Integrin 4 7 Antagonists: Activities, Mechanisms of Action and Therapeutic Prospects

Transcription

1 118 Current Immunology Reviews, 2012, 8, Integrin 4 7 Antagonists: Activities, Mechanisms of Action and Therapeutic Prospects Dulce Soler-Ferran *,1 and Michael J. Briskin 2 1 Former Affiliation: Millennium Pharmaceuticals. Currently at Centers for Therapeutic Innovation (CTI), Pfizer, Inc., 3 Blackfan Circle, Boston, MA 02115, USA 2 Former Affiliation: Millennium Pharmaceuticals and Merrimack Pharmaceuticals; Currently at Biotechnology Consulting, 28 Harbell Street, Lexington, MA 02421, USA Abstract: The 4 7 integrin is a leukocyte homing receptor with selective tissue tropism for the gastrointestinal tract through its interaction with MAdCAM-1, an adhesion receptor expressed on the endothelium of the gut mucosa. Crohn s disease (CD) and ulcerative colitis (UC), two inflammatory bowel diseases resulting from intestinal immunedysregulation, are associated with pronounced infiltration of 4 7 positive lymphocytes. This has triggered the development of inhibitors of the 4 7 /MAdCAM-1 homing pathway. Vedolizumab is a humanized monoclonal antibody selective for 4 7 that demonstrated efficacy in early clinical studies for the treatment of CD and UC and is currently in phase 3 clinical trials. Targeting of 4 7 is also achieved by less selective therapeutic modalities which also block one of the two other leukocyte integrins that share a subunit with 4 7, namely, 4 1 and E 7. Natalizumab is an anti- 4 monoclonal antibody and dual 4 1 and 4 7 antagonist approved for the treatment of multiple sclerosis and CD. Other therapies in development include antibodies targeting the 7 subunit of 4 7 and E 7, MAdCAM-1, and dual 4 small molecule antagonists. This review will focus on the mechanism of action, pharmacology, efficacy and safety properties as well as future opportunities that may arise from this unique class of leukocyte anti-adhesion antagonists. Keywords: 4 7, 4 1, and E 7, integrin, inflammatory bowel disease (IBD), mucosal addressin cell adhesion molecule 1 (MAdCAM-1), natalizumab, progressive multifocal leukoencephalopathy (PML), vedolizumab. INTRODUCTION Chronic inflammatory diseases arise from dysregulated innate and adaptive immune responses and are characterized by large inflammatory infiltrates, which come about as a result of expansion and excessive trafficking of particular leukocyte subsets [1]. Regulation of lymphocyte trafficking into lymphoid and non-lymphoid tissues involves a complex multi-step process that requires the sequential engagement of unique combinations of particular adhesive and signaling molecules on the surface of the lymphocyte with their respective ligands/receptors on the endothelial cell at the sites of leukocyte extravasation [2, 3]. This molecular diversity provides a mechanism for the regional specialization of adaptive immune responses. Targeting the molecular mechanisms that confer tissue-selectivity to the trafficking of lymphocytes is a novel therapeutic strategy for modulating pathogenic adaptive immune responses and treating organ-specific chronic inflammatory diseases without causing significant systemic immunosuppression. Naïve lymphocytes exert their immune surveillance function by circulating continuously between the blood and the lymphoid organs in search of cognate antigens. Upon antigen encounter and activation in different regional lymphoid organs, lymphocytes acquire homing receptors which imprint them with capacities to migrate to the tissues where antigen was first encountered [4]. This selective *Address correspondence to this author at the Centers for Therapeutic Innovation (CTI), Pfizer, Inc., 3 Blackfan Circle, Boston, MA 02115, USA; Tel: ; s: dulce.soler@pfizer.com, dsolerferran@comcast.net tissue-tropism is most elegantly illustrated in the gastrointestinal tract by the imprinting of the gut-homing lymphocyte receptors, the integrin 4 7 and the chemokine receptor CCR9. These homing receptors are induced via interaction with dendritic cells of the inductive gut associated lymphoid tissues (GALT), the Peyer s patches (PPs) and mesenteric lymph nodes (MLNs) [5-7]. The expression of 4 7 is associated with homing to both the large and small intestine while 4 hi 7 CCR9 + lymphocyte populations preferentially migrate to the small intestine [8-10]. The regulation of these receptors during memory acquisition dictates the specification and segregation of intestinal versus systemic (or non intestinal) immune responses. Increased expression of MAdCAM-1 and associated trafficking of 4 hi 7 subsets in the gastrointestinal tract have long suggested that these receptors might play a role in induction and maintenance of intestinal inflammation. The two major forms of inflammatory bowel diseases (IBD) Crohn s disease (CD) and ulcerative colitis (UC), are still fraught with lack of understanding of etiology and mechanism but are both characterized by amplified cellular hi infiltrates consisting of 4 7 lymphocytes and increased expression of MAdCAM-1 [11-15]. It is thought that initiation of these diseases partially results from defects in innate immunity which cause altered adaptive immune responses which lead to the chronic inflammatory states that characterize these debilitating disorders [1, 16]. Crohn s disease can affect the entire gastrointestinal tract; it is characterized by discontinuous lesions, transmural pathology and often granulomas and fistulae. UC pathology however, is restricted to the large bowel and is characterized by /12 $ Bentham Science Publishers

2 Integrin 4 7 Antagonists Current Immunology Reviews, 2012, Vol. 8, No continuous superficial lesions of the mucosal layer. Current management of CD and UC is often unsatisfactory because of either lack of efficacy and durable response or side effects. Recently, anti-tnf biologics have been shown to be effective for induction and maintenance of response in IBD [17]. However, a significant proportion of patients fail to respond or become refractory to anti-tnf therapy. In addition, anti-tnf agents are frequently associated with serious complications [18]. Consequently, there is a need for new therapies for IBD that induce and maintain remission and cause minimal immunosuppression or other complications. Decreasing immune cell infiltration in the chronically inflamed gut tissue by blocking the interaction of the gut homing 4 7 integrin on circulating leukocytes with its endothelial counter receptor, MAdCAM-1, represents a promising novel therapeutic modality for the treatment of IBD. Current antagonists of 4 7 in development include a selective anti- 4 7 monoclonal antibody, vedolizumab; a selective MAdCAM-1 antibody; and dual anti- 4 ( 4 1 and 4 7 ) and anti- 7 ( 4 7 and E 7) monoclonal antibodies. Natalizumab, the dual 4 antibody, is used clinically for the treatment of multiple sclerosis and in the US for Crohn s disease. This review will focus on the current status in the development of these novel anti-adhesive therapeutics as well as their mechanism of action and pharmacologic properties. We will also discuss potential efficacy and safety advantages and disadvantages, small molecule antagonists of 4 7 as well as other potential therapeutic indications for therapies targeting the 4 7 /MAdCAM-1 pathway. and mediate important immune cell functions. 4 1 binding to its endothelial receptor, VCAM-1 (vascular cell adhesion molecule-1) mediates lymphocyte recruitment into several inflamed tissues outside the gastrointestinal tract including the brain, the lung, synovium and the heart. The predominant ligand for 4 7, mucosal addressin cell adhesion molecule (MAdCAM-1), exhibits a more restricted tissue expression. It is preferentially and constitutively expressed at sites of extravasation of intestinal lymphoid tissue and intestinal lamina propria where it mediates the migration of guthoming lymphocyte subsets. A number of studies discussed below suggest that integrin 4 7 interacting with MAdCAM- 1 plays a critical role in lymphocyte recirculation to intestinal tissues but not to non-intestinal tissues. ROLE OF 4 7 AND MADCAM-1 IN LYMPHOCYTE RECRUITMENT TO THE GASTROINTESTINAL TRACT The intestinal adaptive immune system plays an essential role in protecting against constant exposure to pathogens and in maintaining tolerance to commensal antigens and normal flora. Lymphocytes in the intestine are found in the gutassociated lymphoid tissues (GALT) (e.g. Peyer s patches, mesenteric lymph nodes (MNL)), diffused through the lamina propria and embedded in the epithelium (IELs intraepithelial lymphocytes), reaching these sites by extravasation from the vasculature. Leukocyte migration from the circulation into lymphoid and non-lymphoid tissues takes place following a sequence of molecular events (Fig. 1). The first is the tethering or rolling of the leukocyte along the vessel wall through a series of weak and reversible interactions typically mediated by members of the selectin family of proteins and their carbohydrate ligands on the endothelium. This allows for sampling of signaling molecules such as chemokines which activate their respective receptors and induce increased integrin adhesiveness, causing them to firmly bind to their endothelial receptor and arrest the cell. Diapedesis and transendothelial migration then ensues in the direction of a chemotactic gradient [2, 19]. Integrins are heterodimeric cell surface proteins that mediate many important cellular functions including adhesion, migration, proliferation, hematopoiesis and survival by binding to their counter-receptors on cells or extracellular matrix proteins [20, 21]. The 4 integrins, 4 1 and 4 7 are expressed on many types of hematopoietic cells Fig. (1). Classical view of multistep adhesion cascade. Initial lymphocyte endothelial cell interactions characterized by leukocyte rolling, are typically mediated by selectin family members and various carbohydrate containing glycoprotein ligands. Some species of MAdCAM-1 are also decorated with selectin binding determinants and can bind L-selectin. 4 7 can thus mediate leukocyte rolling as well, but upon chemokine triggering it mediates firm adhesion to MAdCAM-1 on the vessel wall after which transmigration ensues. 4 7 Integrin 4 7 initially was termed LPAM-1 (lymphocyte Peyer s patch (PP) adhesion molecule-1) as it was shown to mediate the adhesion of lymphocytes to the high endothelial cells of postcapillary venules (HEVs) of PPs [22, 23]. The critical role of 4 7 in mediating the migration of lymphocytes to Peyer s patches and the small and large intestinal lamina propria was first demonstrated by in vivo homing studies with antibodies specific for the 4 7 heterodimer, and anti- 4, anti- 7 and anti- MAdCAM-1 antibodies [24, 25] and with studies with 7 deficient mice or immune cells. Antibodies to 7 preferentially inhibited in vivo homing to Peyer s patches, partially inhibited homing to MLN, and were ineffective in blocking homing to peripheral nodes; a pattern that is consistent with the progressive

3 120 Current Immunology Reviews, 2012, Vol. 8, No. 2 Soler-Ferran and Briskin decreased expression of MAdCAM-1 at these respective sites. 7 deficient mice are healthy and develop normally but contain hypocellular PPs (but no change in their numbers); they have greatly decreased numbers of B cells, plasma cells, and helper T cells in the lamina propria and decreased numbers of IELs. Firm adhesion and migration of 7 -/- lymphocytes to PPs was mostly abrogated and it was reduced in MLNs [26]. Another study with 7 -/- mice demonstrated a requirement for this integrin in the migration of naïve CD8 + T cells and B cells to PPs but not to MLNs. In this study, migration of activated CD8 + T cells during an anti-viral immune response was impaired in PPs and MLNs, and in large and small intestinal lamina propria [9]. Lastly, a study that showed the requirement of 7 for B lymphocyte migration into isolated lymphoid follicles (ILF) within intestinal mucosa (but not for cryptopatch formation) added further proof to the selectivity of this pathway [27]. An important role for 4 7 in the migration of leukocytes to the gut was also suggested by several studies that established a correlation between 4 7 expression and lymphocyte recirculation into the gut and not into other peripheral tissues, including the lung or the skin [28]. Consistent with a role of 4 7 in intestinal immunity, protective adaptive immune responses to enteric pathogens are also associated with high expression of 4 7 in pathogen-specific lymphocytes [28, 29]. In addition, a subset of dendritic cells (CD103 + ) derived from LP, PPs and MLNs, but not DCs from other microenvironments, induce expression of 4 7 and CCR9 --a small intestine-tropic chemokine receptor for CCL25 (a chemokine expressed by small intestine epithelium)-- on T and B cells in a retinoic acid dependent manner [5-7, 30-33]. The strength of the RA signal appears to regulate large versus small intestinal tropism by inducing expression of 4 7 without or with concomitant CCR9 expression respectively; while 4 hi 7 CCR9 + migrate to the small intestine, 4 hi 7 CCR9 - lymphocytes migrate to and induce inflammation in the large intestine but can also migrate to the small intestine, presumably through activation by other chemokine receptors [10, 34]. Importantly, conservation of this mechanism was also shown as CD103 + DCs with similar functional properties were isolated from human MLNs and from MLNs of CD patients [35]. In human peripheral blood leukocytes, 4 7 is expressed most highly in a subset of memory CD4 + T lymphocytes (CD4 + 4 hi 7 ), which also express low levels of 4 1, and represents approximately a fourth of the entire population [36]. The rest of memory CD4 + T cells do not express 4 7 but the majority express high levels of 4 1. Memory CD8 + T cells have a similar pattern of 4 7 expression, albeit at lower levels, while naïve T and B lymphocytes and NK cells express intermediate to low 4 7. Neutrophils are negative for this integrin; a small percentage of monocytes (15%) express very low levels, while Eosinophils are relatively bright in 4 7 expression [37, 38]. In human intestinal lamina propria, most T and B lymphocytes, blasts and plasma cells were positive for 4 7 expression while most IELs were negative but expressed the other 7 integrin, E 7 as did some dendritic cells [14]. Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1) 4 7 was shown to bind to MAdCAM-1 [39], a cell adhesion molecule of the immunoglobulin (Ig) domain super family of integrin receptors [40-43]. MAdCAM-1 is preferentially, but not exclusively, expressed at anatomic sites of lymphocyte extravasation in gastrointestinal tissues. It is constitutively expressed in high endothelial cells of post-capillary venules of intestinal lymphoid tissues, PPs and mesenteric MLNs, and in endothelial cells of post-capillary venules of the small and large intestinal lamina propria. MAdCAM-1 is also expressed in follicular dendritic cells of Peyer s patches [11, 44]. While MAdCAM-1 is the predominant ligand for 4 7, this integrin can also bind to VCAM-1 and fibronectin but with much lower affinity. These additional interactions do not appear to play a role in extravasation but rather, may be involved in intra-tissue interactions with stromal cells or extracellular matrix. 4-VCAM-1 interactions appear to be dominated by 4 1 even when adhesion is performed with hi 4 7 lymphocytes (which also express low levels of 4 1 ) [4, 45]. MAdCAM-1 gene-deficient mice are healthy, fertile and with no obvious alterations. PPs developed normally until day 3 of birth, but became hypocellular as a result of a homing defect, a phenotype reminiscent of the 7-deficient mice. These mice have decreased numbers of B cells and plasma cells in the LP, likely due to a homing failure of these cells as shown by adoptive transfer experiments. In agreement with this, IgA plasma cells in blood and spleen were elevated and humoral immune responses to oral but not systemic antigen immunizations were compromised, emphasizing the role of MAdCAM-1/ 4 7 in regional immunity [46]. 4 7 /MAdCAM-1 Interactions Mediate Migration of Lymphocytes to Intestinal Lymphoid and Non-Lymphoid Tissue 4 7 /MAdCAM-1 interactions mediate not only firm adhesion but also L-selectin independent lymphocyte rolling [47]. The topographical location of the 4 integrins on microvillus tips makes this interaction possible. In addition, the mucin domain of MAdCAM-1 on HEVs of intestinal lymphoid tissues (but not intestinal non-lymphoid venules) is decorated with L-selectin carbohydrate ligands and can support L-selectin mediated interactions (Fig. 1). This unique property of MAdCAM-1 confers this molecule with the ability to mediate leukocyte rolling and homing of naïve L-selectin + lymphocytes to intestinal lymphoid tissues [48]. Naïve cells, which express low levels of 4 7, extravasate in PPs through rolling mediated by MAdCAM-1 interacting with L-selectin and 4 7, and firm adhesion mediated by hi 4 7 /MAdCAM-1 and LFA-1/ICAM interactions [24]. 4 7 memory lymphocytes can extravasate into GI lymphoid organs or lamina propria by 4 7 /MAdCAM-1 interactions alone [4]. MAdCAM-1 is also expressed on endothelial cells of the gastric mucosa and lymphocyte activation by antigens in the stomach or the intestine microenvironments results in 4 7 expression and homing to the gastric mucosa [49, 50]. In addition to directing the trafficking of lymphocytes, 4 7 has been implicated in GI mucosa trafficking of other leukocytes important in immune responses to parasitic infections, such as certain intestinal helminths. Studies with gene-deficient mice and neutralizing antibodies demonstrated a critical role

4 Integrin 4 7 Antagonists Current Immunology Reviews, 2012, Vol. 8, No for 4 7 in directing the selective migration of mast cell progenitors and eosinophils to the intestine in these settings [51, 52]. 4 7 /MAdCAM-1 Interactions in Intestinal Inflammation and Preclinical Models of IBD MAdCAM-1 expression is increased in CD and UC [11-13] and is generally not associated with other inflamed tissues with a few exceptions such as the inflamed pancreas and liver (these two will be discussed in the section on other therapeutic indications for antagonists of 4 7 ). A number of studies demonstrating effectiveness of function-blocking antibodies selective for 4 7 or MAdCAM-1 in preclinical models of IBD supported a potential therapeutic role in human IBD for antagonists of 4 7 /MAdCAM-1 mediated interactions. Act-1 (the selective 4 7 murine antibody from which vedolizumab was derived) and a dual anti- 4 antibody antagonist, were effective in resolving disease symptoms in cotton top tamarins, a nonhuman primate model of spontaneous colitis [53, 54]. A number of rodent gut inflammation models (via antibody blockade or deficiency of the 4 7 /MAdCAM-1 pathway) resulted in attenuation of disease including: 1) colitis induced in immunodeficient mice by adoptive transfer of CD45RB hi T cells [55-58]; 2) murine DSS-induced colitis [59, 60]; 3) granulomatous colitis in rats [61]; 4) CD8 + T-cell dependent ileitis in the TNF(DeltaARE) mouse [62]; 5) ileitis in SAMP1/Yit mice [63]; 6) and in TNBS-induced colitis (in this case was treated with antisense oligos against MAdCAM-1) [64]. 4 7 /MAdCAM-1 Interactions are not Generally Involved in Migration of Lymphocytes to Non-Intestinal Tissues Despite the role of MAdCAM-1/ 4 7 interactions in intestinal mucosal immunity, they appear to play no or little role in immunity to other mucosal tissues such as the lung or pulmonary tissues, oral, and nasal mucosa, although contradicting reports suggest a potential role in some infections of the genitourinary tract [4, 28, 65, 66]. These interactions may also play a role in oral infections as a study with HIV patients with oropharyngeal candidiasis showed significantly increased expression of MAdCAM-1 in infected patients versus those not infected, as well as expression of 4 7 in infiltrating lymphocytes [67]. In mice, nasal associated lymphoid tissue (NALT) MAdCAM-1 expression appears to be associated with follicular dendritic cells which are not involved in extravasation but perhaps in 4 7 dependent B cell adhesion and activation in germinal centers [68]. Natalizumab is likely effective in the treatment of MS because of inhibition of leukocyte infiltration into the brain by blocking 4 1 binding to VCAM-1 [69]. In the midst of this success, inhibition of effector T cell migration into the brain has also been implicated as one of the potential causes in the reactivation of the JC virus and PML development during natalizumab treatment (discussed in the next section). Although natalizumab inhibits both 4 1 and 4 7 -mediated interactions with VCAM-1 and MAdCAM-1 respectively, the following evidence briefly discussed here, suggests T cell migration into the brain is likely mediated by 4 1 /VCAM-1 but not 4 7 /MAdCAM-1 interactions. Blockade with monoclonal antibodies against 4 and VCAM-1, but not against 7 or 4 7, inhibited development of experimental autoimmune encephalomyelitis (EAE); (the encephalitogenic T cells expressed both 4 integrins) [70-73]. Furthermore, studies in a rat model of EAE showed equal efficacy (and a similar pharmacological effect on circulating lymphocytes) of an 4 1 selective small molecule antagonist and a dual anti- 4 antibody, suggesting the mechanism of action for the dual inhibitor was the inhibition of 4 1 and not 4 7 [74]. In addition, the expression of 1 in encephalitogenic T cells was shown to be required for development of experimental autoimmune encephalomyelitis in mice as 1 was required for T cell trafficking to the brain but not for T cell proliferation or activation [75]. In spite of these data some studies have suggested that antibodies against MAdCAM-1 and 7, as well as 7-deficient mice, partially protected against murine EAE. In these studies however, disease inhibition was greater with anti- 4 blockade when compared to the effect of anti- 7 antibodies. Lastly anti- 4 mabs inhibited EAE induced in the 7 deficient mice suggesting a predominant role for 4 1 [76, 77]. Importantly, no MAdCAM-1 expression was detected in normal or inflamed brain from control and MS human samples [78] or in the CNS of normal humans or cynomolgus monkeys or EAE cynomolgus monkeys [11, 79]. BIOLOGIC THERAPIES TARGETING INTEGRIN 4 7 FUNCTION FOR THE TREATMENT OF INFLAMMATORY BOWEL DISEASE There are currently four monoclonal antibodies targeting blockade of MAdCAM-1 interactions which are either in the clinic or in clinical studies for the treatment of IBD. Of the three antibodies that bind the integrin 4 7 and block its function, only vedolizumab (previously known as MLN0002, MLN02, LDP02; Millennium Pharmaceuticals Inc.: The Takeda Oncology Company, Cambridge, MA) selectively inhibits this heterodimer. The other two antibodies that bind 4 7 also bind either 4 1 (Natalizumab, Tysabri, formerly known as Antegren, Elan Pharmaceuticals [Elan; San Francisco, CA] and Biogen Idec [Biogen; Cambridge, MA]) or E 7 (rhumab beta7, Genentech, CA) (Table 1 and Fig. 2). The fourth monoclonal antibody that was in clinical development, PF , selectively binds to human MAdCAM-1 and inhibits its binding to 4 7 (Pfizer, Groton, CT). Natalizumab (Tysabri) is a humanized IgG4 monoclonal antibody in clinical use for the treatment of relapsing forms of multiple sclerosis and moderate to severe active Crohn s disease. It binds to the 4 subunit of the integrins 4 1 and 4 7 and as mentioned before, blocks adhesion to VCAM-1 and MAdCAM-1, respectively, thereby inhibiting lymphocyte infiltration into the brain (and other tissues) and into GI tract, respectively. Natalizumab also blocks leukocyte cell adhesion to the extracellular matrix by disrupting binding of the 4 integrins to one of the protein components of the ECM, a fibronectin splice variant containing the CS-1 peptide. Natalizumab was shown to be effective for the treatment of multiple sclerosis [80, 81] and

5 122 Current Immunology Reviews, 2012, Vol. 8, No. 2 Soler-Ferran and Briskin Table Antagonist Programs in Clinical Development Company Name Inhibitor Type Target Mechanism of Action Disease Clinical Status Biogen/Elan Natalizumab (Tysabri ) MAb (humanized IgG4) 4 ( 4 1/ 4 7) MS, CD Marketed Millennium/Takeda Vedolizumab (LDP-02, MLN-02, MLN0002) MAb (humanized IgG1) 4 7 UC, CD Phase 3 Genentech/Roche rhumabbeta7 (RG7413) MAb (humanized IgG2) 7 ( 4 1/ 4 7) UC Phase 1 ELAN ELND-004 Small molecule 4 7? UC, CD Phase 1 Ajinomoto AJM-300 Small molecule 4 1/ 4 7 CD Phase 3 Ajinomoto AJM-300 Small molecule 4 1/ 4 7 UC Phase 2 GSK Mitsubishi/Tanabe pharma SB (Firategrast) Small molecule 4 1/ 4 7 MS, CD Phase 2 UCB, Inc./Biogen IDEC CDP-323 Small molecule 4 1/ 4 7 MS Phase 2 Encysive/Schering Plough/Merck TBC-4746 Small molecule 4 1/ 4 7 Asthma Phase 1 gained accelerated FDA approval for the treatment of relapsing forms of MS in November RA/MS Tysabri IBD MLN0002 IBD??/?? a4 b1 a4 b7 ae b7 rhumab Beta7 Fig. (2). Delineation of relevant integrin pairs related to mucosal and non-mucosal homing/retention. The 4 integrin can pair with two beta chains, namely 1 and 7. Expression of 4 1 correlates with homing to non-intestinal lymphoid tissues as well as non-intestinal sites of inflammation, while 4 7 homing is largely restricted to the GI tract. Antibodies that inhibit 4 function, such as Tysabri, can block migration of both of these heterodimers, and as shown in the clinic, have therapeutic utility in a wider range of disease indications; albeit with potentially greater risks of immunosuppression. Blocking 4 7 specifically, as in the case of vedolizumab, will largely target GI homing lymphocytes (as well as other leukocytes that express 4 7 ) and in doing so has more limited therapeutic utility, but with perhaps a greater safety profile. The 7 integrin can also pair with another alpha chain, namely E. E 7 expression is associated with mucosal associated leukocytes including intraepithelial lymphocytes (as well as a subset of dendritic cells that are associated with education of gut-homing lymphocytes, some with potential regulatory properties). MAbs that target the 7 chain alone, like rhumabbeta7, may affect both 4 7 interactions with MAdCAM-1 as well as E 7 interactions with its counter-ligand E-cadherin. Whether or not this approach has greater therapeutic utility than specific 4 7 inhibition remains to be seen. Early clinical studies had also suggested that Natalizumab may be effective for the treatment of Crohn s disease [82, 83]. The phase 3 study ENACT, consisted of an induction trial, ENACT-1 (Efficacy of Natalizumab as Active Crohn s Therapy) and a maintenance trial, ENACT-2 (Evaluation of Natalizumab as Continuous Therapy) [84]. In the induction trial, 905 moderately to severe active Crohn s disease patients were randomly assigned to receive 3 doses at 0, 4 and 8 weeks in a 4:1 ratio of 300 mg of natalizumab or placebo. The primary end point was induction of response as measured by a decrease of 70 points in the CDAI (Crohn s Disease Activity Index) score at week 10. A secondary outcome was disease remission, marked by a CDAI below 150. The natalizumab and placebo groups had similar rates of response (56 percent and 49 percent, respectively; P=0.05) and remission (37 percent and 30 percent, respectively; P=0.12). In contrast, statistically significant differences in rates of response or remission for natalizumab versus placebo were achieved in subgroups of patients with either an elevated concentration of C-reactive protein (CRP) at baseline (660 patients), or that had previously received anti-tnf- therapy (358 patients), or with active disease despite the use of immunosuppressants (330). The rates of response or remission where higher among patients receiving natalizumab that those receiving placebo in the patient groups that did not receive prior anti- TNF- therapy or were not concomitantly treated with immunosuppressants, but these differences were not significant. No differences between the natalizumab and placebo groups were observed in patients with normal concentrations of CRP. For the maintenance trial, 339 patients that had a response to natalizumab in the induction trial were randomly reassigned to either 300 mg of natalizumab or placebo every 4 weeks through week 56. The primary outcome was a sustained response through week 36. A secondary outcome was sustained remission through week 36. The rates of sustained response (61 percent versus 28 percent; P<0.001) and disease remission (44 percent versus 26 percent, P=0.003) through week 36 were significantly higher in the patients that continued on natalizumab versus the ones that were switched to placebo. In another induction phase 3 study, Encore (Efficacy of Natalizumab in Crohn s Disease Response and Remission), 509 patients with moderate to severe active Crohn s disease and active inflammation characterized by elevated concentrations of CRP (> 2.87 mg/l), were randomly assigned to receive 300 mg of natalizumab or placebo in a 1:1 ratio at weeks 0, 4 or 8 [85]. The primary outcome was

6 Integrin 4 7 Antagonists Current Immunology Reviews, 2012, Vol. 8, No induction of response ( 70 point decrease from baseline in CDAI at week 8 and sustained through week 12). Secondary efficacy end points included induction of sustained remission (CDAI <150) and response or remission over time. Rates of sustained response and remission were significantly higher in patients that received natalizumab as compared to those that received placebo (48 percent vs 32 percent; P=0.001 and 26 percent vs 16 percent; P=0.002) respectively. Natalizumab induced response and remission at weeks 4, 8 and 12, demonstrating early and sustained efficacy as induction therapy for active Crohn s disease patients with elevated CRP. Natalizumab was well tolerated in moderate to severe CD patients with no increased rates of serious adverse events. In combined safety data for several MS or CD natalizumab studies there was no increased incidence of lymphomas or other cancers, autoimmune or cardiovascular disease in the natalizumab-treated patients [80-87]. However, one CD patient in an open-label extension study in the ENACT trial, who had been treated with 8 doses of natalizumab over 18 months and received multiple immunosuppressant therapies, and two MS patients in clinical trials, who had been treated with combined natalizumab and interferon beta-1 therapy for over 2 years, developed progressive multifocal leukoencephalopathy (PML), a rare but frequently fatal brain disease caused by the JC polyomavirus [88-92]. Two of these patients died and Tysabri was voluntarily removed from the market in February Early investigations determined the risk for PML to be 1 in 1000 patients. Natalizumab was reintroduced to the market in June 2006 and was also approved for treatment of moderate to severe active Crohn s disease in the United States (but not in Europe) in January Natalizumab is approved only as monotherapy and its distribution restricted by a risk minimization program called TOUCH (Tysabri Outreach Unified Commitment to Health) prescribing program. The program restricts distribution and closely monitors patients for the occurrence of PML and other serious opportunistic infections. New safety information released by the FDA in early February 2010 [93] will be included in the Tysabri label and the patient medication guide to maximize the safe use of Tysabri. This new information is based on 31 new confirmed cases of PML worldwide in natalizumab-treated patients (8 of these have died) since marketing resumption in July 2006 and through January 21, The risk of acquiring PML increases with the number of infusions received. No PML cases have been reported in patients with less than 12 months of therapy. In patients treated for 24 to 36 months (24 to 36 infusions), the overall rates of developing PML worldwide and in the U.S. are 1.3 and 0.8 in 1000 patients respectively; the rate in Europe is 1.9 in 1000; the reason for this difference is not understood. There is scant clinical experience beyond 36 month either from clinical trials or in the postmarketing setting. Approximately, 66,000 people worldwide have received at least one infusion of Tysabri since resumption of marketing and through the end of The new label includes a warning and precaution on the occurrence of IRIS (Immune Reconstitution Inflammatory Syndrome) in patients that develop PML and who undergo subsequent removal of Tysabri. Based on current information, the FDA believes that the clinical benefits of using Tysabri outweigh its potential risks. PML is a rare and often fatal demyelinating disease of the white matter of the brain caused by lytic infection of oligodendrocytes by the JC polyoma virus. Exposure to the JC virus occurs in childhood and is widespread in the adult population as indicated by the high incidence of JC virus seropositivity (~80%). Viral latency and reactivation are poorly understood, but reactivation occurs only in severely immune-compromised patients such as the HIV population, which carries the highest incidence of PML (~3-5%); organ transplant recipients; or those with hematologic malignancies, although the incidence in these populations is much lower than that in the HIV population [94]. In addition to the cases described above for natalizumab, PML in patients with autoimmune disease has newly emerged in the setting of other new biological immune-modulatory therapies. PML cases have been reported in severe plaque psoriasis patients treated with efalizumab (Raptiva ), another anti-integrin antibody (CD11a) which inhibits migration of T cells into tissues, and in patients with rheumatoid arthritis and lymphomas treated with rituximab (Rituxan ), a B-cell lytic anti-cd20 antibody [95, 96]. The mechanisms by which natalizumab may reactivate the JC virus and cause PML are not fully understood but several mechanisms have been proposed. It is likely that a combination of factors related to the mechanism of action of natalizumab and to the biology and pathogenicity of the JC virus come into play. Decreased immunosurveillance in the CNS and other tissues and mobilization of hematopoietic cells with a latent infection from the bone marrow might come together to reactivate the virus. Ransohoff was the first to propose that the effects of nataluzimab on the bone marrow might be one of the key contributing factors to viral reactivation [97]. Cumulative evidence suggests lymphoid tissues and bone marrow cells including B cells and CD34 + hematopoietic stem cells are sites of viral latency and important players in the pathogenesis of JCV [94, ]. Virus recovered from hematopoietic infected cells infects glial cells suggesting that a plausible mechanism for the pathogenesis of JCV brain infection is a reactivation of the bone marrow reservoir under conditions of immune suppression with resulting viremia and brain infiltration of either cell-bound or free virus. Natalizumab administration to primates and humans results in a rapid, sustained and significant elevation in the numbers of all cells that express 4 1 in the circulation including B, CD4 + and CD8 + T lymphocytes, NK cells, monocytes, basophils and eosinophils. Neutrophils-- which do not express 4 1, are not elevated [81, 82, 84, 92]. This leukocytosis might result from inhibition of leukocyte extravasation and retention into multiple tissues including the bone marrow. Inhibition of migration to the bone marrow as well as mobilization of cells retained in the bone marrow by 4 1 /VCAM-1 adhesion is also likely to contribute to this elevation. A wide body of scientific evidence points to a predominant role for 4 1 /VCAM-1 interactions in hematopoiesis, homing and retention of hematopoietic progenitors, B and T lymphocytes, and dendritic cells in the bone marrow [ ]: 1) Conditional VCAM-1 deletion in mice (conventional VCAM-1 deficiency in mice results in

7 124 Current Immunology Reviews, 2012, Vol. 8, No. 2 Soler-Ferran and Briskin embryonic lethality) resulted in decreased numbers of immature B cells and mature B and T cells in the bone marrow with a concomitant increase in immature B cells and leukocytosis in blood. These mice exhibited a profound impairment of bone marrow homing of transferred mature T and B lymphocytes and a defect in T-cell dependent humoral response [106, 107]. 2) Furthermore, 4 deficiency in the hematopoietic compartment of chimeric mice resulted in a severe defect in B cell development as well as compromised general lymphopoiesis. These mice had decreased B cell progenitors in the bone marrow, hematopoietic progenitors in the circulation, and lymphocytosis [108]. 3) Mouse and human hematopoietic progenitors and lymphocyte precursors bind to VCAM-1 expressed on bone marrow stromal cells and anti-vcam-1 and anti- 4 antibodies inhibit lymphopoiesis and erythropoiesis in bone marrow cultures [ ]. Attachment of hematopoietic progenitors to bone marrow stroma also occurs through binding of 4 1 to the connecting segment 1 (CS-1) of alternatively spliced fibronectin in the extracellular matrix (ECM). Binding is blocked by anti- 4 and anti- 1 MAbs. Anti- 1 1 antibodies also block medullary hematopoiesis in vivo following infusion of bone marrow cells [114]. A role for 4 integrin in the attachment of hematopoietic progenitors to stromal cells in the bone marrow was confirmed by the release of progenitors to the circulation upon 4 blockade in mice and primates [105, 115]. Accordingly, natalizumab treatment mobilizes CD34 + hematopoietic progenitors in humans [116, 117]. Furthermore, the bone marrow has been shown to be a major reservoir and site of activation for central memory CD8 + T lymphocytes by dendritic cells and induction of recall responses as well as a site of long-term persistence of antiviral memory cells [103, 104, 118]. Intravital microscopy demonstrated a major role for 4 1 (but not 4 7 ) adhesion to VCAM-1 on bone marrow microvessels in homing of memory CTLs and dendritic cells. Disruption of 4 1 binding to VCAM-1 by a dual 4 antagonist such as natalizumab might result in viral reactivation not only by the release of infected bone marrow B cells and CD34 + progenitors but by interfering with a CTL response in the bone marrow that might be critical to keep the virus in check. Long-term persistence of anti-viral T cell responses might also explain why it takes over a year of Tysabri infusions for the virus to be reactivated. The rate of viral reactivation associated with natalizumab as well as the proposed increased JC viremia associated with natalizumab administration [119] may be explained by one or several of these effects of the mechanism of action of natalizumab. In addition, PML development may occur because of inhibition of CNS immune surveillance. JC virus specific CD8 + Cytotoxic T lymphocyte (CTL) and CD4 + Helper T lymphocyte immune responses correlate with control of PML disease progression [ ]. It has then been proposed that blockade of infiltration of effector virus-specific CTL and CD4 + cells into the brain, the effector site of action, and inhibition of CNS immune surveillance are likely to be key mechanisms contributing to PML development by natalizumab. Consistent with the major role that 4 1 /VCAM-1 interactions play in migration of lymphocytes to the brain, natalizumab has been shown to interfere with lymphocyte infiltration into the brain [69, 73]. Additionally, Stuve and colleagues demonstrated that natalizumab reduces the number of cerebrospinal fluid (CFS) mononuclear cells including CD4 +, CD8 +, B lymphocytes and plasma cells [123]. So it is possible that natalizumab may cause PML from impaired lymphocyte responses in the brain in combination with viral reactivation at other sites. Elan Pharmaceuticals is directing an observational study (CD INFORM NCT ) to determine the incidence of serious infections, malignancies or other SAEs (serious adverse events) in Crohn s disease patients treated with natalizumab. The molecular mechanisms proposed for viral reactivation and PML development by natalizumab implicate 4 1 /VCAM-1 interactions and not 4 7 due to the latter's more restricted tissue-tropism to the GI tract. Consequently, selective 4 7 inhibitor therapies in IBD treatment might carry less risk of opportunistic infections while still achieving a comparable level of efficacy. Vedolizumab is a humanized IgG1 monoclonal antibody selective for the integrin 4 7 currently in phase 3 clinical development for the treatment of UC and CD sponsored by Millennium Pharmaceuticals Inc and the Takeda Pharmaceutical Company. The implied mechanism of action of vedolizumab is the inhibition of adhesion and migration hi of 4 7 memory T lymphocytes to MAdCAM-1 expressed on endothelial cells of the GI tract and associated lymphoid tissues. Vedolizumab was engineered from the murine antibody Act-1 which was shown to bind an epitope that is accessible only to the heterodimer 4 7 and fails to bind to the other two integrins expressing the 4 or 7 subunits, namely 4 1 and E 7, respectively (Fig. 2). Vedolizumab was shown to exhibit the same ligand specificity, affinity and inhibitory potency and selectivity as Act-1 [37]. It binds most brightly to a small subset (approximately 25%) of memory CD4 + T lymphocytes (which contain some T-helper 17 lymphocytes expressing IL17, a cytokine recently implicated in IBD pathogenesis). Vedolizumab does not bind the vast majority of memory helper T cells, nor does it bind neutrophils or the vast majority of monocytes in human peripheral blood. Vedolizumab also binds at high levels to peripheral blood eosinophils and to a lesser extent naive T- helper, naïve and memory cytotoxic T and B lymphocytes as well as natural killer cells and basophils. Vedolizumab binds to peripheral blood 4 hi 7 memory CD4 cells and B cells with high affinity (EC 50 =0.05 μg/ml) and inhibits binding of hi human MAdCAM to whole blood activated 4 7 CD4 memory cells with similar potency [37]. It also blocks 4 7 adhesion to fibronectin but not to VCAM-1. Vedolizumab does not mediate complement dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC). Phase 2 clinical trials have demonstrated therapeutic activity of vedolizumab in ulcerative colitis (UC) and Crohn s disease (CD). Vedolizumab is currently in phase 3 clinical development for the treatment of UC and CD. In an exploratory Phase 2 study in patients with mild to moderately active ulcerative colitis, treatment with 2 mg/kg of vedolizumab was well tolerated and induced complete remission in some patients [124]. A second well-powered phase 2 study demonstrated that treatment with vedolizumab was safe and efficacious in the induction of clinical and endoscopic remission [125]. This randomized, double-blind placebo-controlled multicenter study was carried out in 181 patients with moderately to severely active ulcerative colitis

8 Integrin 4 7 Antagonists Current Immunology Reviews, 2012, Vol. 8, No (ulcerative colitis clinical score from 5 to 9 on a scale ranging from 0 to 12, that included a score of at least 1 for rectal bleeding or stool frequency; in addition patients had to have a baseline modified Baron score (endoscopic evaluation) of at least 2 on a scale of 0 to 4). Patients were randomly assigned to receive intravenous infusions of either 0.5 or 2 mg/kg of body weight of MLN-02 (a vedolizumab precursor with identical potency and binding specificity) or placebo at days 1 and 29. Some patients also received concomitant mesalamine. Patients were evaluated at 6 weeks, the primary study end point, by ulcerative colitis clinical scores (clinical activity) and modified Baron score (endoscopic activity). The primary outcome was clinical remission at week 6, as defined by an ulcerative colitis clinical score of 0 to 1 and a modified Baron score of 0 to 1 with no rectal bleeding. Secondary outcomes included the proportion of patients with a clinical response (a decrease of at least 3 points on the ulcerative colitis clinical score), endoscopically evident remission (modified Baron score of 0), endoscopic response (an improvement of the modified Baron score of at least 2) at weeks 4 and 6. Other secondary end points were changes in the ulcerative colitis clinical scores, modified Baron scores, Riley s histopathology score (from 0 to 7 on biopsies) and the scores on the inflammatory bowel disease questionnaire on health-related quality of life (32 to 224). Clinical remission rates at week 6 were 33 percent, 32 percent and 14 percent for the groups receiving 0.5 mg/kg of MLN-02, 2 mg/kg of MLN-02 and placebo respectively (P=0.03). The corresponding proportion of patients that experienced clinical response (a minimum 3 points improvement in ulcerative colitis clinical scores), were 66 percent, 53 percent and 33 percent (P=0.002). Endoscopically evident remission was achieved by 28 percent, 12 percent and 8 percent of those receiving 0.5 mg of vedolizumab, 2 mg of vedolizumab and placebo, respectively (P=0.007). Furthermore, patients who received 0.5 or 2 mg/kg of MLN- 02 were more likely to improve 2 or more points in their endoscopic scores than patients who received placebo (48 percent and 35 percent respectively, vs 16 percent; P=0.001). Patients receiving MLN-02 had greater improvements in the inflammatory bowel disease questionnaire (IBDQ), the ulcerative colitis clinical score and the Riley score. Moreover, endoscopic, histopathological improvements, and health-related quality of life questionnaire improvements were more frequent among patients that were assigned to active treatment. In summary, treatment with vedolizumab resulted in a clinically significant benefit in the induction of clinical remission in this short study with patients with active ulcerative colitis. No significant differences in adverse effects were observed among the three groups including no deaths, cancers or opportunistic infections. Most common adverse events were exacerbations of ulcerative colitis, nausea and headache. Serious adverse events included an infusion reaction which occurred only in one patient of the 103 that received MLN-02. This patient tested positive for high titers of anti-mln-02 antibodies. Another patient developed a primary cytomegalovirus infection which resolved without treatment and was not consistent with a viral reactivation that might be seen in a setting of compromised immunity. Lobar pneumonia developed postoperatively in a patient and was successfully treated. No differences in laboratory results were identified among the treatment groups. Importantly, no changes were observed in the total blood lymphocyte, T and B lymphocyte counts between patients receiving vedolizumab or placebo, a noteworthy finding that informs on the mechanism of action of MLN-02 as compared to that of natalizumab. Human antihuman antibodies were observed in 44 percent of patients that received MLN-02 with 24 percent (11 percent in the 2 mg/kg group and 38 percent in the 0.5 mg/kg group) developing high enough antibody titers to affect saturation of 4 7 receptors in the memory CD4 + population. Patients who lost saturation of 4 7 receptors in the memory CD4 + population, not surprisingly, lost the statistically significant benefit on efficacy versus patients receiving placebo (12 percent in clinical remission vs 14 percent in the placebo group). In contrast, in the 76 percent of patients with undetectable or low antibody titers, receptor saturation was greater than 90 percent, and the rates of clinical remission were 42 percent versus 14 percent in the placebo group). The serum half -lives were 9 and 12 days for the 0.5 and 2 mg/kg doses, respectively. A phase 2 study in patients with active Crohn s disease suggested a dose dependent beneficial effect of vedolizumab in inducing clinical remission [18]. Vedolizumab was well tolerated in this patient population. Patients with moderate to severe active Crohn s disease (CDAI ranging from 220 to 400 on a scale from 0 to 600) were randomly assigned to receive an intravenous infusion of 0.5 mg/kg vedolizumab, 2 mg/kg vedolizumab or placebo at days 1 and 29. Patients were stratified by mesalamine usage, the only permitted concomitant medication in addition to antibiotics. The primary efficacy endpoint was clinical response, defined as a decrease in the CDAI of 70 points on day 57. Secondary efficacy end points included clinical remission (absolute CDAI 150 points) and enhanced clinical response (defined by a decrease of 100 points in the CDAI) at day 57 among others. The proportions of patients with clinical response at day 57 were 49 percent, 53 percent and 41 percent in the 0.5 mg/kg vedolizumab, 2 mg/kg vedolizumab and placebo groups respectively. These differences however, were not statistically significant. At day 15 a statistically significant difference in the rate of clinical response of the 2 mg/kg vedolizumab group as compared to the placebo group was observed, however this difference was not sustained over time despite a continued rising rate of MLN-02 treated patients achieving response, as the delta was reduced due to placebo patients achieving clinical response at later time points. In addition, the median time to clinical response was significantly different in the 2 mg/kg group as compared to the placebo group (17 days vs 42 days; P=0.04). A clinically relevant benefit of vedolizumab was further suggested by the dose-dependent trend in the more stringent secondary end points of enhanced clinical response and clinical remission. The proportions of patients in clinical remission at day 57 were 30 percent, 37 percent and 21 percent for the 0.5 mg/kg vedolizumab, 2 mg/kg vedolizumab and placebo groups, respectively (P=0.04 for the 2 mg/kg vs placebo comparison). Statistically significant differences in remission rates between the 2 mg/kg vedolizumab and the placebo groups occurred also at days 15 and 29. The proportions of patients achieving the more stringently defined enhanced clinical response at day 57