosis factor a (TNF-a), such as infliximab (Remicade, Janssen Biotech Inc., Horsham, PA, USA) and adalimumab (Humira, AbbVie, Chicago, IL, USA), can be used to induce and maintain remission. 6,7 Overall, the benefit-risk profiles for anti-TNF-a biologics are considered acceptable. However, approximately 10%-30% of IBD patients are primary nonresponders to anti-TNF-a therapy, and another approximately 20%-40% of IBD patients stop responding to anti-TNF-a therapy over time. 8,9 In addition, these TNF-a inhibitors are associated with increased risk of infections and IBD-related complications stemming from inadequate disease control. [10][11][12] In addition to offering better benefit-risk profiles, new innovative and effective therapies will also positively influence the balance of cost-effectiveness by lowering healthcare utilisation, given that IBD was the primary cause for 1.36 and 1.9 million physician visits per year in the United States in 2004 and 2009, respectively, and the first-listed discharge diagnosis in over 100 000 hospitalisations. 13 Hence, there continues to be a need for novel treatments with improved benefit-risk profiles that enhance the long-term prognosis of patients with IBD.
Binding of integrins to their ligands controls leucocyte trafficking and retention within peripheral tissues, such as the gut, and modulates the local inflammatory milieu; thus, integrins are emerging therapeutic targets for the treatment of IBD. Natalizumab (anti-a4 integrin; Tysabri, Biogen, Cambridge, MA, USA) is approved for Crohn's disease in the United States, 14 whereas vedolizumab (an anti-integrin mAb targeting a4b7 heterodimers; Entyvio, Takeda, Osaka, Japan) is on the market for the treatment of both UC and Crohn's disease. 15,16 Etrolizumab (also called rhuMAb b7; Genentech, San Francisco, CA, USA), currently under evaluation in clinical studies for its therapeutic potential in IBD, is an IgG1-humanised monoclonal antibody that specifically targets the b7 subunit of both the a4b7 and aEb7 integrins. Binding to b7 integrin enables etrolizumab to block both leucocyte trafficking to the gut via a4b7 interactions with its endothelial ligand mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1), 17,18 expressed primarily on endothelium of venules of intestinal lamina propria and associated lymphoid tissues 19,20 as well as the retention of lymphocyte subsets within the intestinal mucosa through the interaction of aEb7 with its ligand E-cadherin, expressed on the gut epithelium 21 (Figure 1, Table 1). [22][23][24][25] The objectives of this review are to summarise the available published and unpublished nonclinical and clinical pharmacological, pharmacokinetic (PK) and pharmacodynamic (PD) data for etrolizumab, and to provide a comprehensive understanding of its mechanism of action (MOA) and pharmacological effects. Since etrolizumab has a unique dual MOA associated with inhibition of both the a4b7 and aEb7 pathways, the integrated pharmacological information summarised in this review aims to provide researchers and investigators with a clear view of its unique biology distinct from other anti-integrin drugs in the class.

| The role of b7 integrins and their ligands
Integrins are cell surface glycoprotein receptors that play a role in a wide range of cell-cell and cell-matrix interactions during embryogenesis, thrombosis and leucocyte adhesion, signalling, proliferation and migration. 26 They are composed of heterodimeric, noncovalently interacting a and b subunits that bind distinct cell adhesion molecules (CAMs) on endothelia, epithelia and the extracellular matrix.
This review focuses on leucocyte homing mediated by integrin binding to tissue-specific CAMs to regulate cell recruitment from the peripheral circulation into tissues. 27 a4b7 integrin plays a crucial role in leucocyte homing to the intestinal mucosa and associated lymphoid tissues. a4b7 integrin is constitutively expressed at a modest level on naive T and B cells and at higher levels on intestinal-homing effector/memory T and B cells.
In contrast, effector/memory T and B cells that are activated to home to nonintestinal tissue express low levels of a4b7 integrin.
Additionally, a4b7 integrin is also expressed on NK cells, stimulated monocytes, macrophages, mast cells, eosinophils and basophils. 26,28,29 In the gut, firm adhesion of leucocytes to the mucosal endothelium is initiated by chemokine signals which induce a conformational change in the a4b7 heterodimer on leucocytes that enable binding to MAdCAM-1 with high affinity. This engagement slows leucocyte movement along the endothelial surface, eventually arresting the cell before extravasation through the vascular endothelium to underlying tissue. 27 T lymphocytes bearing a4b7 preferentially bind to endothelial venules expressing MAdCAM-1 in intestinal tissue as well as to the extracellular matrix molecule fibronectin fragment CS-1. [30][31][32] The endothelial cell surface protein vascular cell adhesion molecule-1 (VCAM-1) also interacts with a4b7, albeit with a slightly lower affinity compared with its interaction to MAdCAM-1. 33 These relative binding affinities are not necessarily static, as distinct chemokine-induced b7 phosphorylation states exist that promote a4b7 + cell adhesion via MAdCAM-1 while suppressing binding to VCAM-1 and vice versa. 33 Both the extent of a4b7 + cellular infiltrates and MAdCAM-1 expression are increased in the intestinal tracts of patients with UC or Crohn's disease. 19,34 The TANG ET AL.
| 1441 majority of b7 integrin-expressing cells in the circulating lymphocytes (including T and B cells) are a4b7 + cells. 24 aEb7 integrin is found primarily on intraepithelial lymphocytes (IEL) and lamina propria T lymphocytes within mucosal tissues.
Indeed, mucosal T lymphocytes expressing aE (also known as CD103) are significantly enriched in intestinal tissue compared with lung and the spleen. 35 The finding that more than 90% of IELs and 50% of T lymphocytes in the human intestinal lamina propria express T A B L E 1 Summary of monoclonal antibodies targeting integrins and currently approved or under development for the treatment of IBD Gut epithelium Lamina propria F I G U R E 1 Etrolizumab mechanism of action. Etrolizumab has a dual mechanism of action, inhibiting both a4b7:MAdCAM-1-mediated lymphocyte trafficking to the gut mucosa (arrow #2) and aEb7:E-cadherin-mediated lymphocyte retention in the intraepithelial space (arrow #1). IEL, intraepithelial lymphocytes; VCAM-1, vascular cell adhesion molecule 1; MAdCAM-1, mucosal vascular addressin cell adhesion molecule 1 aE supports a distinct role for this integrin in mucosal immunology. 36,37 aE expression has also been described on dendritic cells, mast cells 28 and innate lymphoid cells (ILCs). 38 The aE integrin is only known to dimerise with the b7 integrin, a process critical for the translocation of aEb7 to the cell surface to enable cell binding to E-cadherin. 21,39 The aEb7 integrin on leucocytes binds specifically to E-cadherin on epithelial cells and is thought to mediate the retention of cells, particularly IELs, in mucosal tissue. 20,21,36,40 Anti-aEb7 antibody treatment has been shown to attenuate the severity of colitis in a mouse experimental colitis model, suggesting a pathogenic role for aEb7 + lymphocytes in experimental models of IBD. 41 Recent data show that there are notable differences between aE + and aE À T cells from healthy control and UC colonic biopsies, with an overall increased proportion of CD3 + aE + T cells and a 2- cytokines IFN-c, TNF-a and IL-17A compared with non-IBD controls. 50 Likewise, T-helper 9 (Th9) lymphocytes, which produce IL-9, a cytokine that inhibits epithelial repair, 51 express high levels of aE relative to other T-helper lymphocytes and are also increased in patients with UC. 52 In addition, cytolytic granules, including granzyme A, secreted by CD8 + aE and CD4 + aE T lymphocytes have been shown to be more prevalent in the gut tissue of patients with IBD compared with non-IBD controls. 43,50,51,53,54 In patients with Crohn's disease, aE + intraepithelial type 1 ILCs have also been found to be elevated. 38 Experiments in a murine anti-CD40-induced colitis model suggest that aE + intraepithelial type 1 ILCs contribute to the inflammatory process. 38  Etrolizumab is a humanised mAb derived from the rat anti-mouse/human b7 mAb FIB504. 55 The humanised version of FIB504 antibody,  (Table 3). These data indicate that etrolizumab is potent in inhibiting in vitro adhesion of a4b7 + bearing cells to human MAdCAM-1 or VCAM-1.
In addition, etrolizumab is highly specific for the b7 subunit of a4b7 and aEb7 and does not bind to a4 or b1 integrin subunits.
Experiments with a4b1 + /a4b7 À Ramos cell line binding to human VCAM-1 showed that etrolizumab did not inhibit a4b1 + cell adhesion at concentrations as high as 100 nmol l À1 (Figure 2), indicating that the migration of T-lymphocyte subsets expressing a4b1 but not a4b7 should not be affected by etrolizumab. In addition, studies in cynomolgus monkeys demonstrated that animals dosed with etrolizumab increased their peripheral blood b7 High CD45RA À "gut homing memory" CD4 + T lymphocytes but had no substantial effect on b7 Low CD45RA À "peripheral homing memory" CD4 + T lymphocytes or b7 Intermediate CD45RA + naive CD4 + T-helper lymphocytes. 56 The increase in peripheral b7 High lymphocytes, presumably due to the inhibition of the trafficking mechanisms of these cells to gut tissue, correlated with the complete peripheral Treatment with the anti-a4b1 integrin antibody natalizumab has been associated with an increased risk of progressive multifocal T A B L E 2 Etrolizumab affinity data a for binding to a4b7 or aEb7 transfected human HEK293 cells or mouse 38C13 cells, or human peripheral blood lymphocytes  Overall rates of adverse events (AEs) in the etrolizumab-treated patients of the EUCALYPTUS study were similar to those in the placebo arm. 24 Notably, rates of infections were similar among all groups. Mild injection site reactions (ISRs) were reported in the 300 mg+LD cohort, but no severe hypersensitivity reactions were seen.  were not found to have significant impact on etrolizumab PK parameters. 66 This population PK model will be refined pending additional data from ongoing phase III studies.

| Pharmacodynamics
Binding of etrolizumab to b7 integrin receptors on the surface of circulating lymphocytes was evaluated by flow cytometry in both phase I and phase II EUCALYPTUS studies. Complete or near-complete occupancy of b7 integrin receptors on circulating b7 High intestinal homing T and B lymphocytes was observed as early as day 2 following etrolizumab dosing. This rapid RO in the peripheral blood was observed in all dose levels (0.3-10 mg/kg IV, 0.5-3.0 mg/kg SC and 100 or 300 mg + LD SC doses) evaluated, but the duration of complete RO was dose dependent during the drug washout period in etrolizumab-treated patients. 24,65 Furthermore, a consistent PK/PD relationship was observed across etrolizumab-treated cohorts, with a minimum etrolizumab serum concentration range of 1-3 lg/mL deemed necessary for full b7 RO on circulating b7 High CD45RA À intestinal homing T cells. 65 Consistent with b7 RO in peripheral blood, complete drug occupancy of both a4b7 and aEb7 receptors was observed in T lymphocyte subsets in colonic biopsy tissues from etrolizumab-treated patients.

| PK/PD modelling outcome
The serum etrolizumab concentration-time profile and the time profile of peripheral blood cells with free b7 receptors (available for ligand binding) from the phase I study were simultaneously modelled using a quasi-steady-state (QSS) target-mediated drug disposition (TMDD) model. 67 The final QSS TMDD model adequately described