HK1230521B - Methods for diagnosing and treating inflammatory bowel disease - Google Patents

Description

Methods for diagnosing and treating inflammatory bowel disease

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 61/971,379, filed March 27, 2014, which is hereby incorporated by reference in its entirety.

Sequence Listing

This application contains a Sequence Listing, which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. This ASCII copy, created on March 5, 2015, is named P5817R1-WO_SL.txt and is 20,156 bytes in size.

Technical Field

Provided are biomarkers that predict responsiveness to integrin beta7 antagonists (including anti-beta7 integrin subunit antibodies) and methods of using such biomarkers. Furthermore, provided are methods of treating gastrointestinal inflammatory disorders (such as inflammatory bowel disease, including ulcerative colitis and Crohn's disease). Also provided are methods of using such predictive biomarkers for treating inflammatory bowel disease, including ulcerative colitis and Crohn's disease.

Background Art

Inflammatory bowel disease (IBD) is a chronic inflammatory autoimmune disorder of the gastrointestinal (GI) tract that manifests clinically as ulcerative colitis (UC) or Crohn's disease (CD). CD is a chronic transmural inflammatory disease with the potential to affect any part of the entire GI tract, while UC is an inflammation of the mucosa of the colon. Both conditions are clinically characterized by frequent bowel movements, malnutrition, and dehydration, with disruption of daily living activities. CD is often complicated by the development of malabsorption, strictures, and fistulas, which may require repeated surgery. Less commonly, UC can be complicated by severe bloody diarrhea and toxic megacolon, which also require surgery. Both IBD conditions are associated with an increased risk of gastrointestinal malignancies. The etiology of IBD is complex, and many aspects of its pathogenesis remain unclear.

The treatment of moderate to severe IBD presents a significant challenge to treating physicians, as conventional therapy with corticosteroids and immunomodulatory therapy (e.g., azathioprine, 6-mercaptopurine, and methotrexate) is associated with side effects and intolerance and has not shown a proven benefit in maintenance therapy (steroids). Monoclonal antibodies targeting tumor necrosis factor alpha (TNF-α), such as infliximab (a chimeric antibody) and adalimumab (a fully human antibody), are currently used to treat CD. Infliximab has also shown efficacy in UC and is approved for use in UC. However, approximately 10%-20% of CD patients are primary non-responders to anti-TNF therapy, and an additional ~20%-30% of CD patients lose response over time (Schnitzler et al., Gut 58:492-500 (2009)). Other adverse events (AEs) associated with anti-TNF include increased rates of bacterial infections (including tuberculosis) and, more rarely, lymphomas and demyelination (Chang et al., Nat Clin Pract Gastroenterol Hepatology 3:220 (2006); Hoentjen et al., World J. Gastroenterol. 15(17):2067 (2009)). Currently, no available therapy achieves sustained remission in more than 20%-30% of IBD patients with chronic disease (Hanauer et al., Lancet 359:1541-49 (2002); Sandborn et al., N Engl J Med 353:1912-25 (2005)). Furthermore, most patients do not achieve sustained steroid-free remission and mucosal healing (clinical outcomes associated with true disease change). Therefore, there is a need to develop more targeted IBD therapies optimized for long-term use: an improved safety profile with sustained remission (especially steroid-free remission) and prevention of long-term complications in a larger proportion of patients, including those who never respond to anti-TNF therapeutics or who lose response over time (TNF-inadequate responders or TNR-IR patients).

Integrins are α/β heterodimeric cell surface glycoprotein receptors that play a role in many cellular processes, including leukocyte adhesion, signaling, proliferation, and migration, as well as in gene regulation (Hynes, R.O., Cell, 1992, 69:11-25; and Hemler, M.E., Annu. Rev. Immunol., 1990, 8:365-368). They are composed of two heterodimeric, non-covalently interacting α and β transmembrane subunits that specifically bind to different cell adhesion molecules (CAMs) on endothelial and epithelial cells, as well as extracellular matrix proteins. In this manner, integrins can function as tissue-specific cell adhesion receptors that assist in the highly regulated recruitment of leukocytes from the blood to nearly all tissue sites, playing a role in leukocyte homing to normal tissues and sites of inflammation (von Andrian et al., N Engl J Med 343:1020-34 (2000)). In the immune system, integrins are involved in leukocyte trafficking, adhesion, and infiltration during inflammation (Nakajima, H. et al., J. Exp. Med., 1994, 179: 1145-1154). Differential expression of integrins regulates the adhesion properties of cells, and different integrins are involved in different inflammatory responses (Butcher, E.C. et al., Science, 1996, 272: 60-66). β7-containing integrins (i.e., α4β7 and αEβ7) are primarily expressed on monocytes, lymphocytes, eosinophils, basophils, and macrophages, but not on neutrophils (Elices, M.J. et al., Cell, 1990, 60: 577-584).

The α4β7 integrin is an important leukocyte homing receptor in cell migration to the intestinal mucosa and associated lymphoid tissues (such as the Peyer's patches in the small intestine, the lymph nodes in the large intestine, and the mesenteric lymph nodes). In the intestine, leukocyte tumbling and tight adhesion to the mucosal endothelium is initiated by signals from chemokines and mediated by mucosal addressin cell adhesion molecule (MAdCAM)-1 related sialyl Lewis X. Chemokine signaling induces the α4β7 integrin to undergo a change from low MAdCAM-1 binding affinity to high MAdCAM-1 binding affinity. The leukocytes then arrest and begin the process of extravasation through the vascular endothelium to the underlying tissues. This extravasation process is believed to occur both in normal immune cell recycling states and in inflammatory conditions (von Andrian et al., supra). The number of α4β7 ⁺ cells in the infiltrate and the expression of the ligand MAdCAM-1 are higher in sites of chronic inflammation such as the intestine of patients with UC or CD (Briskin et al., Am J Pathol 151:97-110 (1997); Souza et al., Gut 45:856-63 (1999)). α4β7 preferentially binds to high endothelial venules that express MAdCAM-1 and vascular cell adhesion molecule (VCAM)-1, as well as the extracellular matrix molecule fibronectin fragment CS-1 (Chan et al., J Biol Chem 267:8366-70 (1992); Ruegg et al., J Cell Biol 17:179-89 (1992); Berlin et al., Cell 74:185-95 (1993)). The α4β7 integrin plays a selective role in leukocyte intestinal tropism together with MAdCAM-1, which is constitutively expressed in intestinal mucosal vasculature, but does not appear to contribute to leukocyte homing to peripheral tissues or the CNS. In contrast, peripheral lymphatic trafficking is associated with the interaction of α4β1 with VCAM-1 (Yednock et al., Nature 356:63-6 (1992); Rice et al., Neurology 64:1336–42 (2005)).

Another member of the β7 integrin family that is expressed only on T lymphocytes and associated with mucosal tissues is the αEβ7 integrin, or CD103. αEβ7 integrin selectively binds to E-cadherin on epithelial cells and has been proposed to play a role in the retention of T cells in the intraepithelial lymphocyte compartment in mucosal tissues (Cepek et al., J Immunol 150:3459-70 (1993); Karecla et al., Eur J Immunol 25:852–6 (1995)). αEβ7 ⁺ cells in the lamina propria have been reported to exhibit cytotoxicity to stressed or infected epithelial cells (Hadley et al., J Immunol 159:3748–56 (1997); Buri et al., J Pathol 206:178–85 (2005)). Expression of αEβ7 is elevated in CD (Elewaut et al., Acta Gastroenterol Belg 61:288–94 (1998); Oshitani et al., Int J Mol Med 12:715–9 (2003)), and anti-αEβ7 antibody treatment has been reported to attenuate experimental colitis in mice, suggesting a role for αEβ7 ⁺ lymphocytes in experimental models of IBD (Ludviksson et al., J Immunol 162:4975–82 (1999)).

It has been reported that administration of anti-αEβ7 monoclonal antibodies prevented and improved immune-induced colitis in IL-2 ^−/− mice, indicating that the onset and maintenance of inflammatory bowel disease depend on the colonic localization of lamina propria CD4 ⁺ lymphocytes expressing αEβ7 (Ludviksson et al., J Immunol. 1999, 162(8):4975-82). It has been reported that anti-α4 antibodies (natalizumab) are effective in the treatment of CD patients (Sandborn et al., N Engl J Med 2005;353:1912-25), and anti-α4β7 antibodies (MLN-02, MLN0002, vedolizumab) are effective in UC patients (Feagan et al., N Engl J Med 2005;352:2499-507). A second anti-α4β7 antibody (AMG181) is also under development, and clinical trials have recently begun (clinicaltrials(dot)gov identifier NCT01164904, September 2012). These studies and findings confirm α4β7 as a therapeutic target and support the idea that the interaction between α4β7 and MAdCAM-1 mediates the pathogenesis of IBD. Therefore, antagonists of the β7 integrin have great potential as therapeutic agents in the treatment of IBD.

Humanized monoclonal antibodies targeting the β7 integrin subunit have been previously described. See, for example, International Patent Publication No. WO2006/026759. One such antibody, rhuMAb β7 (etrolizumab), is derived from the anti-rat mouse/human monoclonal antibody FIB504 (Andrew et al. 1994). It was engineered to contain a human IgG1 heavy chain and a κ1 light chain framework. International Patent Publication No. WO2006/026759. Administration of etrolizumab to human patients according to certain dosing regimens has been previously described. See, for example, International Patent Publication No. WO/2012/135589.

RhuMAb β7 (etrolizumab) binds to α4β7 (Holzmann et al., Cell 56:37-46 (1989); Hu et al., Proc Natl Acad Sci USA 89:8254-8 (1992)) and αEβ7 (Cepek et al., J Immunol 150:3459-70 (1993)), which regulate the trafficking and retention of lymphocyte subsets, respectively, in the intestinal mucosa. Clinical studies have demonstrated the effectiveness of anti-α4 antibodies (natalizumab) in the treatment of CD (Sandborn et al., N Engl J Med 353:1912-25 (2005)), and encouraging results have been reported for anti-α4β7 antibodies (LDP02/MLN02/MLN0002/vedolizumab) in the treatment of UC (Feagan et al., N Engl J Med 352:2499-507 (2005), Feagan et al., N Engl J Med 369(8):699-710 (2013)) and CD (Sandborn et al., N Engl J Med 369(8):711-721 (2013)). These findings help identify α4β7 as a potential therapeutic target and support the hypothesis that the interaction between α4β7 and mucosal addressin cell adhesion molecule 1 (MAdCAM 1) contributes to the pathogenesis of inflammatory bowel disease (IBD).

Unlike natalizumab, which binds to α4 and, therefore, to both α4β1 and α4β7, rhuMAbβ7 specifically binds to the β7 subunit of both α4β7 and αEβ7, but not to the individual α4 or β1 integrins. This is demonstrated by the antibody's inability to inhibit the adhesion of α4β1+α4β7–Ramos cells to vascular cell adhesion molecule 1 (VCAM 1) at concentrations as high as 100 nM. Importantly, this characteristic of rhuMAbβ7 suggests selectivity: T cell subsets that express α4β1 but not β7 should not be directly affected by rhuMAbβ7.

Support for the intestinal-specific effect of rhuMAb β7 on lymphocyte homing comes from several in vivo nonclinical studies. In severe combined immunodeficiency (SCID) mice reconstituted with CD45RB ^high CD4+ T cells (an animal model of colitis), rhuMAb β7 blocked the homing of radiolabeled lymphocytes to the inflamed colon, but did not block homing to the peripheral lymphoid organ spleen. See, for example, International Patent Publication No. WO2006/026759. In addition, rat-mouse chimeric anti-murine β7 (anti-β7, muFIB504) was unable to reduce the histological extent of central nervous system (CNS) inflammation or improve disease survival in myelin basic protein T cell receptor (MBP-TCR) transgenic mice (an animal model of multiple sclerosis) with experimental autoimmune encephalitis (EAE). Id. In addition, in two safety studies in cynomolgus monkeys, rhuMAb β7 induced a moderate increase in the number of peripheral blood lymphocytes, which was primarily caused by a significant (approximately 3- to 6-fold) increase in CD45RA-β7 ^high peripheral blood T cells (a subset phenotypically similar to gut-homing memory/effector T cells in humans). See, for example, International Patent Publication No. WO 2009/140684; Stefanich et al., Br. J. Pharmacol. 162: 1855-1870 (2011). In contrast, rhuMAb β7 had minimal or no effect on the number of CD45RA+β7 peripheral blood T cells (a subset phenotypically similar to naive T cells in humans) and had no effect on the number of CD45RA-β7 ^low peripheral blood T cells (a subset phenotypically similar to peripheral homing memory/effector T cells in humans), confirming the specificity of rhuMAb β7 for the gut-homing lymphocyte subset. International Patent Publication No. WO 2009/140684; Stefanich et al., Br. J. Pharmacol. 162: 1855-1870 (2011).

Although clinical studies have demonstrated the effectiveness of anti-α4 antibodies (natalizumab) for the treatment of CD (Sandborn et al., NEngl J Med 353:1912-25 (2005)), and encouraging results have been reported for anti-α4β7 antibodies (LDP02/MLN02/MLN0002/vedolizumab) in the treatment of UC, there is still a need for further improvement in the treatment of these disorders. For example, natalizumab treatment is associated with confirmed cases of progressive multifocal leukoencephalopathy (PML) in patients with Crohn's disease (separately, multiple sclerosis) who receive concurrent treatment with natalizumab and immunosuppressants. PML is a potentially fatal neurological disorder associated with reactivation and active viral replication of polyomavirus (JC virus) in the brain. No known intervention can reliably prevent PML or adequately treat PML when it occurs. One limitation of vedolizumab therapy is that it is administered intravenously, which can be inconvenient for the patient and can also be associated with unwanted or adverse events (e.g., infusion site reactions). Therefore, there is a need for improved treatments for gastrointestinal inflammatory disorders such as IBD (e.g., ulcerative colitis and Crohn's disease) and more desirable dosing regimens.

It is often unknown before treatment whether a patient will respond to a particular therapeutic agent or class of therapeutic agents. Therefore, when IBD patients, particularly those with UC and CD, seek treatment, considerable trial and error is involved in finding a therapeutic agent that is effective for a particular patient. This trial and error often involves considerable risk and discomfort for the patient in order to find the most effective therapy. Therefore, there is a need for more effective means for determining which patients will respond to which treatments and incorporating such determinations into more effective treatment regimens for IBD patients.

Therefore, it would be highly advantageous to have additional diagnostic methods, including predictive diagnostic methods, that can be used to objectively identify patients most likely to respond to treatment with various IBD therapeutics, including anti-β7 integrin subunit antibodies. Thus, there is a continuing need to identify new biomarkers that correlate with ulcerative colitis, Crohn's disease, and other inflammatory bowel disorders and predict response to anti-β7 integrin subunit antibody therapy. Furthermore, statistically and biologically significant and reproducible information about such correlations can be used as an integral component in efforts to identify specific subpopulations of UC or CD patients (e.g., TNF-IR patients) that are expected to benefit significantly from anti-β7 integrin subunit antibody therapy, for example, when a therapeutic agent is shown in clinical studies to have therapeutic benefit in such specific UC or CD patient subpopulations.

The invention described herein satisfies some of the above needs and provides other advantages.

All references cited herein, including patent applications and publications, are incorporated by reference in their entirety for any purpose.

SUMMARY OF THE INVENTION

The methods of the present invention are based, at least in part, on the discovery that mRNA expression levels of certain genes in a biological sample obtained from a patient (eg, intestinal biopsy or blood) are predictive of responsiveness of patients with gastrointestinal inflammatory disorders to treatment with an integrin beta7 antagonist.

Thus, in one aspect, methods are provided for predicting the response of a patient with a gastrointestinal inflammatory disorder to a treatment comprising an integrin beta 7 antagonist or for predicting the responsiveness of a patient with a gastrointestinal inflammatory disorder to a treatment comprising an integrin beta 7 antagonist. In certain embodiments, a biological sample is obtained from the patient and mRNA expression levels are measured. In some embodiments, expression of at least one, at least two, at least three, or at least four highly expressed predictor genes ("HEPGs") selected from GZMA, KLRB1, FOXM1, CCDC90A, CCL4, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and SLC8A3 is measured in the sample. In some embodiments, the at least one, at least two, at least three, or at least four HEPGs are selected from GZMA, KLRB1, FOXM1, SLC8A3, and ECH1. In some embodiments, another HEPG other than those listed above is measured, which HEPG is ITGAE. In one embodiment, the biological sample is a tissue biopsy sample. In one embodiment, the biopsy tissue is obtained from intestinal tissue. In some such embodiments comprising a tissue biopsy sample or intestinal tissue, the HEPG does not include SLC8A3. In one embodiment, the biological sample is peripheral whole blood. In some such embodiments comprising peripheral blood, the HEPG includes SLC8A3. In one embodiment, the peripheral whole blood is collected in a PAXgene tube. In certain embodiments, the mRNA expression level is measured by RNA sequencing, microarray or PCR. In one embodiment, the PCR method is qPCR. In certain embodiments, the measurement includes amplifying one or more of GZMA, KLRB1, FOXM1, CCDC90A, CCL4L1.2, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and SLC8A3 mRNA, and optionally further includes amplifying ITGAE mRNA and detecting the amplified mRNA to measure the level of the amplified mRNA. In certain embodiments, the mRNA expression level is compared with a reference level. In some embodiments, the mRNA expression level is compared with a reference level of each HEPG measured. In certain embodiments, each reference level is a median. In some embodiments, when the mRNA expression level of at least one, at least two, at least three or at least four HEPGs selected from GZMA, KLRB1, FOXM1, CCDC90A, CCL4, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2 and SLC8A3 in the sample is increased compared to the reference value of each HEPG measured (which in certain embodiments is the median of each reference value), it is predicted that the patient will respond to treatment comprising an integrin beta 7 antagonist. In one embodiment, the reaction is clinical remission. In one embodiment, the reaction is mucosal healing. In one embodiment, the reaction is a clinical response. In certain embodiments, when the absolute Mayo clinical score is ≤2 and no single subscore is >1, it is determined that remission is induced in the patient, which is also referred to as clinical remission. In certain embodiments, mucosal healing is determined to have occurred when the patient is determined to have an endoscopy subscore of 0 or 1 as assessed by flexible sigmoidoscopy. In certain such embodiments, mucosal healing is determined to have occurred in a patient having an endoscopy subscore of 0. In certain embodiments, a clinical response is determined to have occurred when the patient has a 3-point decrease in MCS and a 30% decrease from baseline and a ≥1-point decrease in rectal bleeding subscore or an absolute rectal bleeding score of 0 or 1.

In another aspect, a method of identifying a patient with a gastrointestinal inflammatory disorder as likely to respond to a treatment comprising an integrin beta 7 antagonist is provided. In certain embodiments, the method comprises: (a) measuring the mRNA expression level of at least one, at least two, at least three, or at least four HEPGs selected from the group consisting of GZMA, KLRB1, FOXM1, CCDC90A, CCL4, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and SLC8A3 in a biological sample from the patient; (b) comparing the mRNA expression level measured in (a) with a reference level for each HEPG measured; and (c) identifying the patient as more likely to respond to a treatment comprising an integrin beta 7 antagonist when the mRNA expression level of each HEPG measured in (a) is higher than the reference level for each HEPG measured. In some embodiments, the at least one, at least two, at least three or at least four HEPGs are selected from GZMA, KLRB1, FOXM1, SLC8A3 and ECH1. In some embodiments, another HEPG other than those listed above is measured, the other HEPG being ITGAE. In one embodiment, the patient is a human. In one embodiment, the patient has not been previously treated with an anti-TNF therapeutic agent. In one embodiment, the gastrointestinal inflammatory disorder is inflammatory bowel disease. In one embodiment, the inflammatory bowel disease is ulcerative colitis or Crohn's disease. In one embodiment, the inflammatory bowel disease is ulcerative colitis, and the response is selected from clinical response, mucosal healing and remission. In certain embodiments, induction of remission in the patient is determined when the absolute Mayo clinical score is ≤2 and no single subscore is >1, which is also referred to as clinical remission. In certain embodiments, mucosal healing is determined to have occurred when the patient is determined to have an endoscopic subscore of 0 or 1 assessed by flexible sigmoidoscopy. In certain such embodiments, the patient in whom mucosal healing is determined to have occurred has an endoscopic subscore of 0. In certain embodiments, a clinical response is determined to have occurred when a patient experiences a 3-point decrease in MCS and a 30% decrease from baseline and a ≥1-point decrease in the rectal bleeding subscore or an absolute rectal bleeding score of 0 or 1.

In another aspect, methods of treating a patient suffering from a gastrointestinal inflammatory disorder are provided. In certain embodiments, the method comprises: (a) measuring mRNA expression of at least one, at least two, at least three, or at least four HEPGs selected from the group consisting of GZMA, KLRB1, FOXM1, CCDC90A, CCL4, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and SLC8A3 in a biological sample from the patient; (b) comparing the mRNA expression level of each HEPG measured in (a) with a reference level for each HEPG measured; (c) identifying the patient as more likely to respond to a treatment comprising an integrin beta 7 antagonist when the mRNA expression level of each HEPG measured in (a) is higher than the respective reference level for each HEPG measured; and (d) administering the treatment when the mRNA expression level of each HEPG measured in (a) is higher than the respective reference level for each HEPG measured, thereby treating the gastrointestinal inflammatory disorder. In some embodiments, the at least one, at least two, at least three, or at least four HEPGs are selected from GZMA, KLRB1, FOXM1, SLC8A3, and ECH1. In some embodiments, another HEPG other than those listed above is measured, the other HEPG being ITGAE. In one embodiment, 105 mg of an integrin beta 7 antagonist is administered subcutaneously once every four weeks. In one embodiment, an initial dose of 210 mg of the integrin beta7 antagonist is administered subcutaneously, followed by subsequent doses, each 210 mg subsequent dose of the integrin beta7 antagonist being administered subcutaneously at each of weeks 2, 4, 8, and 12 after the initial dose. In one embodiment, the patient is a human. In one embodiment, the patient has not been previously treated with an anti-TNF therapeutic. In one embodiment, the gastrointestinal inflammatory disorder is inflammatory bowel disease. In one embodiment, the inflammatory bowel disease is ulcerative colitis or Crohn's disease. In one embodiment, the inflammatory bowel disease is ulcerative colitis, and the response is selected from the group consisting of clinical response, mucosal healing, and remission. In certain embodiments, induction of remission in the patient is determined to be ≤2 with no individual subscore >1, also referred to as clinical remission. In certain embodiments, mucosal healing is determined to have occurred when the patient is determined to have an endoscopic subscore of 0 or 1 as assessed by flexible sigmoidoscopy. In certain such embodiments, a patient determined to have experienced mucosal healing has an endoscopic subscore of 0. In certain embodiments, a clinical response is determined to have occurred when a patient experiences a 3-point decrease in MCS and a 30% decrease from baseline and a ≥1-point decrease in the rectal bleeding subscore or an absolute rectal bleeding score of 0 or 1.

In yet another aspect, a method of predicting a response of a patient with a gastrointestinal inflammatory disorder to a treatment comprising an integrin beta 7 antagonist or predicting responsiveness of a patient with a gastrointestinal inflammatory disorder to a treatment comprising an integrin beta 7 antagonist is provided, wherein the expression of at least one, at least two, or at least three low expression predictor genes ("LEPGs") selected from the group consisting of SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7, AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, VNN2, and VNN3 is measured in a sample. In some embodiments, the at least one, at least two, or at least three LEPGs are selected from the group consisting of SLC8A3, TNFSF15, BEST2, VNN2, and CCL2. In some embodiments, at least one, at least two, or at least three LEPGs are selected from SLC8A3, VNN2, and TNFSF15. In one embodiment, the biological sample is a tissue biopsy sample. In one embodiment, the biopsy tissue is obtained from intestinal tissue. In certain such embodiments comprising a tissue biopsy sample or intestinal tissue, the LEPGs include SLC8A3. In one embodiment, the biological sample is peripheral whole blood. In certain such embodiments comprising peripheral blood, the LEPGs do not include SLC8A3. In one embodiment, the peripheral whole blood is collected in a PAXgene tube. In certain embodiments, the mRNA expression level is measured by RNA sequencing, microarray, or PCR. In one embodiment, the PCR method is qPCR. In certain embodiments, the measurement comprises amplifying one or more of SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, VNN2, and VNN3 mRNAs, and detecting the amplified mRNAs to measure the level of the amplified mRNAs. In certain embodiments, the mRNA expression level is compared to a reference level. In some embodiments, the mRNA expression level is compared to a reference level of each LEPG measured. In certain embodiments, the reference level of each LEPG is a median. In some embodiments, when the mRNA expression level of at least one, at least two, or at least three LEPGs selected from SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, VNN2, and VNN3 in the sample is reduced compared to the reference value of each LEPG measured (which in some embodiments is the median of each reference value), the patient is predicted to respond to treatment comprising an integrin beta 7 antagonist. In one embodiment, the response is clinical remission. In one embodiment, the response is mucosal healing. In one embodiment, the response is a clinical response. In certain embodiments, induction of remission in the patient is determined when the absolute Mayo clinical score is ≤2 and no single subscore is >1, which is also referred to as clinical remission. In certain embodiments, mucosal healing is determined to have occurred when the patient is determined to have an endoscopy subscore of 0 or 1 as assessed by flexible sigmoidoscopy. In certain such embodiments, mucosal healing is determined to have occurred in a patient having an endoscopy subscore of 0. In certain embodiments, a clinical response is determined to have occurred when the patient has a 3-point decrease in MCS and a 30% decrease from baseline and a ≥1-point decrease in rectal bleeding subscore or an absolute rectal bleeding score of 0 or 1.

In yet another aspect, methods are provided for identifying a patient suffering from a gastrointestinal inflammatory disorder as likely to respond to a treatment comprising an integrin beta7 antagonist. In certain embodiments, the method comprises: (a) measuring the mRNA expression level of at least one, at least two, or at least three LEPGs selected from SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, VNN2, and VNN3 in a biological sample from the patient; (b) comparing the mRNA expression level measured in (a) with a reference level for each LEPG measured; and (c) identifying the patient as more likely to respond to a treatment comprising an integrin beta 7 antagonist when the mRNA expression level of each LEPG measured in (a) is lower than the reference level for each LEPG measured. In some embodiments, the at least one, at least two, or at least three LEPGs are selected from SLC8A3, TNFSF15, BEST2, VNN2, and CCL2. In some embodiments, the at least one, at least two, or at least three LEPGs are selected from SLC8A3, VNN2, and TNFSF15. In one embodiment, the patient is a human. In one embodiment, the patient has not been previously treated with an anti-TNF therapeutic agent. In one embodiment, the gastrointestinal inflammatory disorder is inflammatory bowel disease. In one embodiment, the inflammatory bowel disease is ulcerative colitis or Crohn's disease. In one embodiment, the inflammatory bowel disease is ulcerative colitis, and the response is selected from clinical response, mucosal healing, and remission. In certain embodiments, remission is determined to have been induced in the patient when the absolute Mayo clinical score is ≤2 and no individual subscore is >1, which is also referred to as clinical remission. In certain embodiments, mucosal healing is determined to have occurred when the patient is determined to have an endoscopic subscore of 0 or 1 assessed by flexible sigmoidoscopy. In certain such embodiments, the patient in whom mucosal healing is determined to have occurred has an endoscopic subscore of 0. In certain embodiments, a clinical response is determined to have occurred when a patient experiences a 3-point decrease in MCS and a 30% decrease from baseline and a ≥1-point decrease in the rectal bleeding subscore or an absolute rectal bleeding score of 0 or 1.

In another aspect, methods of treating a patient suffering from a gastrointestinal inflammatory disorder are provided. In certain embodiments, the methods comprise: (a) measuring in a biological sample from the patient at least one, at least two, or more of the following: SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7, AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, VNN2, and VNN3. (b) comparing the mRNA expression level of each LEPG measured in (a) to a reference level for each LEPG measured; (c) identifying the patient as more likely to respond to a treatment comprising an integrin beta 7 antagonist when the mRNA expression level of each LEPG measured in (a) is lower than the respective reference level for each LEPG measured; and (d) administering the treatment when the mRNA expression level of each LEPG measured in (a) is lower than the respective reference level for each LEPG measured, thereby treating the gastrointestinal inflammatory disorder. In some embodiments, the at least one, at least two, or at least three LEPGs are selected from SLC8A3, TNFSF15, BEST2, VNN2, and CCL2. In some embodiments, the at least one, at least two, or at least three LEPGs are selected from SLC8A3, VNN2, and TNFSF15. In one embodiment, 105 mg of the integrin beta 7 antagonist is administered subcutaneously once every four weeks. In one embodiment, an initial dose of 210 mg of the integrin beta7 antagonist is administered subcutaneously, followed by subsequent doses, each 210 mg subsequent dose of the integrin beta7 antagonist being administered subcutaneously at each of weeks 2, 4, 8, and 12 after the initial dose. In one embodiment, the patient is a human. In one embodiment, the patient has not been previously treated with an anti-TNF therapeutic. In one embodiment, the gastrointestinal inflammatory disorder is inflammatory bowel disease. In one embodiment, the inflammatory bowel disease is ulcerative colitis or Crohn's disease. In one embodiment, the inflammatory bowel disease is ulcerative colitis, and the response is selected from the group consisting of clinical response, mucosal healing, and remission. In certain embodiments, induction of remission in the patient is determined to be ≤2 with no individual subscore >1, also referred to as clinical remission. In certain embodiments, mucosal healing is determined to have occurred when the patient is determined to have an endoscopic subscore of 0 or 1 as assessed by flexible sigmoidoscopy. In certain such embodiments, a patient determined to have experienced mucosal healing has an endoscopic subscore of 0. In certain embodiments, a clinical response is determined to have occurred when a patient experiences a 3-point decrease in MCS and a 30% decrease from baseline and a ≥1-point decrease in the rectal bleeding subscore or an absolute rectal bleeding score of 0 or 1.

In yet another aspect, a method of predicting a response of a patient having a gastrointestinal inflammatory disorder to a treatment comprising an integrin beta7 antagonist or predicting responsiveness of a patient having a gastrointestinal inflammatory disorder to a treatment comprising an integrin beta7 antagonist is provided, wherein at least one of the group consisting of GZMA, KLRB1, FOXM1, CCDC90A, CCL4, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and SLC8A3 is measured in a biological sample. In some embodiments, the expression of at least two, at least three, or at least four HEPGs is measured, and the expression of at least one, at least two, or at least three LEPGs selected from SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, VNN2, and VNN3 is measured in the sample. In some embodiments, the at least one, at least two, at least three, or at least four HEPGs are selected from GZMA, KLRB1, FOXM1, SLC8A3, and ECH1, and the at least one, at least two, or at least three LEPGs are selected from SLC8A3, TNFSF15, BEST2, VNN2, and CCL2. In some embodiments, at least one, at least two, or at least three LEPGs are selected from SLC8A3, VNN2, and TNFSF15. In some embodiments, the mRNA expression level of each HEPG is compared to the reference level of each HEPG measured, and the mRNA expression level of each LEPG is compared to the reference level of each LEPG measured. In some embodiments, when the mRNA expression level of each HEPG measured is increased compared to the reference level of each HEPG measured, and when the mRNA expression level of each LEPG measured is decreased compared to the reference level of each LEPG measured, the patient is predicted to respond to the treatment. In one embodiment, the biological sample is a tissue biopsy sample. In one embodiment, the biopsy tissue is obtained from intestinal tissue. In certain such embodiments comprising tissue biopsy samples or intestinal tissue, the HEPG does not include SLC8A3, and the LEPG includes SLC8A3. In one embodiment, the biological sample is peripheral whole blood. In certain such embodiments comprising peripheral blood, the HEPG includes SLC8A3, and the LEPG does not include SLC8A3. In one embodiment, the peripheral whole blood is collected in a PAXgene tube. In certain embodiments, the mRNA expression level is measured by RNA sequencing, microarray or PCR. In one embodiment, the PCR method is qPCR. In certain embodiments, the measurement comprises amplifying one or more of GZMA, KLRB1, FOXM1, CCDC90A, CCL4L1.2, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2 and SLC8A3 mRNA, and optionally further comprises amplifying ITGAE mRNA, and includes amplifying one or more of SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, VNN2, and VNN3 mRNA, and detecting the amplified mRNA to measure the level of the amplified mRNA. In certain embodiments, the mRNA expression level of each gene measured is compared with the reference level of the gene measured (which is the median level in some embodiments). In one embodiment, the response is clinical remission. In one embodiment, the response is mucosal healing. In one embodiment, the response is a clinical response. In certain embodiments, when the absolute Mayo clinical score is ≤2 and no single subscore is >1, it is determined that remission is induced in the patient, which is also referred to as clinical remission. In certain embodiments, mucosal healing is determined to have occurred when the patient is determined to have an endoscopy subscore of 0 or 1 as assessed by flexible sigmoidoscopy. In certain such embodiments, mucosal healing is determined to have occurred in a patient having an endoscopy subscore of 0. In certain embodiments, a clinical response is determined to have occurred when the patient has a 3-point decrease in MCS and a 30% decrease from baseline and a ≥1-point decrease in rectal bleeding subscore or an absolute rectal bleeding score of 0 or 1.

In another aspect, a method is provided for identifying a patient with a gastrointestinal inflammatory disorder as likely to respond to a treatment comprising an integrin beta7 antagonist, comprising: (a) measuring the mRNA expression level of at least one, at least two, at least three, or at least four HEPGs selected from the group consisting of GZMA, KLRB1, FOXM1, CCDC90A, CCL4, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and SLC8A3 in a biological sample from the patient; (b) comparing the mRNA expression level measured in (a) to a reference level of each HEPG measured; and (c) measuring the mRNA expression level of at least one, at least two, at least three, or at least four HEPGs selected from the group consisting of SLC8A3, TNFSF15, BEST2, CCL2, C (e) when the mRNA expression level of each HEPG measured in (a) is higher than the respective reference level of each HEPG measured, and when the mRNA expression level of each LEPG measured in (c) is lower than the respective reference level of each LEPG measured, the patient is identified as being more likely to respond to a treatment comprising an integrin beta 7 antagonist. In some embodiments, the at least one, at least two, at least three, or at least four HEPGs are selected from GZMA, KLRB1, FOXM1, SLC8A3, and ECH1, and the at least one, at least two, or at least three LEPGs are selected from SLC8A3, TNFSF15, BEST2, VNN2, and CCL2. In some embodiments, at least one, at least two, or at least three LEPGs are selected from SLC8A3, VNN2, and TNFSF15. In some embodiments, another HEPG other than those listed above is measured, which HEPG is ITGAE. In one embodiment, the patient is a human. In one embodiment, the patient has not been previously treated with an anti-TNF therapeutic agent. In one embodiment, the gastrointestinal inflammatory disorder is inflammatory bowel disease. In one embodiment, the inflammatory bowel disease is ulcerative colitis or Crohn's disease. In one embodiment, the inflammatory bowel disease is ulcerative colitis, and the response is selected from clinical response, mucosal healing, and remission. In certain embodiments, induction of remission in the patient is determined when the absolute Mayo clinical score is ≤2 and no single subscore is >1, which is also referred to as clinical remission. In certain embodiments, mucosal healing is determined to have occurred when the patient is determined to have an endoscopy subscore of 0 or 1 as assessed by flexible sigmoidoscopy. In certain such embodiments, mucosal healing is determined to have occurred in a patient having an endoscopy subscore of 0. In certain embodiments, a clinical response is determined to have occurred when the patient has a 3-point decrease in MCS and a 30% decrease from baseline and a ≥1-point decrease in rectal bleeding subscore or an absolute rectal bleeding score of 0 or 1.

In yet another aspect, a method of treating a patient suffering from a gastrointestinal inflammatory disorder is provided, wherein the method comprises: (a) measuring at least one of GZMA, KLRB1, FOXM1, CCDC90A, CCL4, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and SLC8A3 in a biological sample from the patient; (b) comparing the mRNA expression level of each HEPG measured in (a) with a reference level of each HEPG measured ("HEPG reference level"); the method further comprising: (c) measuring the mRNA expression level of a gene selected from the group consisting of SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, IN, in a biological sample from the patient; (d) comparing the mRNA expression level of each LEPG measured in (c) with a reference level of each LEPG measured ("LEPG reference level"); (e) identifying the patient as more likely to respond to a treatment comprising an integrin beta 7 antagonist when the mRNA expression level of each HEPG measured in (a) is higher than the HEPG reference level and the mRNA expression level of each LEPG measured in (c) is lower than the LEPG reference level; and (f) administering the treatment when the mRNA expression level of each HEPG measured in (a) is higher than the HEPG reference level and the mRNA expression level of each LEPG measured in (c) is lower than the LEPG reference level, thereby treating the gastrointestinal inflammatory disorder. In some embodiments, the at least one, at least two, at least three, or at least four HEPGs are selected from GZMA, KLRB1, FOXM1, SLC8A3, and ECH1, and the at least one, at least two, or at least three LEPGs are selected from SLC8A3, TNFSF15, BEST2, VNN2, and CCL2. In some embodiments, at least one, at least two, or at least three LEPGs are selected from SLC8A3, VNN2, and TNFSF15. In some embodiments, another HEPG other than those listed above is measured, which HEPG is an ITGAE. In one embodiment, 105 mg of integrin beta 7 antagonist is administered subcutaneously once every four weeks. In one embodiment, a starting dose of 210 mg integrin beta 7 antagonist is administered subcutaneously, followed by subsequent doses, each subsequent dose of 210 mg integrin beta 7 antagonist is administered subcutaneously at each time point of 2, 4, 8, and 12 weeks after the starting dose. In one embodiment, the patient is human. In one embodiment, the patient has not been previously treated with an anti-TNF therapeutic. In one embodiment, the gastrointestinal inflammatory disorder is inflammatory bowel disease. In one embodiment, the inflammatory bowel disease is ulcerative colitis or Crohn's disease. In one embodiment, the inflammatory bowel disease is ulcerative colitis and the response is selected from clinical response, mucosal healing and remission. In certain embodiments, remission is determined to be induced in the patient when the absolute Mayo clinical score is ≤2 and no single subscore is >1, which is also referred to as clinical remission. In certain embodiments, mucosal healing is determined to have occurred when it is determined that the patient has an endoscopic subscore of 0 or 1 assessed by flexible sigmoidoscopy. In certain such embodiments, a patient in whom mucosal healing is determined to have occurred has an endoscopic subscore of 0. In certain embodiments, a clinical response is determined to have occurred when the patient experiences a 3-point reduction in MCS and a 30% reduction from baseline and a ≥1-point reduction in rectal bleeding subscore or an absolute rectal bleeding score of 0 or 1.

In yet another aspect, an integrin beta 7 antagonist is provided for use in treating a patient with a gastrointestinal inflammatory disorder. In certain embodiments, the patient is treated or selected for treatment when the mRNA expression level of at least one gene selected from GZMA, KLRB1, FOXM1, CCDC90A, CCL4L1.2, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and SLC8A3 is above a reference level. In one embodiment, another gene other than those listed above is measured, and the other gene is ITGAE. In certain embodiments, the patient is treated or selected for treatment when the mRNA expression level of at least one gene selected from SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, VNN2, and VNN3 is below a reference level. In one embodiment, the reference level for each gene measured is the median. In one embodiment, the integrin beta7 antagonist is used to treat the patient, wherein 105 mg is administered subcutaneously once every four weeks. In one embodiment, an initial dose of 210 mg of the integrin beta7 antagonist is administered subcutaneously, followed by subsequent doses, each 210 mg of the integrin beta7 antagonist is administered subcutaneously at each of weeks 2, 4, 8, and 12 after the initial dose.

In another aspect, an in vitro use of at least one substance that specifically binds to a biomarker selected from the group consisting of GZMA, KLRB1, FOXM1, CCDC90A, CCL4L1.2, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and SLC8A3 is provided. In one embodiment, the substance that binds to another biomarker other than those listed above is measured, the other biomarker being ITGAE. In certain embodiments, the at least one substance is used to identify or select patients with a gastrointestinal inflammatory disorder as likely to respond to a treatment comprising an integrin beta7 antagonist, wherein an mRNA expression level above a reference level identifies or selects the patient as more likely to respond to the treatment. In one embodiment, the reference level is a median. In certain embodiments, the in vitro use comprises a kit.

In another aspect, an in vitro use of at least one substance that specifically binds to a biomarker selected from the group consisting of SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, VNN2, and VNN3 is provided. In certain embodiments, the at least one substance is used to identify or select a patient with a gastrointestinal inflammatory disorder as likely to respond to a treatment comprising an integrin beta7 antagonist, wherein an mRNA expression level below a reference level identifies or selects the patient as more likely to respond to the treatment. In one embodiment, the reference level is a median. In certain embodiments, the in vitro use comprises a kit.

In yet another aspect, methods of treating a gastrointestinal inflammatory disorder in a patient are provided. In certain embodiments, a therapeutically effective amount of an integrin beta7 antagonist is administered to the patient when a biological sample obtained from the patient is determined to express elevated mRNA expression levels of one or more of certain genes. In some embodiments, the sample is determined to express elevated levels of GZMA, KLRB1, FOXM1, CCDC90A, CCL4L1.2, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, or SLC8A3 compared to the median. In some embodiments, the sample is determined to express elevated mRNA levels of two, three, or four of GZMA, KLRB1, FOXM1, CCDC90A, CCL4L1.2, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and SLC8A3, compared to the median level of the same mRNA. In some embodiments, the sample is determined to express elevated mRNA levels of another gene other than those listed above, the other gene being ITGAE. In certain embodiments, patients are selected for treatment based on elevated mRNA expression levels of certain genes in a biological sample compared to the median of the same gene or genes. In one embodiment, the biological sample is a tissue biopsy sample. In certain such embodiments comprising a biopsy, SLC8A3 expression is not determined. In one embodiment, the biological sample is peripheral whole blood. In certain such embodiments comprising peripheral blood, increased expression of SLC8A3 is determined. In one embodiment, the peripheral whole blood is collected in PAXgene tubes. In certain embodiments, the mRNA expression level is measured by RNA sequencing, microarray, or PCR. In one embodiment, the PCR method is qPCR. In certain embodiments, the measurement comprises amplifying one or more of GZMA, KLRB1, FOXM1, CCDC90A, CCL4L1.2, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and SLC8A3 mRNA, and optionally further comprises amplifying ITGAE mRNA and detecting the amplified mRNA to measure the level of the amplified mRNA. In certain embodiments, administration of the integrin beta7 antagonist results in one or more of the following: (1) a 3-point reduction in MCS and a 30% reduction from baseline, with a ≥1-point reduction in the rectal bleeding subscore or an absolute rectal bleeding score of 0 or 1; (2) an endoscopic subscore of 0 or 1; (3) an MCS ≤ 2, with no individual subscore > 1. In one embodiment, 105 mg of the integrin beta7 antagonist is administered subcutaneously once every four weeks. In one embodiment, a 210 mg initial dose of the integrin beta7 antagonist is administered subcutaneously, followed by subsequent doses, each 210 mg subsequent dose of the integrin beta7 antagonist being administered subcutaneously at each time point of weeks 2, 4, 8, and 12 after the initial dose.

In yet another aspect, methods of treating a gastrointestinal inflammatory disorder in a patient are provided. In certain embodiments, a therapeutically effective amount of an integrin beta7 antagonist is administered to the patient when a biological sample obtained from the patient is determined to express reduced mRNA expression levels of one or more of certain genes. In some embodiments, the sample is determined to express reduced mRNA expression of SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7, AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, VNN2, or VNN3 compared to the median. In some embodiments, the sample is determined to express reduced mRNA levels of two or three of SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7, AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, VNN2, and VNN3, as compared to the median level of the same mRNA. In certain embodiments, patients are selected for treatment based on reduced mRNA expression levels of certain genes in a biological sample as compared to the median for one or more of the same genes. In some embodiments, patients are selected for treatment based on decreased expression of SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, VNN2, or VNN3 compared to the median. In some embodiments, patients are selected for treatment based on decreased mRNA expression of two or three of SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, VNN2, and VNN3, compared to the median of the same mRNA. In one embodiment, the biological sample is a tissue biopsy sample. In certain such embodiments comprising a tissue biopsy sample, the method comprises decreased SLC8A3 expression compared to a reference value or median. In one embodiment, the biological sample is peripheral whole blood. In certain such embodiments comprising peripheral blood, SLC8A3 expression is not decreased compared to a reference value or median. In one embodiment, the peripheral whole blood is collected in a PAXgene tube. In certain embodiments, the mRNA expression level is measured by RNA sequencing, microarray or PCR. In one embodiment, the PCR method is qPCR. In certain embodiments, the measurement comprises amplifying one or more of SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, VNN2 and VNN3 mRNA and detecting the amplified mRNA to measure the level of the amplified mRNA. In certain embodiments, administration of the integrin beta7 antagonist results in one or more of the following: (1) a 3-point reduction in MCS and a 30% reduction from baseline, with a ≥1-point reduction in the rectal bleeding subscore or an absolute rectal bleeding score of 0 or 1; (2) an endoscopic subscore of 0 or 1; (3) an MCS ≤ 2, with no individual subscore > 1. In one embodiment, 105 mg of the integrin beta7 antagonist is administered subcutaneously once every four weeks. In one embodiment, a 210 mg initial dose of the integrin beta7 antagonist is administered subcutaneously, followed by subsequent doses, each 210 mg subsequent dose of the integrin beta7 antagonist being administered subcutaneously at each time point of weeks 2, 4, 8, and 12 after the initial dose.

In certain of the above embodiments, the gastrointestinal inflammatory disorder is inflammatory bowel disease, in certain such embodiments, the inflammatory bowel disease is ulcerative colitis (UC) or Crohn's disease (CD), in certain such embodiments, the integrin beta7 antagonist is a monoclonal anti-beta7 antibody. In certain such embodiments, the anti-beta7 antibody is selected from a chimeric antibody, a human antibody, and a humanized antibody. In certain embodiments, the anti-beta7 antibody is an antibody fragment. In certain embodiments, the anti-beta7 antibody comprises six hypervariable regions (HVRs), wherein:

(i) HVR-L1 comprises amino acid sequence A1-A11, wherein A1-A11 is RASESVDTYLH (SEQ ID NO: 1); RASESVDSLLH (SEQ ID NO: 7); RASESVDTLLH (SEQ ID NO: 8); or RASESVDDLLH (SEQ ID NO: 9); or a variant of SEQ ID NO: 1, 7, 8 or 9 (SEQ ID NO: 9). NO:26), wherein amino acid A2 is selected from the group consisting of A, G, S, T, and V, and/or amino acid A3 is selected from the group consisting of S, G, I, K, N, P, Q, R, and T, and/or amino acid A4 is selected from the group consisting of E, V, Q, A, D, G, H, I, K, L, N, and R, and/or amino acid A5 is selected from the group consisting of S, Y, A, D, G, H, I, K, N, P, R, T, and V, and/or amino acid A6 is selected from the group consisting of V, R, I, A, G, K, L, M, and Q, and/or amino acid A7 is selected from the group consisting of D, V, S, A, E, G, H, I, K, L, N, P, S, and T, and/or amino acid A8 is selected from the group consisting of D, G, N, E, T, P, and S, and/or amino acid A9 is selected from the group consisting of L, Y, I, and M, and/or amino acid A10 is selected from the group consisting of L, A, I, M, and V, and/or amino acid A11 is selected from the group consisting of H, Y, F, and S;

(ii) HVR-L2 comprises amino acid sequence B1-B8, wherein B1-B8 is KYASQSIS (SEQ ID NO:2); RYASQSIS (SEQ ID NO:20); or Xaa YASQSIS (SEQ ID NO:21, wherein Xaa represents any amino acid); or a variant of SEQ ID NO:2, 20 or 21 (SEQ ID NO:27), wherein amino acid B1 is selected from the group consisting of K, R, N, V, A, F, Q, H, P, I, L, Y and Xaa (wherein Xaa represents any amino acid), and/or amino acid B4 is selected from the group consisting of S and D, and/or amino acid B5 is selected from the group consisting of Q and S, and/or amino acid B6 is selected from the group consisting of S, D, L and R, and/or amino acid B7 is selected from the group consisting of I, V, E and K;

(iii) HVR-L3 comprises amino acid sequence C1-C9, wherein C1-C9 is QQGNSLPNT (SEQ ID NO:3); or a variant of SEQ ID NO:3 (SEQ ID NO:28), wherein amino acid C8 is selected from the group consisting of N, V, W, Y, R, S, T, A, F, H, IL, and M;

(iv) HVR-H1 comprises amino acid sequence D1-D10, wherein D1-D10 is GFFITNNYWG (SEQ ID NO: 4);

(v) HVR-H2 comprises amino acid sequence E1-E17, wherein E1-E17 is GYISYSGSTSYNPSLKS (SEQ ID NO:5); or a variant of SEQ ID NO:5 (SEQ ID NO:29), wherein amino acid E2 is selected from the group consisting of Y, F, V, and D, and/or amino acid E6 is selected from the group consisting of S and G, and/or amino acid E10 is selected from the group consisting of S and Y, and/or amino acid E12 is selected from the group consisting of N, T, A, and D, and/or amino acid 13 is selected from the group consisting of P, H, D, and A, and/or amino acid E15 is selected from the group consisting of L and V, and/or amino acid E17 is selected from the group consisting of S and G; and

(vi) HVR-H3 comprises the amino acid sequence of F2-F11, wherein F2-F11 are MTGSSGYFDF (SEQ ID NO:6) or RTGSSGYFDF (SEQ ID NO:19); or comprises the amino acid sequence of F1-F11, wherein F1-F11 are AMTGSSGYFDF (SEQ ID NO:16); ARTGSSGYFDF (SEQ ID NO:17); or AQTGSSGYFDF (SEQ ID NO:18); or a variant of SEQ ID NO:6, 16, 17, 18, or 19 (SEQ ID NO:30), wherein amino acid F2 is R, M, A, E, G, Q, S, and/or amino acid F11 is selected from F and Y.

In certain embodiments, the anti-beta7 antibody comprises three heavy chain hypervariable region (HVR-H1-H3) sequences and three light chain hypervariable region (HVR-L1-L3) sequences, wherein:

(i) HVR-L1 comprises SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9;

(ii) HVR-L2 comprises SEQ ID NO: 2;

(iii) HVR-L3 comprises SEQ ID NO: 3;

(iv) HVR-H1 comprises SEQ ID NO: 4;

(v) HVR-H2 comprises SEQ ID NO: 5; and

(vi) HVR-H3 comprises SEQ ID NO: 6 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 19.

In certain embodiments, the anti-beta7 antibody comprises three heavy chain hypervariable region (HVR-H1-H3) sequences and three light chain hypervariable region (HVR-L1-L3) sequences, wherein:

(i) HVR-L1 comprises SEQ ID NO: 9;

(ii) HVR-L2 comprises SEQ ID NO: 2;

(iii) HVR-L3 comprises SEQ ID NO: 3;

(iv) HVR-H1 comprises SEQ ID NO: 4;

(v) HVR-H2 comprises SEQ ID NO: 5; and

(vi) HVR-H3 comprises SEQ ID NO: 19. In certain embodiments, the anti-beta7 antibody comprises a variable light chain comprising the amino acid sequence of SEQ ID NO:31 and a variable heavy chain comprising the amino acid sequence of SEQ ID NO:32.

In certain embodiments, the anti-beta7 antibody is etrolizumab, also known as rhuMAb beta7.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and 1B show an alignment of the sequences of the variable light and heavy chains against the following consensus sequences and anti-beta7 subunit antibody sequences: light chain human subgroup κI consensus sequence (Figure 1A, SEQ ID NO: 12); heavy chain human subgroup III consensus sequence (Figure 1B, SEQ ID NO: 13); rat anti-mouse beta7 antibody (Fib504) variable light chain (Figure 1A, SEQ ID NO: 10); rat anti-mouse beta7 antibody (Fib504) variable heavy chain (Figure 1B, SEQ ID NO: 11); and humanized antibody variants: humanized hu504K grafted variable light chain (Figure 1A, SEQ ID NO: 14), humanized hu504K grafted variable heavy chain (Figure 1B, SEQ ID NO: NO:15), variants hu504-5, hu504-16, and hu504-32 (for variants hu504-5, hu504-16, and hu504-32, the amino acid variations from the humanized hu504K graft are shown in Figure 1A (light chain) (SEQ ID NOs:22-24, respectively, in order of appearance) and Figure 1B (heavy chain) (SEQ ID NO:25)).

Figure 2 shows the variable light chain region (Figure 2A) (SEQ ID NO:31) and variable heavy chain region (Figure 2B) (SEQ ID NO:32) of etrolizumab.

FIG3 shows the study plan for the Phase II clinical study described in Example 1.

FIG4 shows the study plan for the Phase II open-label extension clinical study described in Example 1.

Figure 5 shows the odds ratios for the indicated differentially expressed genes measured by qPCR adjusted for estimates of differences in efficacy based on concurrent medications and prior anti-TNF exposure in all patients described in Example 2. Shown are differential expression of genes enriched for remission.

Figure 6 shows odds ratios for the indicated differentially expressed genes measured by qPCR adjusted for estimates of efficacy differences based on concurrent medications and prior anti-TNF exposure in anti-TNF naive patients as described in Example 2. Shown are differential expression of genes enriched for remission.

FIG. 7 is a heat map showing two-way clustering ( FIG. 7A ) and correlation ( FIG. 7B ) of selected genes described in Example 2. FIG.

Figure 8 shows the proportions (percentages) of patients stratified by baseline gene expression levels in intestinal biopsies (low (below the median) vs. high (at or above the median)) described in Example 2 and treated with placebo (black bars), 100 mg/dose etrolizumab (speckled bars), or 300 mg/dose etrolizumab plus a loading dose (LD) (striped bars). (Figure 8A) Proportion of patients stratified by granzyme A (GZMA) gene expression; (Figure 8B) Proportion of patients stratified by KLRB1 gene expression; (Figure 8C) Proportion of patients stratified by FOXM1 gene expression; (Figure 8D) Proportion of patients stratified by integrin αE (ITGAE) gene expression. Left panel: all patients; right panel: patients not previously treated with any antagonist of tumor necrosis factor (TNF) activity (anti-TNF naive).

Figure 9 shows that baseline granzyme A (GZMA) expression above the median level is enriched for responsiveness to etrolizumab treatment as described in Example 2. (Figure 9A) Proportion (percentage) of TNF antagonist-naive patients in remission at Week 10, demonstrating mucosal healing at Week 10, or showing clinical response at Week 10, stratified by baseline GZMA gene expression level in intestinal biopsies (low (below median) vs. high (at or above median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (speckled bars), or 300 mg/dose etrolizumab (striped bars). (Figure 9B) Percentage of patients from an open-label extension study who had failed or had an inadequate response to prior TNF antagonist therapy (TNF-IR) who were in clinical remission (left) or showed clinical response (right) 4-6 weeks after continuing treatment with etrolizumab, stratified by baseline GZMA gene expression in intestinal biopsies (low (below median) vs. high (at or above median)). (Figure 9C) GZMA gene expression relative to GAPDH expression in baseline biopsies obtained from patients enrolled in the Phase II etrolizumab trial and identified as TNF antagonist-naive (anti-TNF naive) or TNF-IR (anti-TNF failure). Black solid circles: non-responders at week 10; open circles: responders at week 10. Dashed lines indicate medians. (Figure 9D) GZMA variability is shown for TNF antagonist-naive patients (left panel) and TNF-IR patients (right panel) in the placebo group at baseline, day 43, and day 71. Each line represents a single patient at each time point tested.

Figure 10 shows the enrichment of baseline KLRB1 expression above the median level for responsiveness to etrolizumab treatment as described in Example 2. (Figure 10A) Proportion (percentage) of TNF antagonist-naive patients in remission at Week 10, demonstrating mucosal healing at Week 10, or demonstrating a clinical response at Week 10, stratified by baseline KLRB1 gene expression level in intestinal biopsy (low (below median) vs. high (at or above median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (spotted bars), or 300 mg/dose etrolizumab (striped bars). (Figure 10B) Percentage of TNF-IR patients from the open-label extension study in clinical remission (left) or demonstrating a clinical response (right) 4-6 weeks after continued treatment with etrolizumab, stratified by baseline KLRB1 gene expression in intestinal biopsy (low (below median) vs. high (at or above median)). (Figure 10C) KLRB1 gene expression relative to GAPDH expression in baseline biopsies obtained from patients enrolled in the Phase II etrolizumab trial and identified as TNF antagonist-naive (anti-TNF naive) or TNF-IR (anti-TNF failure). Black solid circles: non-responders at week 10; open circles: responders at week 10. Dashed line indicates the median. (Figure 10D) KLRB1 variability is shown in TNF antagonist-naive patients (left panel) and TNF-IR patients (right panel) in the placebo group at baseline, day 43, and day 71. Each line represents a single patient at each time point tested.

Figure 11 shows the enrichment of baseline FOXM1 expression above the median level for responsiveness to etrolizumab treatment as described in Example 2. (Figure 11A) Proportion (percentage) of TNF antagonist-naive patients in remission at Week 10, demonstrating mucosal healing at Week 10, or demonstrating a clinical response at Week 10, stratified by baseline FOXM1 gene expression level in intestinal biopsy (low (below median) vs. high (at or above median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (speckled bars), or 300 mg/dose etrolizumab (striped bars). (Figure 11B) Percentage of TNF-IR patients from the open-label extension study in clinical remission (left) or demonstrating a clinical response (right) 4-6 weeks after continued treatment with etrolizumab, stratified by baseline FOXM1 gene expression in intestinal biopsy (low (below median) vs. high (at or above median)). (FIG. 11C) FOXM1 gene expression relative to GAPDH expression in baseline biopsies obtained from patients enrolled in the Phase II etrolizumab trial and identified as TNF antagonist-naive (anti-TNF naive) or TNF-IR (anti-TNF failure). Black solid circles: non-responders at week 10; open circles: responders at week 10. Dashed line indicates the median. (FIG. 11D) FOXM1 variability is shown in TNF antagonist-naive patients (left panel) and TNF-IR patients (right panel) in the placebo group at baseline, day 43, and day 71. Each line represents a single patient at each time point tested.

Figure 12 shows the enrichment of baseline ITGAE expression above the median level for responsiveness to etrolizumab treatment as described in Example 2. (Figure 12A) Proportion (percentage) of TNF antagonist-naive patients in remission at Week 10, showing mucosal healing at Week 10, or showing clinical response at Week 10, stratified by baseline ITGAE gene expression level in intestinal biopsy (low (below median) vs. high (at or above median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (spotted bars), or 300 mg/dose etrolizumab (striped bars). (Figure 12B) Percentage of TNF-IR patients from the open-label extension study in clinical remission (left) or showing clinical response (right) 4-6 weeks after continued treatment with etrolizumab, stratified by baseline ITGAE gene expression in intestinal biopsy (low (below median) vs. high (at or above median)). (FIG. 12C) ITGAE gene expression relative to GAPDH expression in baseline biopsies obtained from patients enrolled in the Phase II etrolizumab trial and identified as TNF antagonist-naive (anti-TNF naive) or TNF-IR (anti-TNF failure). Black solid circles: non-responders at week 10; open circles: responders at week 10. Dashed lines indicate medians. (FIG. 12D) ITGAE variability is shown in TNF antagonist-naive patients (left panel) and TNF-IR patients (right panel) in the placebo group at baseline, day 43, and day 71. Each line represents a single patient at each time point tested.

Figure 13 shows the proportions (percentages) of patients stratified by baseline gene expression levels in intestinal biopsies (low (below the median) vs. high (at or above the median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (speckled bars), or 300 mg/dose etrolizumab + LD (striped bars) as described in Example 2. (Figure 13A) Proportion of patients stratified by SLC8A3 gene expression; (Figure 13B) Proportion of patients stratified by TNFSF15 gene expression; (Figure 13C) Proportion of patients stratified by CCL2 gene expression; (Figure 13D) Proportion of patients stratified by BEST2 gene expression.

Figure 14 shows that baseline SLC8A3 expression below the median level in the screening biopsy tissue described in Example 2 is enriched for responsiveness to etrolizumab treatment. (Figure 14A) Proportion (percentage) of TNF antagonist-naive patients in remission at Week 10, showing mucosal healing at Week 10, or showing clinical response at Week 10, stratified by baseline SLC8A3 gene expression level in intestinal biopsy tissue (low (below median) vs. high (at or above median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (spotted bars), or 300 mg/dose etrolizumab (striped bars). (Figure 14B) Percentage of TNF-IR patients from the open-label extension study in clinical remission (left) or showing clinical response (right) 4-6 weeks after continued treatment with etrolizumab, stratified by baseline SLC8A3 gene expression in intestinal biopsy tissue (low (below median) vs. high (at or above median)). (FIG. 14C) SLC8A3 gene expression relative to GAPDH expression in baseline biopsies obtained from patients enrolled in the Phase II etrolizumab trial and identified as TNF antagonist-naive (anti-TNF naive) or TNF-IR (anti-TNF failure). Black solid circles: non-responders at week 10; open circles: responders at week 10. Dashed line indicates the median. (FIG. 14D) SLC8A3 variability is shown in TNF antagonist-naive patients (left panel) and TNF-IR patients (right panel) in the placebo group at baseline, day 43, and day 71. Each line represents a single patient at each time point tested.

Figure 15 shows that baseline TNFSF15 expression below the median level is enriched for responsiveness to etrolizumab treatment as described in Example 2. (Figure 15A) Proportion (percentage) of TNF antagonist-naive patients in remission at Week 10, showing mucosal healing at Week 10, or showing clinical response at Week 10, stratified by baseline TNFSF15 gene expression level in intestinal biopsy (low (below median) vs. high (at or above median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (spotted bars), or 300 mg/dose etrolizumab (striped bars). (Figure 15B) Percentage of TNF-IR patients from the open-label extension study in clinical remission (left) or showing clinical response (right) 4-6 weeks after continued treatment with etrolizumab, stratified by baseline TNFSF15 gene expression in intestinal biopsy (low (below median) vs. high (at or above median)). (FIG. 15C) TNFSF15 gene expression relative to GAPDH expression in baseline biopsies obtained from patients enrolled in the Phase II etrolizumab trial and identified as TNF antagonist-naive (anti-TNF naive) or TNF-IR (anti-TNF failure). Black solid circles: non-responders at Week 10; open circles: responders at Week 10. Dashed line indicates the median. (FIG. 15D) TNFSF15 variability is shown in TNF antagonist-naive patients (left panel) and TNF-IR patients (right panel) in the placebo group at baseline, Day 43, and Day 71. Each line represents a single patient at each time point tested.

Figure 16 shows the enrichment of baseline CCL2 expression below the median level for responsiveness to etrolizumab treatment as described in Example 2. (Figure 16A) Proportion (percentage) of TNF antagonist-naive patients in remission at Week 10, showing mucosal healing at Week 10, or showing clinical response at Week 10, stratified by baseline CCL2 gene expression level in intestinal biopsy tissue (low (below median) vs. high (at or above median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (spotted bars), or 300 mg/dose etrolizumab (striped bars). (Figure 16B) Percentage of TNF-IR patients from the open-label extension study in clinical remission (left) or showing clinical response (right) 4-6 weeks after continued treatment with etrolizumab, stratified by baseline CCL2 gene expression in intestinal biopsy tissue (low (below median) vs. high (at or above median)). (FIG. 16C) CCL2 gene expression relative to GAPDH expression in baseline biopsies obtained from patients enrolled in the Phase II etrolizumab trial and identified as TNF antagonist-naive (anti-TNF naive) or TNF-IR (anti-TNF failure). Black solid circles: non-responders at week 10; open circles: responders at week 10. Dashed lines indicate medians. (FIG. 16D) CCL2 variability is shown in TNF antagonist-naive patients (left panel) and TNF-IR patients (right panel) in the placebo group at baseline, day 43, and day 71. Each line represents a single patient at each time point tested.

Figure 17 shows that baseline BEST2 expression below the median level is enriched for responsiveness to etrolizumab treatment as described in Example 2. (Figure 17A) Proportion (percentage) of TNF antagonist-naive patients in remission at Week 10, showing mucosal healing at Week 10, or showing clinical response at Week 10, stratified by baseline BEST2 gene expression level in intestinal biopsy tissue (low (below median) vs. high (at or above median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (spotted bars), or 300 mg/dose etrolizumab (striped bars). (Figure 17B) Percentage of TNF-IR patients from the open-label extension study in clinical remission (left) or showing clinical response (right) 4-6 weeks after continued treatment with etrolizumab, stratified by baseline BEST2 gene expression in intestinal biopsy tissue (low (below median) vs. high (at or above median)). (FIG. 17C) BEST2 gene expression relative to GAPDH expression in baseline biopsies obtained from patients enrolled in the Phase II etrolizumab trial and identified as TNF antagonist-naive (anti-TNF naive) or TNF-IR (anti-TNF failure), black solid circles: non-responders at week 10; open circles: responders at week 10, dotted lines represent medians. (FIG. 17D) BEST2 variability is shown for TNF antagonist-naive patients (left panel) and TNF-IR patients (right panel) in the placebo group at baseline, day 43, and day 71. Each line represents a single patient at each time point tested.

Figure 18 shows (Figure 18A) the proportion (percentage) of TNF antagonist-naive patients in remission at Week 10, showing mucosal healing at Week 10, or showing a clinical response at Week 10, stratified by baseline ECH1 gene expression level in intestinal biopsy (low (below median) vs. high (at or above median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (spotted bars), or 300 mg/dose etrolizumab (striped bars) as described in Example 2. (Figure 18B) The percentage of TNF-IR patients from the open-label extension study who were in clinical remission (left) or showed a clinical response (right) 4-6 weeks after continued treatment with etrolizumab, stratified by baseline ECH1 gene expression in intestinal biopsy (low (below median) vs. high (at or above median)). (FIG. 18C) ECH1 gene expression relative to GAPDH expression in baseline biopsies obtained from patients enrolled in the Phase II etrolizumab trial and identified as TNF antagonist-naive (anti-TNF naive) or TNF-IR (anti-TNF failure). Black solid circles: non-responders at Week 10; open circles: responders at Week 10. Dashed lines indicate medians. (FIG. 18D) ECH1 variability is shown in TNF antagonist-naive patients (left panel) and TNF-IR patients (right panel) in the placebo group at baseline, Day 43, and Day 71. Each line represents a single patient at each time point tested.

Figure 19 shows (Figure 19A) the proportion (percentage) of TNF antagonist-naive patients in remission at Week 10, showing mucosal healing at Week 10, or showing a clinical response at Week 10, stratified by baseline VNN2 gene expression level in intestinal biopsy (low (below median) vs. high (at or above median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (spotted bars), or 300 mg/dose etrolizumab (striped bars) as described in Example 2. (Figure 19B) The percentage of TNF-IR patients from the open-label extension study who were in clinical remission (left) or showed a clinical response (right) 4-6 weeks after continued treatment with etrolizumab, stratified by baseline VNN2 gene expression in intestinal biopsy (low (below median) vs. high (at or above median)). (FIG. 19C) VNN2 gene expression relative to GAPDH expression in baseline biopsies obtained from patients enrolled in the Phase II etrolizumab trial and identified as TNF antagonist-naive (anti-TNF naive) or TNF-IR (anti-TNF failure). Black solid circles: non-responders at Week 10; open circles: responders at Week 10. Dashed lines indicate medians. (FIG. 19D) VNN2 variability is shown for TNF antagonist-naive patients (left panel) and TNF-IR patients (right panel) in the placebo group at baseline, Day 43, and Day 71. Each line represents a single patient at each time point tested.

Figure 20 shows that baseline peripheral blood ITGAE gene expression above the median level described in Example 2 (baseline in this context refers to the average of the screening and day 1 values) is enriched for responsiveness to etrolizumab treatment. (Figure 20A) Proportion (percentage) of patients who were in remission at week 10, showed mucosal healing at week 10, or showed clinical response at week 10, stratified by baseline peripheral blood ITGAE gene expression level (low (below the median) vs. high (at or above the median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (spotted bars), or 300 mg/dose etrolizumab (striped bars). (Figure 20B) Percentage of TNF-IR patients from the open-label extension study who were in clinical remission (left) or showed clinical response (right) 4-6 weeks after continued treatment with etrolizumab, stratified by baseline peripheral blood ITGAE gene expression (low (below the median) vs. high (at or above the median)). (FIG. 20C) ITGAE gene expression relative to GAPDH expression in peripheral blood from patients enrolled in the Phase II etrolizumab trial and identified as TNF antagonist-naive (anti-TNF naive) or TNF-IR (anti-TNF failure). Black solid circles: non-responders at week 10; open circles: responders at week 10. Dashed lines indicate medians. (FIG. 20D) ITGAE gene expression variability is shown for TNF antagonist-naive patients (left panel) and TNF-IR patients (right panel) in the placebo group at baseline, day 43, and day 71. Each line represents a single patient at each time point tested.

Figure 21 shows the enrichment of baseline peripheral blood ECH1 gene expression above the median level for responsiveness to etrolizumab treatment as described in Example 2. (Figure 21A) Proportion (percentage) of TNF antagonist-naive patients in remission at Week 10, showing mucosal healing at Week 10, or showing clinical response at Week 10, stratified by baseline peripheral blood ECH1 gene expression level (low (below the median) vs. high (at or above the median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (spotted bars), or 300 mg/dose etrolizumab (striped bars). (Figure 21B) Percentage of TNF-IR patients from the open-label extension study in clinical remission (left) or showing clinical response (right) 4-6 weeks after continued treatment with etrolizumab, stratified by baseline peripheral blood ECH1 gene expression (low (below the median) vs. high (at or above the median)). (FIG. 21C) ECH1 gene expression relative to GAPDH expression in baseline biopsies obtained from patients enrolled in the Phase II etrolizumab trial and identified as TNF antagonist-naive (anti-TNF naive) or TNF-IR (anti-TNF failure). Black solid circles: non-responders at Week 10; open circles: responders at Week 10. Dashed lines indicate medians. (FIG. 21D) Variability in ECH1 gene expression in peripheral blood is shown for TNF antagonist-naive patients (left panel) and TNF-IR patients (right panel) in the placebo group at baseline, Day 43, and Day 71. Each line represents a single patient at each time point tested.

Figure 22 shows (Figure 22A) the proportion (percentage) of TNF antagonist-naive patients in remission at Week 10, showing mucosal healing at Week 10, or showing a clinical response at Week 10, stratified by baseline peripheral blood FOXM1 gene expression level (low (below the median) vs. high (at or above the median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (spotted bars), or 300 mg/dose etrolizumab (striped bars) as described in Example 2. (Figure 22B) The percentage of TNF-IR patients from the open-label extension study who were in clinical remission (left) or showed a clinical response (right) 4-6 weeks after continued treatment with etrolizumab, stratified by baseline peripheral blood FOXM1 gene expression (low (below the median) vs. high (at or above the median)). (FIG. 22C) Baseline FOXM1 gene expression relative to GAPDH expression in peripheral blood obtained from patients enrolled in the Phase II etrolizumab trial and identified as TNF antagonist-naive (anti-TNF naive) or TNF-IR (anti-TNF failure). Black solid circles: non-responders at week 10; open circles: responders at week 10. The dotted line represents the median. (FIG. 22D) Variability in FOXM1 gene expression in peripheral blood is shown for TNF antagonist-naive patients (left panel) and TNF-IR patients (right panel) in the placebo group at baseline, day 43, and day 71. Each line represents a single patient at each time point tested.

Figure 23 shows (Figure 23A) the proportion (percentage) of TNF antagonist-naive patients in remission at Week 10, showing mucosal healing at Week 10, or showing a clinical response at Week 10, stratified by baseline peripheral blood GZMA gene expression level (low (below the median) vs. high (at or above the median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (spotted bars), or 300 mg/dose etrolizumab (striped bars) as described in Example 2. (Figure 23B) The percentage of TNF-IR patients from the open-label extension study who were in clinical remission (left) or showed a clinical response (right) 4-6 weeks after continued treatment with etrolizumab, stratified by baseline peripheral blood GZMA gene expression (low (below the median) vs. high (at or above the median)). (FIG. 23C) GZMA gene expression relative to GAPDH expression in baseline peripheral blood samples obtained from patients enrolled in the Phase II etrolizumab trial and identified as TNF antagonist-naive (anti-TNF naive) or TNF-IR (anti-TNF failure). Black solid circles: non-responders at week 10; open circles: responders at week 10. Dashed line represents the median. (FIG. 23D) Variability in GZMA gene expression in peripheral blood is shown for TNF antagonist-naive patients (left panel) and TNF-IR patients (right panel) in the placebo group at baseline, day 43, and day 71. Each line represents a single patient at each time point tested.

Figure 24 shows (Figure 24A) the proportion (percentage) of TNF antagonist-naive patients in remission at Week 10, showing mucosal healing at Week 10, or showing a clinical response at Week 10, stratified by baseline peripheral blood KLRB1 gene expression level (low (below the median) vs. high (at or above the median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (spotted bars), or 300 mg/dose etrolizumab (striped bars) as described in Example 2. (Figure 24B) The percentage of TNF-IR patients from the open-label extension study who were in clinical remission (left) or showed a clinical response (right) 4-6 weeks after continued treatment with etrolizumab, stratified by baseline KLRB1 gene expression in peripheral blood samples (low (below the median) vs. high (at or above the median)). (FIG. 24C) KLRB1 gene expression relative to GAPDH expression in baseline peripheral blood samples obtained from patients enrolled in the Phase II etrolizumab trial and identified as TNF antagonist-naive (anti-TNF naive) or TNF-IR (anti-TNF failure). Black solid circles: non-responders at Week 10; open circles: responders at Week 10. Dashed line indicates the median. (FIG. 24D) Variability in KLRB1 gene expression in peripheral blood is shown for TNF antagonist-naive patients (left panel) and TNF-IR patients (right panel) in the placebo group at baseline, Day 43, and Day 71. Each line represents a single patient at each time point tested.

Figure 25 shows the responsiveness of peripheral blood SLC8A3 gene expression enriched above the median level to etrolizumab treatment as described in Example 2. (Figure 25A) Proportion (percentage) of TNF antagonist-naive patients in remission at Week 10, showing mucosal healing at Week 10, or showing clinical response at Week 10, stratified by baseline SLC8A3 gene expression level in peripheral blood (low (below the median) vs. high (at or above the median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (spotted bars), or 300 mg/dose etrolizumab (striped bars). (Figure 25B) Percentage of TNF-IR patients from the open-label extension study in clinical remission (left) or showing clinical response (right) 4-6 weeks after continued treatment with etrolizumab, stratified by baseline SLC8A3 gene expression in peripheral blood (low (below the median) vs. high (at or above the median)). (FIG. 25C) Peripheral blood gene expression of SLC8A3 relative to GAPDH expression in patients enrolled in the Phase II etrolizumab trial and identified as TNF antagonist-naive (anti-TNF naive) or TNF-IR (anti-TNF failure). Black solid circles: non-responders at week 10; open circles: responders at week 10. Dashed line represents the median. (FIG. 25D) Variability in peripheral blood SLC8A3 gene expression is shown for TNF antagonist-naive patients (left panel) and TNF-IR patients (right panel) in the placebo group at baseline, day 43, and day 71. Each line represents a single patient at each time point tested.

Figure 26 shows the enrichment of peripheral blood TNFSF15 expression below the median level for responsiveness to etrolizumab treatment as described in Example 2. (Figure 26A) Proportion (percentage) of TNF antagonist-naive patients in remission at Week 10, showing mucosal healing at Week 10, or showing clinical response at Week 10, stratified by baseline peripheral blood TNFSF15 gene expression level (low (below the median) vs. high (at or above the median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (spotted bars), or 300 mg/dose etrolizumab (striped bars). (Figure 26B) Percentage of TNF-IR patients from the open-label extension study in clinical remission (left) or showing clinical response (right) 4-6 weeks after continued treatment with etrolizumab, stratified by baseline peripheral blood TNFSF15 gene expression (low (below the median) vs. high (at or above the median)). ( FIG. 26C ) Peripheral blood gene expression of TNFSF15 relative to GAPDH expression in patients enrolled in the Phase II etrolizumab trial and identified as TNF antagonist-naive (anti-TNF naive) or TNF-IR (anti-TNF failure). Black solid circles: non-responders at Week 10; open circles: responders at Week 10. Dashed line represents the median. ( FIG. 26D ) Variability in peripheral blood TNFSF15 gene expression is shown for TNF antagonist-naive patients (left panel) and TNF-IR patients (right panel) in the placebo group at baseline, Day 43, and Day 71. Each line represents a single patient at each time point tested.

Figure 27 shows the enrichment of peripheral blood VNN2 gene expression below the median level for responsiveness to etrolizumab treatment as described in Example 2. (Figure 27A) Proportion (percentage) of TNF antagonist-naive patients in remission at Week 10, showing mucosal healing at Week 10, or showing clinical response at Week 10, stratified by baseline VNN2 gene expression level in peripheral blood (low (below the median) vs. high (at or above the median)) and treated with placebo (black bars), 100 mg/dose etrolizumab (spotted bars), or 300 mg/dose etrolizumab (striped bars). (Figure 27B) Percentage of TNF-IR patients from the open-label extension study in clinical remission (left) or showing clinical response (right) 4-6 weeks after continued treatment with etrolizumab, stratified by baseline peripheral blood VNN2 gene expression (low (below the median) vs. high (at or above the median)). ( FIG. 27C ) Peripheral blood VNN2 gene expression relative to GAPDH expression in patients enrolled in the Phase II etrolizumab trial and identified as TNF antagonist-naive (anti-TNF naive) or TNF-IR (anti-TNF failure). Black solid circles: non-responders at Week 10; open circles: responders at Week 10. Dashed lines indicate medians. ( FIG. 27D ) Variability in peripheral blood VNN2 gene expression is shown for TNF antagonist-naive patients (left panel) and TNF-IR patients (right panel) in the placebo group at baseline, Day 43, and Day 71. Each line represents a single patient at each time point tested.

Figure 28 shows gene expression levels in tissue biopsy samples from untreated patient samples (cohort 1, consisting of healthy controls (HC) (n=14)), patients with ulcerative colitis (UC) (n=30), and patients with Crohn's disease (CD) (n=60) as described in Example 2. In this observational cohort, non-inflamed tissue biopsies were collected from the sigmoid colon of healthy controls and UC patients, and from the ascending or descending colon and ileum of healthy controls and CD patients. Paired inflamed and non-inflamed biopsies were collected from UC and CD patients with active focal disease and adjacent non-inflamed areas. ITGAE (Figure 28A), GZMA (Figure 28B), VNN2 (Figure 28C), ECH1 (Figure 28D), KLRB1 (Figure 28E), SLC8A3 (Figure 28F), TNFSF15 (Figure 28G), and FOXM1 (Figure 28H) gene expression relative to GAPDH is shown. Statistical differences are not shown in these figures.

Figure 29 shows the gene expression levels in peripheral blood from untreated patient samples (cohort 2, consisting of healthy controls (n = 10)) and patients who underwent resection for ulcerative colitis (UC) (n = 32) or Crohn's disease (CD) (n = 32) as described in Example 2. ITGAE (Figure 29A), GZMA (Figure 29B), VNN2 (Figure 29C), ECH1 (Figure 29D), KLRB1 (Figure 29E), SLC8A3 (Figure 29F), TNFSF15 (Figure 29G), and FOXM1 (Figure 29H) gene expression measured relative to GAPDH is shown. * = p < 0.05, ** = p < 0.01, **** = p < 0.0001 using the Mann Whitney test.

Figure 30 shows the effect of etrolizumab on integrin αE-positive (αE+) cells in the intestinal crypt epithelium, stratified by biomarker, in the etrolizumab Phase II study described in Example 2. αE+ cells associated with the intestinal crypt epithelium were counted before and after treatment with etrolizumab or placebo in patients enrolled in the etrolizumab Phase II study. Baseline colon biopsy qPCR median values were used as cutoffs to categorize patients as granzyme A ^high (GZMA) or αE ^high (in each case, high means at or above the median) or granzyme A ^low (GZMA) or αE ^low (in each case, low means below the median). Baseline distribution of αE+ cells in the intestinal crypt epithelium by baseline colon biopsy granzyme A (Figure 30A) and αE (Figure 30B) gene expression status (stratified). Changes in αE+ cells in the intestinal crypt epithelium before and after etrolizumab (open circles) or placebo (black solid triangles) treatment, stratified by baseline biopsy tissue expression levels of granzyme A (Figure 30C) and αE genes (Figure 30D). **p<0.01. Scr = screening.

Figure 31 shows increased granzyme A (GZMA) gene expression in αE+ T cells from UC patients described in Example 2. (Figure 31A) T cells were isolated from colon biopsy tissue from an untreated patient sample (Cohort 3) and sorted and purified based on surface expression of CD4, CD8, and integrin αE. CD4+αE+ T cells from UC patients had increased granzyme A gene expression compared to sorted and purified CD4+αE- T cells from UC patients and CD4+αE+ T cells from non-IBD control individuals. (Figure 31B) Granzyme A and integrin αE gene expression were significantly correlated in sorted and purified CD4+αE+ T cells from UC patients, but not in sorted and purified CD8+αE+ T cells. (Figure 31C) Baseline colon tissue gene expression of granzyme A and αE was significantly correlated in the etrolizumab Phase II study. The dotted line represents the qPCR median cutoff value. (FIG. 31D) In baseline colon biopsies from the etrolizumab Phase II study, granzyme A gene expression was significantly correlated with the number of αE+ cells in the epithelium and lamina propria. (FIG. 31E) Representative images showing co-immunofluorescence staining of granzyme A and αE in colon biopsies from UC patients in untreated patient samples (Cohort 3). *P<0.05, ****P<0.0001.

Detailed Description of the Invention

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons (New York, N.Y. 1994) and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 4th ed., John Wiley & Sons (New York, N.Y. 1992) provide one skilled in the art with a general guide to many of the terms used in this application.

definition

For purposes of interpreting this specification, the following definitions shall apply and, whenever appropriate, terms used in the singular shall also include the plural, and vice versa. In the event of a conflict between any definition set forth below and any document incorporated herein by reference, the definition set forth below shall control.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of proteins; reference to "a cell" includes a mixture of cells, etc.

Ranges provided in this specification and the appended claims include both endpoints and all points between the endpoints. Thus, for example, a range of 2.0 to 3.0 includes 2.0, 3.0, and all points between 2.0 and 3.0.

"Treatment" and its grammatical variations refer to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed for prophylaxis or during the course of clinical pathology. Desirable therapeutic effects include preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis.

"Treatment regimen" refers to the dosage, frequency of administration, or duration of treatment, in combination with or without a second drug.

An "effective therapeutic regimen" is one that will provide a beneficial response in a patient receiving such treatment.

"Altering therapy" refers to changing the treatment regimen, including changing the dosage, frequency of administration, or duration of treatment, and/or adding a second drug.

"Patient response" or "patient responsiveness" can be assessed using any endpoint that demonstrates benefit to the patient, including, but not limited to: (1) some degree of inhibition of disease progression, including slowing and complete blockade; (2) a reduction in the number of disease attacks and/or symptoms; (3) a reduction in lesion size; (4) inhibition (i.e., reduction, slowing, or complete arrest) of disease cell infiltration into adjacent peripheral organs and/or tissues; (5) inhibition (i.e., reduction, slowing, or complete arrest) of disease spread; (6) a reduction in an autoimmune, immune, or inflammatory response, which may, but need not, result in regression or ablation of disease lesions; (7) some degree of alleviation of one or more symptoms associated with the disorder; (8) an increase in the length of time after treatment where disease is not present; and/or (9) a decrease in mortality at a given time point after treatment. The term "responsiveness" refers to a measurable response, including complete response (CR) and partial response (PR).

A "complete response" or "CR" means that all signs of inflammation disappear or resolve in response to treatment. This does not always mean that the disease is cured.

A "partial response" or "PR" refers to a decrease in the severity of inflammation by at least 50% in response to treatment.

A "beneficial response" in a patient to treatment with an integrin beta7 antagonist and similar terms refer to a clinical or therapeutic benefit conferred on a patient at risk for or suffering from a gastrointestinal inflammatory disorder from or as a result of treatment with an antagonist, such as an anti-integrin beta7 antibody. Such benefit includes a cellular or biological response, a complete response, a partial response, stable disease (no progression or relapse), or a response with a later relapse in a patient from or as a result of treatment with the antagonist.

A patient "remains responsive to treatment" when the patient's responsiveness does not decrease over time during treatment.

As used herein, "no response" or "lack of response" or similar terms refers to the lack of a complete response, a partial response, or a beneficial response to treatment with an anti-integrin beta7 antagonist.

As used herein, the term "sample" or "test sample" refers to a composition obtained from or derived from an individual of interest that contains cellular entities and/or other molecular entities to be characterized and/or identified, for example, based on physical, biochemical, chemical and/or physiological characteristics. For example, the phrase "disease sample" and variations thereof refer to any sample obtained from an individual of interest that is expected or known to contain cellular entities and/or molecular entities to be characterized. The sample can be obtained from a tissue of the individual of interest or from the individual's peripheral blood. For example, the sample can be obtained from blood and other fluid samples and tissue samples of biological origin, such as biopsy tissue samples or tissue cultures or cells derived therefrom. The source of the tissue sample can be solid tissue, such as from a fresh, frozen and/or preserved organ or tissue sample, biopsy tissue or aspirate; blood or any blood component; body fluid; cells from any time during the individual's gestation or development; or plasma. The term "sample" or "test sample" includes biological samples that have been manipulated in any manner after their acquisition, such as by treatment with reagents, stabilization, or enrichment for certain components (such as proteins or polynucleotides), or embedded in a semi-solid or solid matrix for sectioning purposes. For the purposes of this article, a "section" of a tissue sample means a single portion or piece of a tissue sample, for example, a thin slice of tissue or cells cut from a tissue sample. Samples include, but are not limited to, whole blood, blood-derived cells, serum, plasma, lymph, synovial fluid, cell extracts, and combinations thereof.

" reference sample " used herein refers to any sample, standard or level for comparison purposes. In one embodiment, the reference sample is obtained from the health and/or non-diseased part (for example, tissue or cell) of the same individual or patient's body. In another embodiment, the reference sample is obtained from the untreated tissue and/or cell of the same individual or patient's body. In another embodiment, the reference sample is obtained from the health and/or non-diseased part (for example, tissue or cell) of the individual's body that is not the individual or patient. In even another embodiment, the reference sample is obtained from the untreated tissue and/or cell part of the individual that is not the individual or patient's body.

"β7 integrin antagonist" or "β7 antagonist" refers to any molecule that inhibits one or more biological activities of β7 integrin or blocks the binding of β7 integrin to one or more of its binding molecules. Antagonists of the present invention can be used to modulate one or more aspects of β7 binding, including but not limited to binding to the α4 integrin subunit, binding to the αE integrin subunit, binding of α4β7 integrin to MAdCAM, VCAM-1 or fibronectin, and binding of αEβ7 integrin to E-cadherin. These effects can be modulated by any biologically relevant mechanism, including disrupting the binding of ligands to β7 subunits or to α4β7 or αEβ7 dimer integrins, and/or by disrupting the binding between α and β integrin subunits, such that the formation of dimeric integrins is inhibited. In one embodiment of the present invention, the β7 antagonist is an anti-β7 integrin antibody (or anti-β7 antibody). In one embodiment, the anti-β7 integrin antibody is a humanized anti-β7 integrin antibody, more specifically, a recombinant humanized monoclonal anti-β7 antibody, such as rhuMAb β7, also known as etrolizumab. In some embodiments, the anti-β7 antibody of the invention is an anti-integrin β7 antagonist antibody that inhibits or blocks the binding of the β7 subunit to the α4 integrin subunit, to the αE integrin subunit, the binding of the α4β7 integrin to MAdCAM, VCAM-1 or fibronectin, and the binding of the αEβ7 integrin to E-cadherin.

"Beta7 subunit" or "β7 subunit" refers to the human β7 integrin subunit (Erle et al. (1991) J. Biol. Chem. 266: 11009-11016). The β7 subunit binds to the α4 integrin subunit (such as the human α4 subunit) (Kilger and Holzmann (1995) J. Mol. Biol. 73: 347-354). The α4β7 integrin is reported to be expressed on most mature lymphocytes, as well as a small population of thymocytes, myeloid cells, and mast cells (Kilshaw and Murant (1991) Eur. J. Immunol. 21:2591-2597; Gurish et al. (1992) 149:1964-1972; and Shaw, S.K. and Brenner, M.B. (1995) Semin. Immunol. 7:335). The β7 subunit also associates with the αE subunit (e.g., human αE integrin subunit) (Cepek, K.L. et al. (1993) J. Immunol. 150:3459). The αEβ7 integrin is expressed on intestinal epithelial lymphocytes (iIEL) (Cepek, K.L. (1993) supra).

By "alphaE subunit" or "alphaE integrin subunit" or "αE subunit" or "αE integrin subunit" or "CD103" is meant the integrin subunit that binds to the β7 integrin on intraepithelial lymphocytes, which αEβ7 integrin mediates binding of iELs to E-cadherin-expressing intestinal epithelium (Cepek, K.L. et al. (1993) J. Immunol. 150:3459; Shaw, S.K. and Brenner, M.B. (1995) Semin. Immunol. 7:335).

"MAdCAM" or "MAdCAM-1" are used interchangeably in the context of the present invention and refer to the protein mucosal addressin cell adhesion molecule-1, which is a single-chain polypeptide comprising a short cytoplasmic tail, a transmembrane region, and an extracellular sequence consisting of three immunoglobulin-like domains. The cDNAs for mouse, human, and cynomolgus macaque MAdCAM-1 have been cloned (Briskin et al., (1993) Nature, 363:461-464; Shyjan et al., (1996) J. Immunol. 156:2851-2857).

"VCAM-1" or "vascular cell adhesion molecule-1" or "CD106" refers to the ligands of α4β7 and α4β1 that are expressed on activated endothelium and are important in endothelial-lymphocyte interactions, such as lymphocyte binding and migration during inflammation.

"CD45" refers to a protein of the protein tyrosine phosphatase (PTP) family. PTPs are known to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. This PTP comprises an extracellular domain, a single transmembrane segment, and two tandem intracytoplasmic catalytic domains, and is therefore classified as a receptor-type PTP. This gene is specifically expressed in hematopoietic cells. This PTP has been shown to be an essential regulator of T-cell and B-cell antigen receptor signaling. It acts by direct interaction with components of the antigen receptor complex or by activating multiple Src family kinases required for antigen receptor signaling. This PTP also inhibits JAK kinases and therefore acts as a regulator of cytokine receptor signaling. Four alternatively spliced transcript variants encoding different isoforms of this gene have been reported (Tchilian EZ, Beverley PC (2002). "CD45 in memory and disease." Arch. Immunol. Ther. Exp. (Warsz.) 50(2):85-93; Ishikawa H, Tsuyama N, Abroun S et al. (2004). "Interleukin-6, CD45 and the src-kinases in myeloma cell proliferation." Leuk. Lymphoma 44(9):1477-81).

There are multiple isoforms of CD45: CD45RA, CD45RB, CD45RC, CD45RAB, CD45RAC, CD45RBC, CD45RO, CD45R(ABC). CD45 is also heavily glycosylated. CD45R is the longest protein and migrates at 200 kDa when isolated from T cells. B cells also express CD45R with a more heavily glycosylated structure, bringing the molecular weight to 220 kDa, hence the name B220; the 220 kDa B cell isoform. B220 expression is not limited to B cells but can also be expressed on activated T cells, a subset of dendritic cells, and other antigen-presenting cells. Stanton T, Boxall S, Bennett A et al. (2004). "CD45 variant alleles: possibly increased frequency of a novel exon4CD45 polymorphism in HIV seropositive Ugandans." Immunogenetics 56(2):107-10.

"Gut-homing lymphocytes" refers to a subset of lymphocytes that have the characteristic of selectively homing to the intestinal lymph nodes and tissues but not to the peripheral lymph nodes and tissues. The lymphocytes of this subset are characterized by a unique expression pattern of a combination of multiple cell surface molecules, including but not limited to a combination of CD4, CD45RA, and β7. Generally, peripheral blood CD4 ⁺ lymphocytes can be further divided into at least two subsets based on the markers CD45RA and β7, CD45RA ^{- β7high} and CD45RA ^{- β7low} CD4 ⁺ cells. CD45RA ^{- β7high} CD4 ⁺ cells preferentially home to the intestinal lymph nodes and tissues, while CD45RA ^{- β7low} CD4 ⁺ cells preferentially home to the peripheral lymph nodes and tissues (Rott et al. 1996; Rott et al. 1997; Williams et al. 1998; Rosé et al. 1998; Williams and Butcher 1997; Butcher et al. 1999). Thus, gut-homing lymphocytes are a distinct subset of lymphocytes identified as CD45RA ^{- β7highCD4 +} in a flow cytometric assay. Methods for identifying this group of lymphocytes are known in the art.

The symbol "+" used herein with respect to cell surface markers indicates positive expression of the cell surface marker. For example, CD4 ⁺ lymphocytes are a group of lymphocytes that express CD4 on their cell surface.

The symbol "-" used herein with respect to cell surface markers indicates negative expression of the cell surface marker. For example, ^CD45RA- lymphocytes are a group of lymphocytes that do not express CD45RA on their cell surface.

The "amount" or "level" of a biomarker can be determined using methods known in the art and disclosed herein (eg, flow cytometric analysis or qPCR).

A "change in the amount or level of a biomarker" is compared to a reference/comparator amount of that biomarker. In certain embodiments, the change is greater than about 10%, greater than about 30%, greater than about 50%, greater than about 100%, or greater than about 300% as a function of the value of the reference or comparison amount. For example, the reference or comparison amount can be the amount of the biomarker prior to treatment, more specifically, the baseline or pre-dose amount.

As used herein, the phrase "substantially the same as" means that the degree of change is not significant, such that one skilled in the art would not consider the change to be statistically significant or a biologically meaningful change in the context of the biological characteristic measured by that value (e.g., the serum level of drug required to saturate the drug's target receptor). For example, serum drug concentrations required to saturate the receptor that differ less than about 2-fold from each other, or less than about 3-fold from each other, or less than about 4-fold from each other are considered to be substantially the same.

"Gastrointestinal inflammatory disorders" are a group of chronic disorders that cause inflammation and/or ulcers in the mucosa. These disorders include, for example, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, indeterminate colitis, and infectious colitis), mucositis (e.g., oral mucositis, gastrointestinal mucositis, nasal mucositis, and proctitis), necrotizing enterocolitis, and esophagitis.

"Inflammatory bowel disease" or "IBD" are used interchangeably herein to refer to bowel diseases that cause inflammation and/or ulcers and include, but are not limited to, Crohn's disease and ulcerative colitis.

"Crohn's disease (CD)" and "ulcerative colitis (UC)" are chronic inflammatory bowel diseases of unknown cause. Unlike ulcerative colitis, Crohn's disease can affect any part of the intestine. The most prominent feature of Crohn's disease is the granular, reddish-purple edematous thickening of the intestinal wall. As the inflammation progresses, these granulomas often lose their limiting borders and become integrated with the surrounding tissue. Diarrhea and intestinal obstruction are the main clinical features. Like ulcerative colitis, the course of Crohn's disease can be continuous or relapsing, mild or severe, but unlike ulcerative colitis, Crohn's disease cannot be cured by resection of the affected segment of the intestine. Most people with Crohn's disease require surgery at some point, but subsequent relapses are common and usually require continued medical management.

Crohn's disease can affect any part of the digestive tract from mouth to anus, but it usually occurs in the ileocolonic, small intestine, or colon-anorectal regions. Histopathologically, the disease is characterized by discontinuous granulomatomas, crypt abscesses, fissures, and aphthous ulcers. The inflammatory infiltrate is mixed and consists of lymphocytes (both T and B cells), plasma cells, macrophages, and neutrophils. There is a disproportionate increase in plasma cells, macrophages, and neutrophils that secrete IgM and IgG.

The anti-inflammatory drugs sulfasalazine and 5-aminosalicylic acid (5-ASA) are used to treat mild active Crohn's disease of the colon and are often used in an attempt to maintain remission of the disease. Metroidazole and ciprofloxacin have similar efficacy to sulfasalazine and are particularly used to treat perianal disease. In more severe cases, corticosteroids can effectively treat active exacerbations and sometimes maintain remission. Azathioprine and 6-mercaptopurine have also been used in patients who require long-term corticosteroid administration. It has been suggested that these drugs may play a role in long-term prevention. Unfortunately, there is a very long delay (up to 6 months) before they become effective in some patients. Antidiarrheal drugs can also provide symptom relief in some patients. Nutritional therapy or an elemental diet can improve the patient's nutritional status and induce symptomatic improvement in acute illness, but it does not induce sustained clinical remission. Antibiotics are used to treat secondary small intestinal bacterial overgrowth and to treat suppurative complications.

"Ulcerative colitis (UC)" damages the large intestine. The course of the disease can be persistent or relapsing, mild or severe. The earliest lesions are inflammatory infiltrates that form abscesses at the base of the intestinal glands. The coalescence of these bulging and ruptured crypts tends to separate the overlying mucosa from its blood supply, leading to ulcer formation. Symptoms of the disease include abdominal cramps, lower abdominal pain, rectal bleeding, and frequent, watery stools consisting primarily of blood, pus, and mucus with very few fecal particles. Acute, severe, or chronic, persistent ulcerative colitis may require total colectomy.

The clinical features of UC are highly variable, with onset either insidious or sudden, and may include diarrhea, tenesmus, and recurrent rectal bleeding. Fulminant involvement of the entire colon, toxic megacolon, and life-threatening emergencies may occur. Extraintestinal manifestations include arthritis, pyoderma gangrenosum, uveitis, and erythema nodosum.

Treatment of UC includes sulfasalazine and related salicylate-containing medications for mild cases and corticosteroids for severe cases. Topical application of salicylates or corticosteroids is sometimes effective, especially when the disease is confined to the distal intestine, and is associated with reduced side effects compared with systemic use. Supportive measures, such as iron and antidiarrheal agents, are sometimes indicated. Azathioprine, 6-mercaptopurine, and methotrexate are also sometimes recommended for refractory corticosteroid-dependent cases.

An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

The term "patient" as used herein refers to any individual for whom treatment is desired. In certain embodiments, the patient herein is a human.

As used herein, a "subject" is typically a human. In certain embodiments, the subject is a non-human mammal. Exemplary non-human mammals include laboratory animals, domesticated animals, pet animals, sports animals, and livestock animals, such as mice, cats, dogs, horses, and cattle. Typically, the subject is eligible for treatment, such as treatment of a gastrointestinal inflammatory disorder.

The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), and may also include certain antibody fragments (as described in more detail herein). The antibody can be human, humanized and/or affinity matured.

"Antibody fragments" comprise only a portion of an intact antibody, wherein the portion preferably retains at least one, preferably most or all, of the functions typically associated with the portion when present in an intact antibody. In one embodiment, the antibody fragment comprises the antigen-binding site of an intact antibody, thereby retaining the ability to bind antigens. In another embodiment, the antibody fragment (e.g., an antibody fragment comprising an Fc region) retains at least one of the biological functions typically associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half-life regulation, ADCC function, and complement fixation. In one embodiment, the antibody fragment is a monovalent antibody having an in vivo half-life substantially similar to that of an intact antibody. For example, such an antibody fragment may comprise an antigen-binding arm connected to an Fc sequence that imparts in vivo stability to the fragment.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of one or more chains is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

The "humanized" form of non-human (e.g., mouse) antibodies is a chimeric antibody containing the minimum sequence from a non-human immunoglobulin. Generally, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues from the hypervariable region of the receptor are replaced with residues from the hypervariable region of the receptor with desired specificity, affinity, and ability from a non-human species (e.g., mouse, rat, rabbit, or non-human primate). In some cases, the framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, the humanized antibody may comprise residues not found in the receptor antibody or the donor antibody. These modifications are carried out to further improve antibody performance. Generally, the humanized antibody will comprise substantially all of at least one, typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

A "human antibody" is an antibody that comprises an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or has been prepared using any of the techniques for preparing human antibodies disclosed herein. Such techniques include screening human-derived combinatorial libraries, such as phage display libraries (see, e.g., Marks et al., J. Mol. Biol., 222:581-597 (1991) and Hoogenboom et al., Nucl. Acids Res., 19:4133-4137 (1991)); using human myeloma and mouse-human heteromyeloma cell lines to produce human monoclonal antibodies (see, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 55-93 (Marcel Dekker, Inc., New York, NY); and the like. York, 1987; and Boerner et al., J. Immunol., 147:86 (1991)); and producing monoclonal antibodies in transgenic animals (e.g., mice) capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)). This definition of a human antibody specifically excludes humanized antibodies comprising antigen-binding residues from a non-human animal.

An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminating components of its natural environment are substances that would interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody is purified to: (1) greater than 95% by weight, and often greater than 99% by weight, as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. An isolated antibody includes the antibody in situ within recombinant cells because at least one component of the antibody's natural environment will not be present. However, an isolated antibody will generally be prepared by at least one purification step.

As used herein, the terms "hypervariable region," "HVR," or "HV" refer to regions of an antibody variable domain that are hypervariable in sequence and/or form structurally defined loops. Typically, antibodies contain six hypervariable regions; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Many hypervariable region descriptions are in use and are encompassed herein. The Kabat complementarity determining regions (CDRs) are the most commonly used based on sequence variability (Kaba et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers to the positioning of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable regions are based on analysis of available complex crystal structures. The residues from each of these HVRs are indicated below.

The hypervariable region may comprise an "extended hypervariable region" as follows: 24-36 or 24-34 (L1), 46-56 or 49-56 or 50-56 or 52-56 (L2), and 89-97 (L3) in VL and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these definitions, the variable domain residues are numbered according to Kabat et al., supra.

"Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

A "human consensus framework" is a framework that represents the most common amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the sequence subgroup is a subgroup as in Kabat et al. In one embodiment, for VL, the subgroup is subgroup κI as in Kabat et al. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al.

"Affinity matured" antibodies are antibodies that have one or more changes in one or more of their CDRs that result in an improvement in the antibody's affinity for the antigen compared to a parent antibody without those changes. In certain embodiments, affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by methods known in the art. Marks et al. Bio/Technology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Barbas et al., Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1996); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992) describe random mutagenesis of CDR and/or framework residues.

As used herein, the phrases "substantially similar" or "substantially identical" mean that the degree of similarity between two values (typically one associated with an antibody of the invention and the other associated with a reference/comparator antibody) is sufficiently high that one skilled in the art would consider that the difference between the two values has little or no biological and/or statistical significance in the context of the biological characteristic being measured by the value.

"Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by methods well known in the art, including those described herein. Low-affinity antibodies generally bind to the antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind to the antigen faster and tend to maintain longer-lasting binding. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention.

The term "variable" in relation to antibodies or immunoglobulins refers to the fact that the sequence of certain portions of the variable domains differs greatly between antibodies and is used for the binding and specificity of each specific antibody to its specific antigen. However, variability is not evenly distributed throughout the variable domains of an antibody. It is concentrated in three segments called hypervariable regions in the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FRs, which primarily adopt a β-pleated sheet configuration and are connected by three hypervariable regions that form loops connecting the β-pleated sheet structure and, in some cases, form part of the β-pleated sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chains, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant region is not directly involved in binding an antibody to an antigen, but exhibits various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces the F(ab') ₂ fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

"Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define the antigen-binding site on the surface of the _VH - _VL dimer. The six hypervariable regions collectively confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv, which contains only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that several residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the one or more cysteine residues of the constant domains have at least one free sulfhydryl group. F(ab') ₂ antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The "light chains" of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

Antibodies (immunoglobulins) can be assigned to different classes depending on the amino acid sequence of their heavy chain constant domains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, several of which can be further divided into subclasses (isotypes), such as IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA _2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described in, for example, Cellular and Mol. Immunology, 4th edition (WB Saunders, Co., 2000) by Abbas et al. Antibodies can be part of a larger fusion molecule formed by the covalent or non-covalent binding of the antibody to one or more other proteins or polypeptides.

The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, not an antibody fragment as defined below. These terms particularly refer to antibodies having heavy chains that contain an Fc region.

For purposes herein, a "naked antibody" is an antibody that is not conjugated to a cytotoxic moiety or a radiolabel.

The term "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain are variable, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or from the amino acid residue at position Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 of the EU numbering system) of the Fc region can be removed, for example, during the production or purification of the antibody, or removed by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody. Thus, the composition of the intact antibody can include antibody populations with all K447 residues removed, antibody populations with no K447 residue removed, and antibody populations with a mixture of antibodies containing and not containing the K447 residue.

Unless otherwise indicated, the numbering of residues in immunoglobulin heavy chains herein is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), which is expressly incorporated herein by reference. The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed, for example, using the various assays disclosed herein.

A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; native sequence human IgG4 Fc region; and naturally occurring variants thereof.

A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification. In certain embodiments, the variant Fc region has at least one amino acid substitution in the native sequence Fc region or in the Fc region of a parent polypeptide, such as from about one to about ten amino acid substitutions, and in certain embodiments from about one to about five amino acid substitutions, compared to a native sequence Fc region or to the Fc region of a parent polypeptide. In certain embodiments, the variant Fc region herein will have at least about 80% homology to a native sequence Fc region and/or to the Fc region of a parent polypeptide, or at least about 90% homology thereto, or at least about 95% homology thereto.

Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes". There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

"Antibody-dependent cytotoxicity" and "ADCC" refer to cell-mediated reactions in which nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) expressing Fc receptors (FcRs) recognize bound antibodies on target cells, subsequently causing the lysis of target cells. The main cells (NK cells) used to mediate ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). In order to assess the ADCC activity of the target molecule, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or 5,821,337, can be performed. Effector cells used for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, eg, in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

"Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. In certain embodiments, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from their natural sources, such as blood or PBMCs as described herein.

The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In certain embodiments, an FcR is a native sequence human FcR. In addition, an FcR is an FcR (gamma receptor) that binds to an IgG antibody and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see Annu. Rev. Immunol. 15: Review in 203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" herein encompasses other FcRs, including those yet to be identified. The term also includes neonatal receptors that are responsible for transferring maternal IgG to fetal FcRn (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulating immunoglobulin homeostasis. Antibodies with improved binding and extended half-life to the neonatal Fc receptor (FcRn) are described in WO00/42072 (Presta, L.) and US2005/0014934A1 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions therein that improve binding to FcRn. For example, the Fc region may have a substitution at one or more of positions 238, 250, 256, 265, 272, 286, 303, 305, 307, 311, 312, 314, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428, or 434 (Eu numbering of residues). In certain embodiments, the Fc region-containing antibody variant with improved FcRn binding comprises amino acid substitutions at one, two, or three of positions 307, 380, and 434 (Eu numbering of residues) of its Fc region.

" Single-chain Fv " or " scFv " antibody fragment comprises the _VH and _VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In certain embodiments, the Fv polypeptide further comprises a polypeptide linker between the _VH and _VL domains that enables the scFv to form a desired structure for antigen binding. A review of scFv is shown in Plückthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994). HER2 antibody scFv fragments are disclosed in WO93/16185, U.S. Patent No. 5,571,894 and U.S. Patent No. 5,587,458.

The term "diabody" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a variable heavy chain domain ( _VH ) connected to a variable light chain domain ( _VL ) in the same polypeptide chain ( _VH - _VL ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

"Affinity matured" antibodies are antibodies with one or more alterations in one or more of their hypervariable regions that result in an improvement in the antibody's affinity for the antigen compared to a parent antibody without those alterations. In certain embodiments, affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by methods known in the art. Marks et al. Bio/Technology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Barbas et al., Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992) describe random mutagenesis of CDR and/or framework residues.

"Amino acid sequence variant" antibodies herein are antibodies having an amino acid sequence that is different from that of the main species antibody. In certain embodiments, the amino acid sequence variants will have at least about 70% homology with the main species antibody, or they will have at least about 80% or at least about 90% homology with the main species antibody. The amino acid sequence variants have substitutions, deletions and/or additions at certain positions within or adjacent to the amino acid sequence of the main species antibody. Examples of amino acid sequence variants herein include acidic variants (such as deamidated antibody variants), basic variants, antibodies with amino terminal leader sequence extensions (e.g., VHS-) on one or both light chains, antibodies with C-terminal lysine residues on one or both heavy chains, and the like, and include combinations of variations in the amino acid sequence of heavy and/or light chains. Particularly important antibody variants herein are antibodies that, relative to the main species antibody, comprise an amino terminal leader sequence extension on one or both light chains, optionally further comprising other amino acid sequence and/or glycosylation differences.

"Glycosylation variant" antibodies herein are antibodies having one or more carbohydrate moieties attached to the antibody that are different from the one or more carbohydrate moieties attached to the main species of the antibody. Examples of glycosylation variants herein include antibodies having a G1 or G2 oligosaccharide structure attached to its Fc region instead of a G0 oligosaccharide structure, antibodies having one or two carbohydrate moieties attached to one or two light chains thereof, antibodies without carbohydrates attached to one or two heavy chains of the antibody, and the like, as well as combinations of glycosylation changes. Where the antibody has an Fc region, the oligosaccharide structure may be attached to one or both heavy chains of the antibody, for example, at residue 299 (298, Eu numbering of residues).

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of a cell and/or causes the destruction of a cell. The term is intended to include radioisotopes (e.g., At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and radioisotopes of Lu), chemotherapeutic agents and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.

The term "cytokine" is a general term for proteins released by one cell population that act as intercellular mediators on another cell. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; Müllerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factors, such as NGF-β; platelet-derived growth factor; transforming growth factors (TGFs), such as TGF-α and TGF-β. -β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons, such as interferon-α, -β and -γ; colony stimulating factors (CSF), such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF); interleukins (IL), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; tumor necrosis factor, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL). The term cytokine as used herein includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of native sequence cytokines.

The term "immunosuppressant" as used herein with respect to adjunctive therapy refers to a substance that acts to suppress or mask the immune system of the subject being treated herein. This would include substances that inhibit cytokine production, downregulate or inhibit the expression of autoantigens, or mask MHC antigens. Examples of such substances include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Patent No. 4,665,077); nonsteroidal anti-inflammatory drugs (NSAIDs); ganciclovir; tacrolimus; glucocorticoids, such as cortisol or aldosterone; anti-inflammatory agents, such as cyclooxygenase inhibitors; 5-lipoxygenase inhibitors; or leukotriene receptor antagonists; purine antagonists, such as azathioprine or mycophenolate mofetil (MMF); alkylating agents, such as Cyclophosphamide; bromocriptine; danazol; dapsone; glutaraldehyde (which masks MHC antigens, as described in U.S. Pat. No. 4,120,649); anti-idiotypic antibodies to MHC antigens and MHC fragments; cyclosporine; 6-mercaptopurine; steroids, such as corticosteroids or glucocorticoids or glucocorticoid analogs, for example, prednisone, methylprednisolone, including SOLU-MEDROL.RTM, methylprednisolone sodium succinate, and dexamethasone; dihydrofolate reductase inhibitors, such as methotrexate (oral or subcutaneous); antimalarials, such as chloroquine and hydroxychloroquine; sulfasalazine; leflunomide; cytokine or cytokine receptor antibodies or antagonists, including anti-interferon-α, -β, or -γ antibodies, anti-tumor necrosis factor (TNF)-α antibodies (infliximab (REMICADE.RTM.) or adalimumab), anti-TNF-α immunoadhesins (etanercept), anti-TNF-β antibodies, anti-interleukin-2 (IL-2) antibodies and anti-IL-2 receptor antibodies, and anti-interleukin-6 (IL-6) receptor antibodies and antagonists; anti-LFA-1 antibodies, including anti-CD11a and anti-CD18 antibodies; anti-L3T4 antibodies; heterologous antilymphocyte globulins; pan-T antibodies, anti-CD3 or anti-CD4/CD4a antibodies; soluble peptides containing the LFA-3 binding domain (WO010103). 90/08187, published July 26, 1990); streptokinase; transforming growth factor-β (TGF-β); streptodornase; RNA or DNA from the host; FK506; RS-61443; chlorambucil; deoxyspergualin; rapamycin; T-cell receptor (Cohen et al., U.S. Patent No. 5,114,721); T cell receptor fragments (Offner et al., Science, 251:430-432 (1991); WO 90/11294; Ianeway, Nature, 341:482 (1989); and WO 91/01133); BAFF antagonists, such as BAFF or BR3 antibodies or immunoadhesins and zTNF4 antagonists (for review, see Mackay and Mackay, Trends Immunol., 23:113-5 (2002), see also definition below); biological agents that interfere with T cell helper signaling, such as anti-CD40 receptor or anti-CD40 ligand (CD154), including blocking antibodies to CD40-CD40 ligand (e.g., Durie et al., Science, 261:1328-30 (1993); Mohan et al., J. Immunol., 154:1470-80 (1995)) and CTLA4-Ig (Finck et al., Science, 265:1225-7 (1994)); and T-cell receptor antibodies (EP 340,109), such as T10B9.

As used herein, the term "ameliorate" refers to the reduction, decrease, or elimination of a condition, disease, disorder, or phenotype, including an abnormality or symptom.

A "symptom" of a disease or disorder (eg, an inflammatory bowel disease, such as ulcerative colitis or Crohn's disease) is any pathological phenomenon or deviation from normal structure, function, or sensation experienced by an individual and indicative of disease.

The expression "therapeutically effective amount" refers to an amount effective for preventing, ameliorating, or treating a disease or disorder (e.g., inflammatory bowel disease, such as ulcerative colitis or Crohn's disease). For example, a "therapeutically effective amount" of an antibody refers to an amount of the antibody that is effective for preventing, ameliorating, or treating a specified disease or disorder. Similarly, a "therapeutically effective amount" of a combination of an antibody and a second compound refers to an amount of the antibody and an amount of the second compound that, when combined, are effective for preventing, ameliorating, or treating a specified disease or disorder.

It should be understood that the term "combination" of two compounds does not mean that the compounds must be administered in a mixture with each other. Therefore, the treatment or use of this combination covers a mixture of compounds or separate administration of compounds, and includes administration on the same day or on different days. Therefore, the term "combination" means that two or more compounds are used for treatment alone or in a mixture with each other. When, for example, an antibody and a second compound are administered to an individual in combination, whether the antibody and the second compound are administered to the individual alone or in a mixture, when the second compound is present in the individual, the antibody is also present in the individual. In certain embodiments, a compound other than the antibody is administered after the antibody.

For purposes herein, "tumor necrosis factor-α (TNF-α)" refers to the human TNF-α molecule comprising the amino acid sequence described in Pennica et al., Nature, 312:721 (1984) or Aggarwal et al., JBC, 260:2345 (1985).

The term "TNF-α inhibitor" is used interchangeably herein with "anti-TNF-α therapeutic agent" to refer to a substance that inhibits the biological function of TNF-α to some extent, generally by binding to TNF-α and neutralizing its activity. Examples of TNF inhibitors specifically contemplated herein are etanercept, infliximab, adalimumab, golimumab (SIMPONI™), and certolizumab pegol.

"Corticosteroid" refers to any of several synthetic or naturally occurring substances with the general chemical structure of a steroid that mimics or amplifies the effects of naturally occurring corticosteroids. Examples of synthetic corticosteroids include prednisone, prednisolone (including methylprednisolone), dexamethasone, triamcinolone, and betamethasone.

"Antagonist" refers to a molecule that can neutralize, block, inhibit, abolish, reduce or interfere with the activity of a specific or specified protein (including its binding to one or more receptors in the case of a ligand, or to one or more ligands in the case of a receptor). Antagonists include antibodies and antigen-binding fragments thereof, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmaceutical preparations and metabolites thereof, transcription and translation control sequences, etc. Antagonists also include small molecule inhibitors of proteins, fusion proteins, receptor molecules and derivatives that specifically bind to proteins to chelate their binding to their targets, antagonistic variants of proteins, antisense molecules directed against proteins, RNA aptamers and anti-protein ribozymes.

"Self-injection device" refers to a medical device used for self-administration of a therapeutic agent, for example, by a patient or a home caregiver. Self-injection devices include automatic injection devices and other devices designed for self-administration.

As used herein, " oligonucleotide " refers to a short single-stranded polynucleotide having a length of at least about 7 nucleotides and a length of less than about 250 nucleotides. Oligonucleotides can be synthetic. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.

The term "primer" refers to a single-stranded polynucleotide that is capable of hybridizing to a nucleic acid and allowing polymerization of a complementary nucleic acid, typically by providing a free 3'-OH group.

The term "amplification" refers to a method for producing one or more copies of a reference nucleic acid sequence or its complement. Amplification can be linear or exponential (e.g., PCR). A "copy" does not necessarily mean perfect sequence complementarity or identity relative to the template sequence. For example, a copy can contain nucleotide analogs (e.g., deoxyinosine), internal sequence changes (e.g., sequence changes introduced by primers that can hybridize to the template but are not completely complementary), and/or sequence errors that occur during amplification.

The term "detecting" includes any means of detection, including direct detection and indirect detection.

By "increased expression" or "increased level" is meant that expression of an mRNA or protein in a patient is increased relative to a control or reference level, such as an individual not suffering from an autoimmune disease (e.g., IBD), or relative to a predetermined threshold or cutoff value, or relative to the median of a population of patients and/or individuals.

"Low expression" or "low expression level" or "reduced expression" refers to a decrease in expression of an mRNA or protein in a patient relative to a control or reference level, such as an individual not suffering from an autoimmune disease (e.g., IBD), or relative to a predetermined threshold or cutoff value, or relative to the median of a population of patients and/or individuals.

The term "multiplex PCR" refers to a single PCR reaction performed with more than one primer set on nucleic acid obtained from a single source (eg, a patient) for the purpose of amplifying two or more DNA sequences in a single reaction.

As used herein, the term "biomarker" refers to an indicator of a patient's phenotype (e.g., a pathological state or possible responsiveness to a therapeutic agent) that can be detected in a biological sample of the patient. Biomarkers include, but are not limited to, DNA, RNA, protein, carbohydrate, or glycolipid-based molecular markers.

As used herein, the term "diagnosis" refers to the identification or classification of a molecular or pathological state, disease, or condition. For example, "diagnosis" can refer to identifying a specific type of gastrointestinal inflammatory disorder, or classifying a specific subtype of a gastrointestinal inflammatory disorder, by the tissue/organ involved (e.g., inflammatory bowel disease), or by other characteristics (e.g., a subpopulation of patients characterized by responsiveness to treatment (e.g., treatment with an integrin beta7 antagonist), or by molecular characteristics (e.g., a subtype of expression of a specific gene or one or a combination of proteins encoded by that gene).

The term "aided diagnosis" is used herein to refer to methods that assist in making a clinical decision about the presence or nature of a particular type of symptom or condition. For example, a method for aiding in the diagnosis of IBD may include measuring the expression of certain genes in a biological sample from an individual.

The term "prognosis" is used herein to refer to the prediction of the likelihood of disease symptoms of a gastrointestinal inflammatory disorder, including, for example, relapse, flare-up, and drug resistance.

The term "prediction" is used herein to refer to the possibility that a patient will respond advantageously or adversely to a drug (therapeutic agent) or a drug group or a treatment regimen. In one embodiment, prediction relates to the degree of those responses. In one embodiment, prediction relates to whether a patient will survive or improve after treatment (such as specific therapeutic agent treatment) and/or the probability of survival or improvement, or to no disease recurrence within a certain period. The prediction method of the present invention can be used clinically to make treatment decisions by selecting the most appropriate treatment modality for any specific patient. In predicting whether a patient may advantageously respond to a treatment regimen (such as a given treatment regimen, including, for example, administering a given therapeutic agent or combination, surgical intervention, steroid therapy, etc.) or whether a patient may survive for a long time after a treatment regimen or alleviate or continue to alleviate, the prediction method of the present invention is a valuable tool.

A "control subject" is a healthy individual who has not been diagnosed with a particular disease (eg, IBD) and is not afflicted with any of the signs or symptoms associated with that disease.

"Correlate" or "correlated" means comparing the performance and/or results of a first analysis or procedure to the performance and/or results of a second analysis or procedure in any manner. For example, the results of a first analysis or procedure can be used to conduct a second procedure, and/or the results of a first analysis or procedure can be used to determine whether a second analysis or procedure should be conducted. With respect to embodiments of gene expression analysis or procedures, the results of a gene expression analysis or procedure can be used to determine whether a particular treatment regimen should be conducted.

As used herein, the term "comparison" refers to comparing the level of a biomarker in a sample from an individual or patient with a reference level of the biomarker specified elsewhere in this description. It should be understood that comparison, as used herein, generally refers to the comparison of corresponding parameters or values, for example, comparing an absolute amount with an absolute reference amount, comparing a concentration with a reference concentration, or comparing an intensity signal obtained from a biomarker in a sample with an intensity signal of the same type obtained from a reference sample. The comparison can be performed manually or with the assistance of a computer. Thus, the comparison can be performed by a computing device (e.g., a computing device of a system disclosed herein). The values of the biomarker level measured or detected in a sample from an individual or patient and the reference level can be compared, for example, to each other, and the comparison can be performed automatically by a computer program executing a comparison algorithm. The computer program performing this evaluation will provide the desired evaluation in a suitable output format. For computer-assisted comparisons, the value of the measured amount can be compared by the computer program with the value corresponding to a suitable reference stored in a database. The computer program can further evaluate the results of the comparison, i.e., automatically provide the desired evaluation in a suitable output format. For computer-assisted comparisons, the value of the measured amount can be compared by the computer program with the value corresponding to a suitable reference stored in a database. The computer program can further evaluate the results of the comparison, ie automatically provide the desired evaluation in a suitable output format.

As used herein, the phrase "recommended treatment" refers to the use of the generated assays for the level or presence in a patient sample of one or more of GZMA, KLRB1, FOXM1, CCDC90A, CCL4L1.2, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, VNN2, VNN3 mRNA, and optionally further for ITGAE. The information or data generated herein may include information or data regarding the level or presence of mRNA to identify patients suitable for treatment with the therapy or those not suitable for treatment with the therapy. In some embodiments, the therapy comprises an integrin beta7 antagonist, including an anti-integrin beta7 antibody, such as etrolizumab. In some embodiments, the phrase "recommending a treatment/therapy" includes identifying a patient in need of an effective amount of an integrin beta7 antagonist to be administered. In some embodiments, recommending a treatment includes recommending an amount of the integrin beta7 antagonist to be administered. As used herein, the phrase "recommending a treatment" may also refer to using the information or data generated to recommend or select a therapy comprising an integrin beta7 antagonist for a patient identified or selected as more or less likely to respond to a therapy comprising an integrin beta7 antagonist. The information or data used or generated may be in any form, whether written, oral, or electronic. In some embodiments, using the information or data generated includes communicating, displaying, reporting, storing, sending, transferring, providing, delivering, distributing, or a combination thereof. In some embodiments, communicating, displaying, reporting, storing, sending, transferring, providing, delivering, distributing, or a combination thereof is performed by a computing device, an analyzer unit, or a combination thereof. In some other embodiments, the communication, display, reporting, storage, sending, transferring, providing, delivering, distributing, or a combination thereof is performed by a laboratory or medical professional. In some embodiments, the information or data comprises comparing the level of one or more of GZMA, KLRB1, FOXM1, CCDC90A, CCL4L1.2, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, VNN2, VNN3 mRNA, and optionally further comprising ITGAE mRNA, to a reference level. In some embodiments, the information or data includes an indication that one or more of the identified mRNAs is present in the sample at an increased or decreased level. In some embodiments, the information or data includes an indication that the patient is suitable for treatment with the therapy comprising an integrin beta7 antagonist, including an anti-integrin beta7 antibody, such as etrolizumab, or is not suitable for treatment with the therapy.

"Package insert" means instructions customarily included in commercial packages of therapeutic products or medications, that contain information about the indications, usage, dosage, administration, contraindications, other therapeutic products to be combined with the packaged product, and/or warnings concerning the use of such therapeutic products or medications.

A "kit" is an article of manufacture (e.g., packaging or container) comprising at least one reagent (e.g., a drug) for treating IBD (e.g., UC or Crohn's disease) or a probe for specifically detecting a biomarker gene or protein of the invention. In certain embodiments, the article of manufacture is marketed, distributed, or sold as a unit for performing the methods of the invention.

"Target audience" is the group of people or entities to whom a specific drug is marketed or intended to be marketed, through sales or advertising, especially for a specific use, treatment or indication, such as individual patients, groups of patients, readers of newspapers, medical literature and magazines, television or internet viewers, radio or internet listeners, physicians, pharmaceutical companies, etc.

The term "serum sample" refers to any serum sample obtained from an individual. Methods for obtaining serum from mammals are well known in the art.

The term "whole blood" refers to any whole blood sample obtained from an individual. Typically, whole blood contains all blood components, such as cellular components and plasma. Methods for obtaining whole blood from mammals are well known in the art.

In relation to the response of an individual or patient to one or more drugs (therapeutic agents) previously administered to them, the expressions "no response to," "no response," and grammatical variations thereof describe individuals or patients who, when administered such drug(s), do not show any or sufficient signs of treatment for the disorder being treated, or who show a clinically unacceptably high degree of toxicity to such drug(s), or who do not maintain signs of treatment after the first administration of such drug(s), the word treatment used in this context being as defined herein. The phrase "no response" includes descriptions of those individuals who are resistant to the one or more drugs previously administered and/or refractory to the one or more drugs previously administered, and includes cases where the individual or patient progresses while receiving the one or more drugs administered to him or her, and where the individual or patient progresses within 12 months (e.g., within 6 months) after completing a regimen involving one or more drugs to which he or she no longer responds. No response to one or more drugs therefore includes individuals who still have active disease after previous or current treatment with the one or more drugs. For example, a patient may have active disease activity after about 1 to 3 months, or 3 to 6 months, or 6 to 12 months of treatment with the one or more drugs to which they are no longer responsive. This responsiveness can be assessed by a clinician skilled in treating the disorder in question.

For the purpose of non-responsiveness to one or more drugs, an individual who has experienced a "clinically unacceptably high level of toxicity" from a previous or current drug or drugs has experienced one or more negative side effects or adverse events associated therewith that are considered significant by an experienced clinician, e.g., serious infection, congestive heart failure, demyelination (leading to multiple sclerosis), significant hypersensitivity reaction, neuropathological event, high autoimmunity, cancer such as endometrial cancer, non-Hodgkin's lymphoma, breast cancer, prostate cancer, lung cancer, ovarian cancer or melanoma, tuberculosis (TB), etc.

The "amount" or "level" of a biomarker associated with an improved clinical benefit in a patient suffering from a disease or disorder or a prediction of response to a particular therapeutic agent or treatment regimen is the level detectable in a biological sample. These can be measured by methods known to those skilled in the art and disclosed herein. The expression level or amount of the assessed biomarker can be used to determine the response or predicted response to a treatment or therapeutic agent.

The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of a polynucleotide or amino acid product or protein in a biological sample. "Expression" generally refers to the process by which the information encoded by a gene is converted into a structure that exists and operates in a cell. Thus, "expression" of a gene as used herein refers to transcription into a polynucleotide, translation into a protein, or even post-translational modification of a protein. Fragments of transcribed polynucleotides, translated proteins, or post-translationally modified proteins are also considered to be expressed, whether they are derived from transcripts produced by alternative splicing or degraded transcripts, or from post-translational processing of the protein (e.g., by proteolysis). "Expressed genes" include those that are transcribed into polynucleotides (e.g., mRNA) and then translated into protein, as well as those that are transcribed into RNA but not translated into protein (e.g., transfer RNA and ribosomal RNA).

Various other terms are defined or otherwise characterized herein.

Compositions and methods

A. β7 integrin antagonists

Methods of treating gastrointestinal inflammatory disorders in individuals (e.g., humans) by administering antagonists of beta7 integrin are provided. Examples of potential antagonists include oligonucleotides that bind to fusions of immunoglobulins with beta7 integrin, particularly antibodies, including, but not limited to, polyclonal and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized forms of such antibodies or fragments, as well as human antibodies and antibody fragments. Alternatively, the potential antagonist may be a closely related protein, e.g., a mutant form of beta7 integrin, which recognizes the ligand but does not confer an effect, thereby competitively inhibiting the action of the beta7 integrin.

Another potential β7 integrin antagonist is an antisense RNA or DNA construct prepared using antisense technology, wherein, for example, the antisense RNA or DNA molecule acts by hybridizing with the targeted mRNA to directly block the translation of the mRNA and prevent protein translation. Antisense technology can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which are based on the binding of polynucleotides to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence encoding the β7 integrin herein is used to design antisense RNA oligonucleotides of about 10 to 40 base pairs in length. The DNA oligonucleotide is designed to be complementary to the region of the gene involved in transcription (triple helix - see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al., Science, 241:456 (1988); Dervan et al., Science, 251:1360 (1991)), thereby preventing transcription and the production of β7 integrin. Antisense RNA oligonucleotides hybridize with mRNA in vivo and block the translation of mRNA molecules into β7 integrin protein (antisense - Okano, Neurochem., 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, Fla., 1988)). The above oligonucleotides can also be delivered to cells so that antisense RNA or DNA can be expressed in vivo to inhibit the production of PRO polypeptides. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation start site, for example, between about -10 and +10 positions of the target gene nucleotide sequence, are typical.

Other potential antagonists include small molecules that bind to the active site, ligand, or binding molecule binding site, thereby blocking the normal biological activity of β7 integrin. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules, typically soluble peptides, and synthetic non-peptidyl organic or inorganic compounds.

Ribozymes are enzymatic RNA molecules that can catalyze the specific cleavage of RNA. Ribozymes work by specifically hybridizing to complementary target RNA sequences, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within potential RNA targets can be identified by known techniques. For further details, see, for example, Rossi, Current Biology, 4:469-471 (1994) and PCT Publication No. WO 97/33551 (published September 18, 1997).

The nucleic acid molecule in the triple helix formation that is used to suppress transcription should be single-stranded, and be made up of deoxynucleotide. Design the base composition of these oligonucleotides like this so that it promotes triple helix formation by the Hoogsteen base pairing rule, and it generally requires purine or pyrimidine sequence that is quite large on a chain of the duplex. Further details are referring to for example PCT Publication No. WO 97/33551. These small molecules can be identified by any one or more and/or by any other screening techniques well known to those skilled in the art in the screening assay discussed above.

Antagonist screening assays are designed to identify compounds that bind to or complex with the β7 integrin encoded by the genes identified herein, or that interfere with the interaction of the encoded polypeptide with other cellular proteins. Such screening assays will include assays capable of high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.

Assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art.

B. Anti-β7 integrin antibodies

In one embodiment, the beta7 integrin antagonist is an anti-beta7 antibody.Exemplary antibodies include polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, bispecific antibodies, and heteroconjugate antibodies, etc., as described below.

1. Polyclonal Antibodies

Polyclonal antibodies can be prepared in animals by multiple subcutaneous (SC) or intraperitoneal (IP) injections of the relevant antigen and _an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized (e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor) using a bifunctional or derivatizing agent (e.g., maleimidobenzoylsulfosuccinimide ester (conjugated through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl 2, or R ¹ N═C═NR where R and R ¹ are different alkyl groups).

By combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites to immunize animals against antigens, immunogenic conjugates or derivatives. One month later, the animals are boosted with 1/5 to 1/10 of the initial amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7 to 14 days, the animals are bled and the antibody titer of the serum is measured. The animals are boosted until the titer plateaus. In certain embodiments, animals are boosted with conjugates of the same antigen, but the antigen is conjugated to different proteins and/or conjugated by different cross-linking agents. Conjugates can also be prepared in recombinant cell cultures as protein fusions. In addition, immune responses are suitably enhanced with aggregating agents such as alum.

2. Monoclonal Antibodies

Monoclonal antibodies may be made by the hybridoma method first described by Kohler et al., Nature, 256 (1975) 495, or may be made by recombinant DNA methods (see, eg, US Pat. No. 4,816,567).

In the hybridoma method, mice or other suitable host animals (such as hamsters) are immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein being used for immunization. Alternatively, lymphocytes can be immunized in vitro. After immunization, the lymphocytes are separated and then fused with a myeloma cell line using a suitable fusing agent (such as polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and cultured in an appropriate culture medium that may contain one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as the fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the selective culture medium for hybridomas will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of cells lacking HGPRT.

In certain embodiments, fusion partner myeloma cells are those that are efficiently fused, support stable high levels of antibody production by selected antibody-producing cells, and are sensitive to the selective culture medium selected for unfused parental cells. In certain embodiments, myeloma cell lines are mouse myeloma cell lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 and derivatives available from American Type Culture Collection, Rockville, Md. USA, such as X63-Ag8-653 cells. Human myeloma and mouse-human heteromyeloma (heteromyeloma) cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, 51-63 pages (Marcel Dekker, Inc., New York, 1987)).

The culture medium in which the hybridoma cells are grown is assayed for the production of monoclonal antibodies against the antigen. In certain embodiments, the binding specificity of monoclonal antibodies produced by the hybridomas is determined by immunoprecipitation or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis as described in Munson et al., Anal. Biochem., 107:220 (1980). Once hybridoma cells producing antibodies with desired specificity, affinity and/or activity are identified, the clones can be subcloned by limiting dilution and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Culture media suitable for this purpose include, for example, D-MEM or RPMI-1640 culture media. In addition, hybridoma cells can be cultured in animals as ascites tumors, for example, by intraperitoneal injection of cells into mice. Monoclonal antibodies secreted by the subclones are suitably separated from culture media, ascites or serum by conventional antibody purification methods (e.g., affinity chromatography (e.g., using protein A or protein G Sepharose) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, etc.).

The DNA encoding the monoclonal antibody is easily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells can be used as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into a host cell such as an Escherichia coli (E. coli) cell, a monkey COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not otherwise produce immunoglobulin proteins to obtain the synthesis of the monoclonal antibody in the recombinant host cell. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pluckthun, Immunol. Revs. 130: 151-188 (1992).

In another embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using techniques such as those described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies using phage libraries, respectively. Subsequent publications describe the generation of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993)). Therefore, these techniques are viable options for isolating monoclonal antibodies in addition to traditional monoclonal antibody hybridoma techniques.

The DNA encoding the antibody can be modified to produce chimeric or fusion antibody polypeptides, for example, by replacing the homologous mouse sequences with human heavy and light chain constant domain (CH and CL) sequences (U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851 (1984)), or by fusing the immunoglobulin coding sequence with all or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide). The non-immunoglobulin polypeptide sequences can replace the constant domains of the antibody, or they can replace the variable domains of one antigen-binding site of the antibody to produce a chimeric bivalent antibody comprising one antigen-binding site specific for one antigen and another antigen-binding site specific for a different antigen.

Exemplary anti-β7 antibodies are Fib504, Fib 21, 22, 27, 30 (Tidswell, M. J Immunol. 1997 Aug 1; 159(3): 1497-505) or humanized derivatives thereof. Humanized antibodies to Fib504 are disclosed in detail in U.S. Patent Publication No. 20060093601 (issued as U.S. Patent No. 7,528,236), the contents of which are incorporated by reference in their entirety (see also discussion below).

3. Human and humanized antibodies

The anti-β7 integrin antibodies of the present invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab') ₂ , or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulins. Humanized antibodies include human immunoglobulins (recipient antibodies) in which residues from the complementary determining regions (CDRs) of the recipient are replaced with residues from CDRs (donor antibodies) of non-human species (e.g., mouse, rat, or rabbit) with the desired specificity, affinity, and capacity. In some cases, framework residues of the human immunoglobulin are replaced with corresponding non-human residues. Humanized antibodies may also contain residues that are not found in the recipient antibody or in the imported CDR or framework sequences. Generally, humanized antibodies will comprise substantially all of at least one, usually two, variable domains, wherein all or substantially all of the CDR regions correspond to those of the non-human immunoglobulin, and all or substantially all of the FR regions are those of the human immunoglobulin consensus sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art. Typically, a humanized antibody has one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from the "import" variable domain. Humanization can essentially be performed by replacing the corresponding human antibody sequence with a rodent CDR or CDR sequence according to the method of Winter and colleagues [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)]. Therefore, this type of "humanized" antibody is a chimeric antibody (U.S. Patent No. 4,816,567), in which substantially less than a complete human variable domain is replaced with a corresponding sequence from a non-human species. In fact, humanized antibodies are usually human antibodies, in which some CDR residues are replaced with residues from similar sites in rodent antibodies, and some FR residues may be replaced. When the antibody is expected to be used for human therapeutic purposes, the selection of the human variable domains (both light and heavy chains) for preparing humanized antibodies is very important for reducing antigenicity and HAMA reaction (human anti-mouse antibodies). According to the so-called "best fit" method, the sequence of the variable domains of rodent antibodies is screened for the entire library of known human variable domain sequences. The human V domain sequence closest to the V domain sequence of rodents is then identified, and the human framework region (FR) therein is accepted for humanized antibodies (Sims et al., J.Immunol.151:2296 (1993); Chothia et al., J.Mol.Biol.,196:901 (1987)). Another method uses the specific framework region of the consensus sequence of all human antibodies derived from the light chain or heavy chain of a specific subtype. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol. 151: 2623 (1993)). It is also important to retain high affinity for the antigen and other favorable biological properties to humanize the antibody. To achieve this goal, according to certain embodiments, humanized antibodies are prepared by analyzing the parent sequence and a variety of conceptual humanized products using three-dimensional models of the parent sequence and the humanized sequence. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences are also available. Inspection of these displays allows analysis of the possible role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of the residues that affect the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the receptor sequence and the input sequence so as to achieve the desired antibody characteristics, such as increased affinity for one or more target antigens. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

Various forms of humanized anti-β7 antibodies are contemplated. For example, the humanized antibody can be an antibody fragment, such as a Fab, which is optionally conjugated to one or more cytotoxic agents to produce an immunoconjugate. Alternatively, the humanized antibody can be a complete antibody, such as a complete IgG1 antibody.

Exemplary humanized anti-β7 antibodies include, but are not limited to, rhuMAb β7, which is a humanized monoclonal antibody against integrin subunit β7 and is derived from rat anti-mouse/human monoclonal antibody FIB504 (Andrew et al., 1994 J Immunol 153:3847-61). It has been engineered to include human immunoglobulin IgG1 heavy chain and κ1 light chain frameworks and is produced by Chinese hamster ovary cells. This antibody binds to two integrins, α4β7 (Holzmann et al. 1989 Cell, 1989; 56: 37-46; Hu et al. 1992 Proc Natl Acad Sci USA 1992; 89: 8254-8) and αEβ7 (Cepek et al. 1993 J Immunol 1993; 150: 3459-70), which regulate the trafficking and retention of lymphocyte subsets in the gastrointestinal tract and are involved in inflammatory bowel diseases (IBD), such as ulcerative colitis (UC) and Crohn's disease (CD). rhuMAbβ7 is a potent in vitro blocker of cellular interactions between α4β7 and its ligands (mucosal addressin cell adhesion molecule-1 [MAdCAM]-1, vascular cell adhesion molecule [VCAM]-1, and fibronectin), as well as the interaction between αEβ7 and its ligand (E-cadherin). rhuMAb β7 reversibly binds with similar high affinity to β7 on lymphocytes from rabbits, cynomolgus monkeys, and humans. It also binds with high affinity to mouse β7. The amino acid sequence, preparation, and use of rhuMAb β7 and its variants are disclosed in detail in U.S. Patent Application Publication No. 20060093601 (issued as U.S. Patent No. 7,528,236), the contents of which are incorporated herein by reference in their entirety.

Figures 1A and 1B show an alignment of the sequences of the variable light and heavy chains for the following: light chain human subgroup κI consensus sequence (Figure 1A, SEQ ID NO: 12); heavy chain human subgroup III consensus sequence (Figure 1B, SEQ ID NO: 13); rat anti-mouse β7 antibody (Fib504) variable light chain (Figure 1A, SEQ ID NO: 10); rat anti-mouse β7 antibody (Fib504) variable heavy chain (Figure 1B, SEQ ID NO: 11); and humanized antibody variants: humanized hu504K grafted variable light chain (Figure 1A, SEQ ID NO: 14), humanized hu504K grafted variable heavy chain (Figure 1B, SEQ ID NO: NO:15), variants hu504-5, hu504-16, and hu504-32 (for variants hu504-5, hu504-16, and hu504-32, the amino acid variations grafted relative to humanized hu504K are shown in Figure 1A (light chain) (SEQ ID NOs:22-24, respectively, in order of appearance) and Figure 1B (heavy chain) (SEQ ID NO:25)).

4. Human Antibodies

As an alternative to humanization, human antibodies can be produced. For example, it is now possible to produce transgenic animals (e.g., mice) that, upon immunization, are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, homozygous deletion of the antibody heavy chain joining region ( _JH ) gene in chimeric and germline mutant mice has been described, resulting in complete inhibition of endogenous antibody production. Transferring the human germline immunoglobulin gene array into such germline mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno. 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all to GenPharm); U.S. Pat. No. 5,545,807; and WO 97/17852.

Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 [1990]) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene libraries from non-immune donors. According to this technology, the antibody V domain genes are cloned into the major or minor coat protein genes of filamentous phage (such as M13 or fd) in frame and displayed on the surface of phage particles as functional antibody fragments. Because filamentous particles contain single-stranded DNA copies of the phage genome, selection based on antibody functional properties also leads to the selection of genes encoding antibodies that display those properties. Therefore, phage simulates some characteristics of B cells. Phage display can be carried out in a variety of forms as summarized in, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. V gene libraries from unimmunized human donors can be constructed, and antibodies against a diverse array of antigens (including self-antigens) can be isolated essentially according to the techniques described in Marks et al., J. Mol. Biol. 222: 581-597 (1991) or Griffith et al., EMBO J. 12: 725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies can also be generated by in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

5. Antibody fragments

In some cases, it is advantageous to use antibody fragments rather than whole antibodies. The smaller size of the fragments allows for rapid clearance and can result in better access to solid tumors.

Various techniques have been developed to produce antibody fragments. Typically, these fragments are derived by proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be directly produced by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage library discussed above. Alternatively, Fab'-SH fragments can be directly recovered from Escherichia coli and chemically coupled to form F(ab') ₂ fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According to another method, F(ab') ₂ fragments can be directly isolated from recombinant host cell cultures. Other techniques for producing antibody fragments are apparent to skilled practitioners. In other embodiments, the selected antibody is a single-chain Fv fragment (scFv). See WO 93/16185; US Patent No. 5,571,894; and US Patent No. 5,587,458. Antibody fragments may also be "linear antibodies," as described, for example, in US Patent No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

6. Bispecific Antibodies

Bispecific antibodies are antibodies with binding specificity to at least two different epitopes. Exemplary bispecific antibodies can be combined with two different epitopes of β7 integrin as described herein. Other such antibodies can combine TAT binding sites with binding sites for another protein. Alternatively, anti-β7 integrin arms can be combined with the arm of triggering molecules (such as T cell receptor molecules (such as CD3), or IgG Fc receptors (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16)) in conjunction with leukocytes, to concentrate and position cellular defense mechanisms in cells expressing TAT. Bispecific antibodies can also be used to position cytotoxic agents to cells expressing TAT. These antibodies have TAT binding arms and the arm of cytotoxic agents (such as saporin, anti-interferon-α, vinca alkaloids, ricin A chain, methotrexate or radioactive isotope haptens) in conjunction with cytotoxic agents. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (eg, F(ab') ₂ bispecific antibodies).

The method for preparing bispecific antibodies is known in the art. The traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, wherein the two chains have different specificities (Millstein et al., Nature 305:537-539 (1983)). Due to the random distribution of immunoglobulin heavy chains and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule (usually carried out by affinity chromatography step) is very troublesome, and the product yield is low. Similar methods are disclosed in WO 93/08829 and Traunecker et al., EMBO J.10:3655-3659 (1991).

According to different methods, the antibody variable domain with the desired binding specificity (antibody-antigen binding site) is fused with the immunoglobulin constant domain sequence. In certain embodiments, the fusion is fused with an Ig heavy chain constant domain comprising at least a portion of the hinge region, CH2 region, and CH3 region. In certain embodiments, the first heavy chain constant region ( _CH1 ) comprising the site necessary for light chain binding is present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusion and (if desired) the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism. When the three polypeptide chains in unequal proportions used for construction provide the optimal yield of the desired bispecific antibody, this provides great flexibility for adjusting the mutual ratio of the three polypeptide fragments in the embodiment. However, when expressing at least two polypeptide chains in equal proportions results in high yield, or when the ratio has no significant effect on the yield of the desired chain combination, the coding sequences of two or all three polypeptide chains may be inserted into a single expression vector.

In certain embodiments, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations because the immunoglobulin light chain is only present in half of the bispecific molecule, providing an easy way to separate. This method is disclosed in WO94/04690. For further details on the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology 121: 210 (1986).

According to another method described in U.S. Patent number 5,731,168, the interface between antibody molecules can be transformed to maximize the percentage of heterodimers recovered from recombinant cell culture. In certain embodiments, the interface comprises at least part of the _CH3 domain. In this method, one or more little amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (for example tyrosine or tryptophan). Compensation "holes" of the same or similar size as one or more large side chains are produced on the interface of the second antibody molecule by replacing large amino acid side chains with smaller amino acid side chains (for example alanine or threonine). This provides a mechanism that increases the productive rate of heterodimers and exceeds other undesirable end products (such as homodimers).

Bispecific antibodies include cross-linked antibodies or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. For example, it has been proposed that such antibodies target immune system cells to unwanted cells (U.S. Patent No. 4,676,980) and are used to treat HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Heteroconjugate antibodies can be produced using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980, along with many cross-linking techniques.

The technology for producing bispecific antibodies from antibody fragments has also been described in the literature. For example, bispecific antibodies can be prepared by chemical linkage. Brennan et al., Science 229:81 (1985) described a method for producing F(ab') ₂ fragments by proteolytic cleavage of intact antibodies. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize the adjacent dithiol groups and prevent the formation of intermolecular disulfide. The Fab' fragments produced are then converted into thionitrobenzoate (TNB) derivatives: one of the Fab'-TNB derivatives is then converted into a Fab'-sulfhydryl group by reduction with mercaptoethylamine, and mixed with other Fab'-TNB derivatives in equimolar amounts to form bispecific antibodies. The bispecific antibodies produced can be used as a material for selectively immobilizing enzymes.

Recent advances have facilitated the direct recovery of Fab'-SH fragments from Escherichia coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) described the production of fully humanized bispecific antibody F(ab') ₂ molecules. Each Fab' fragment was secreted from E. coli and subjected to directed chemical coupling in vitro to form a bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing ErbB2 receptors and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets. Various techniques have also been described for preparing and isolating bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers are reduced at the hinge region to form monomers, which are then oxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) provides an alternative mechanism for preparing bispecific antibody fragments. The fragments contain a _VH connected to a _VL by a linker that is too short to allow pairing between the two domains on the same chain. Therefore, the _VH and _VL domains of one fragment are forced to pair with the complementary _VL and _VH domains of the other fragment, thereby forming two antigen-binding sites. Another strategy for preparing bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

7. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently bound antibodies. For example, such antibodies have been proposed to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980] and to treat HIV infection [WO 91/00360; WO92/200373; EP 03089]. It is contemplated that the antibodies can be prepared in vitro using methods known in synthetic protein chemistry (including those involving cross-linking agents). For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of reagents suitable for this purpose include iminothiolates and methyl-4-mercaptobutyrimidate and, for example, those disclosed in U.S. Patent No. 4,676,980.

8. Multivalent Antibodies

Multivalent antibodies can be internalized (and/or decomposed) faster than bivalent antibodies by cells expressing the antigens to which the antibodies bind. The antibodies of the present invention can be multivalent antibodies (e.g., tetravalent antibodies) with three or more antigen-binding sites (except IgM species), which can be easily prepared by recombinant expression of nucleic acids encoding the polypeptide chains of the antibodies. The multivalent antibody can comprise a dimerization domain and three or more antigen-binding sites. In certain embodiments, the dimerization domain comprises an Fc region or a hinge region (or consists of an Fc region or a hinge region). In this case, the antibody will comprise an Fc region and three or more antigen-binding sites at the amino terminus of the Fc region. In certain embodiments, the multivalent antibody herein comprises three to about eight, but typically four, antigen-binding sites (or consists of three to about eight, but typically four, antigen-binding sites). The multivalent antibody comprises at least one polypeptide chain (and typically two polypeptide chains), wherein the one or more polypeptide chains comprise two or more variable domains. For example, the one or more polypeptide chains may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is a polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, the one or more polypeptide chains may comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein may further comprise at least two (and typically four) light chain variable domain polypeptides. For example, the multivalent antibody herein may comprise about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated herein comprise a light chain variable domain and, optionally, further comprise a CL domain.

9. Effector Function Modification

It may be desirable to modify the antibodies of the present invention with respect to effector functions, for example, to enhance the antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cellular cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions into the Fc region of the antibody. Alternatively or in addition, one or more cysteine residues may be introduced into the Fc region to allow the formation of interchain disulfide bonds in this region. The homodimeric antibodies thus produced may have improved internalization capacity and/or enhanced complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176: 1191-1195 (1992) and Shopes, BJ, Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53: 2560-2565 (1993). Alternatively, antibodies can be engineered to have dual Fc regions, thereby providing enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989). In order to increase the serum half-life of antibodies, a salvage receptor binding epitope can be incorporated into antibodies (particularly antibody fragments) as described, for example, in U.S. Patent No. 5,739,277. The term "salvage receptor binding epitope" as used herein refers to an epitope of the Fc region of an IgG molecule (e.g., IgG ₁ , IgG ₂ , IgG ₃ , or IgG ₄ ) that is responsible for increasing the in vivo serum half-life of an IgG molecule.

10. Immunoconjugates

The antagonists or antibodies used in the methods herein are optionally conjugated to another substance, such as a cytotoxic agent or cytokine.

Conjugation will typically be achieved through covalent attachment, the exact nature of which will be determined by the attachment site on the targeting molecule and the integrin beta7 antagonist or antibody polypeptide. Typically, non-peptide agents are modified by the addition of linkers that allow conjugation to the anti-beta7 integrin antibody through amino acid side chains, carbohydrate chains, or reactive groups introduced on the antibody by chemical modification. For example, drugs can be attached through the epsilon amino group of a lysine residue, through a free alpha amino group, through disulfide exchange with a cysteine residue, or by oxidation of 1,2-diols in carbohydrate chains with periodate to allow attachment of drugs containing a variety of nucleophiles via Schiff base linkage. See, for example, U.S. Patent No. 4,256,833. Protein modifying agents include amine-reactive agents (e.g., reactive esters, isothiocyanates, aldehydes, and sulfonyl halides), thiol-reactive agents (e.g., haloacetyl derivatives and maleimides), and carboxylic acid and aldehyde-reactive agents. The integrin beta7 antagonist or antibody polypeptide can be covalently linked to the peptide agent using a bifunctional cross-linker. Heterobifunctional reagents are more commonly used and allow controlled coupling of two different proteins by using two different reactive moieties (e.g., amine reactive plus thiol, iodoacetamide, or maleimide). The use of such linking reagents is well known in the art. See, for example, Brinkley, supra, and U.S. Patent No. 4,671,958. Peptide linkers can also be used. In an alternative embodiment, the anti-beta7 integrin antibody polypeptide can be linked to the peptide portion by preparation of a fusion polypeptide.

Examples of other bifunctional protein coupling agents include N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyladipate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., 2,6-toluene diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

11. Immunoliposomes

The anti-β7 integrin antibodies disclosed herein can also be formulated as immunoliposomes. "Liposomes" are small vesicles composed of various types of lipids, phospholipids, and/or surfactants that are used to deliver drugs to mammals. The components of the liposomes are typically arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing antibodies are prepared by methods known in the art, such as Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA 77:4030 (1980); U.S. Patent Nos. 4,485,045 and 4,544,545; and WO 97/38731 published on October 23, 1997. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

Particularly useful liposomes can be produced by the reverse phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE).The liposomes are extruded through filters of defined pore size to produce liposomes with the desired diameter.

Fab' fragments of the antibodies of the present invention can be conjugated to liposomes by disulfide exchange reactions as described in Martin et al., J. Biol. Chem. 257: 286-288 (1982). A chemotherapeutic agent can optionally be contained within the liposomes. See Gabizon et al., J. National Cancer Inst. 81(19): 1484 (1989).

12. Vectors, host cells and recombinant methods for antibody production

Also provided are isolated nucleic acids encoding the anti-beta7 antibodies or polypeptide agents described herein, vectors and host cells comprising the nucleic acids, and recombinant techniques for producing the antibodies.

In order to recombinantly produce antibodies, the nucleic acid encoding it can be separated and inserted into a reproducible vector for further cloning (amplification of DNA) or for expression. In another embodiment, antibodies can be produced by homologous recombination as described in, for example, U.S. Patent number 5,204,244 (clearly incorporated herein by reference). The DNA encoding monoclonal antibodies is easy to separate and sequence-check (for example, by using oligonucleotide probes that can specifically bind to the genes of the heavy chain and light chain of the encoding antibody) using conventional methods. Many vectors are available. Vector components generally include but are not limited to one or more of the following: signal sequence, origin of replication, one or more marker genes, enhancer element, promoter and transcription termination sequence, for example, as described in U.S. Patent number 5,534,615, authorized on July 9, 1996, which is clearly incorporated herein by reference.

Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryotic, yeast, or higher eukaryotic cells described above. Prokaryotes suitable for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, Shigella, and Bacilli, such as B. subtilis and B. licheniformis (e.g., DD 12, published April 12, 1989). 266,710 ), Pseudomonas, such as Pseudomonas aeruginosa, and Streptomyces. An E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are also suitable. These examples are illustrative and non-limiting.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for anti-β7 integrin antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used lower eukaryotic host microorganism. However, many other genera, species and strains are commonly available and used herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesei (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

Suitable host cells for expressing glycosylated anti-β7 antibodies are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains and variants and corresponding permissive insect host cells have been identified from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. A variety of viral strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as viruses of the present invention, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become routine. Examples of useful mammalian host cell lines are SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma cell line (Hep G2).

Host cells are transformed with the above-described expression or cloning vectors for anti-beta7 integrin antibody production and cultured in conventional nutrient media modified as needed to induce promoters, select transformants, or amplify the gene encoding the desired sequence.

Host cells used to produce the anti-β7 integrin antibodies of the present invention can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing host cells. In addition, any of the culture media described in Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., Anal. Biochem. 102:255 (1980); U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. 30,985 can be used as culture media for host cells. Hormones and/or other growth factors (such as insulin, transferrins or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN ^™ drugs), trace elements (defined as inorganic compounds usually present at a final concentration in the micromolar range) and glucose or equivalent energy sources can be supplemented in any of these substratums as needed. Any other necessary supplements can also be included at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc. are those previously used for selected expression host cells and will be apparent to those of ordinary skill in the art.

When using recombinant technology, antibodies can be produced intracellularly, produced in the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, as a first step, particle debris (host cells or cleavage fragments) is removed by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) have described a method for separating antibodies secreted into the periplasmic space of Escherichia coli. In brief, the cell paste is melted for about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF). Cell debris is removed by centrifugation. When the antibody is secreted into the culture medium, the supernatant from this type of expression system is usually first concentrated with a commercially available protein concentration filter (such as Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors (such as PMSF) can be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

The antibody composition prepared from cells can be purified by, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, which is a typical purification technique. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are also available. Compared to the flow rate and processing time that can be achieved with agarose, mechanically stable matrices (such as controlled pore glass or poly (styrene divinyl) benzene) allow faster flow rates and shorter processing times. When the antibody comprises a _CH3 domain, purification is performed using Bakerbond ABX ^™ resin (JT Baker, Phillipsburg, NJ). Depending on the antibody to be recovered, other techniques for protein purification (e.g., fractionation on an ion exchange column, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE ^™ , chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation) may also be used. Following any one or more preliminary purification steps, the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5 and 4.5, typically at low salt concentrations (e.g., about 0 to 0.25 M salt).

C. Pharmaceutical preparations

Therapeutic formulations comprising the therapeutic agent, antagonist or antibody of the invention are prepared for storage by mixing the antibody of the desired purity with an optional physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) in the form of an aqueous solution, a lyophilized or other dry preparation. Useful carriers, excipients or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers such as phosphate, citrate, histidine and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other sugars, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as TWEEN.TM., PLURONICS.TM., or polyethylene glycol (PEG).

The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, generally those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

The active ingredient can also be entrapped in microcapsules (e.g., hydroxymethylcellulose microcapsules or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively), prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980).

The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the immunoglobulin of the present invention, which are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Patent number 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT.TM. (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprorelin acetate) and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable the release of molecules for more than 100 days, while some hydrogels release proteins for shorter periods of time. When encapsulated immunoglobulins are stored in the body for extended periods, they can denature or aggregate due to exposure to humidity at 37°C, leading to loss of biological activity and possible changes in immunogenicity. Depending on the mechanism involved, rational strategies can be devised for stabilization. For example, if the aggregation mechanism is found to be through the formation of intermolecular S-S bonds via thiodisulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling water content, using appropriate additives, and developing specific polymer matrix compositions.

D. Application

The treating physician will be able to determine the appropriate dosage for an individual, either weight-based or uniform, and will follow the instructions on the label. The preparation and administration regimens for commercially available second therapeutic agents and other compounds administered in combination with the integrin beta7 antagonist can be used according to the manufacturer's instructions or determined empirically by the skilled practitioner.

For the prevention or treatment of disease, the appropriate dosage of the integrin beta7 antagonist and any second therapeutic agent or other compound administered in combination with the non-excluded antibody will depend on the type of gastrointestinal inflammatory disorder to be treated (e.g., IBD, UC, CD), the severity and course of the disease, whether the integrin beta7 antagonist or combination is being administered for prevention or treatment, previous treatment, the patient's clinical history and response to the integrin beta7 antagonist or combination, and the judgment of the attending physician. In certain embodiments, the integrin beta7 antagonist is administered once a week, or once every two weeks, or once every four weeks, or once every six weeks, or once every eight weeks, for one month (4 weeks), or two months, three months, or six months, or 12 months, or 18 months, or 24 months, or for a long period of time throughout the patient's life. In certain embodiments, treatment is self-administered by the patient.

Depending on the type and severity of the disease, about 0.5 mg/kg to 4.0 mg/kg of anti-β7 antibody is a candidate initial dose for administration to the patient, for example, by one or more separate administrations, or by continuous infusion. For repeated administration over several days or longer, depending on the condition, treatment is continued until the desired suppression of disease symptoms occurs. However, other dosage regimens may be used.

For example, in certain embodiments, a uniform dose of an anti-β7 antibody is administered to a patient. A uniform dose is a specific amount of an anti-β7 antibody administered to each patient without regard to body weight. Depending on the type and severity of the disease, a uniform dose of an anti-β7 antibody between about 50 mg and 450 mg is administered to the patient, which may be one or more separate injections or infusions or administrations. This uniform dose may be administered intravenously or subcutaneously or by other routes described herein. In certain embodiments, the uniform dose is 50 mg, or 100 mg, or 105 mg, or 150 mg, or 200 mg, or 210 mg, or 300 mg, or 315 mg, or 400 mg, or 420 mg, or 450 mg.

In certain embodiments, an initial uniform loading dose of anti-beta7 antibody is followed by one or more maintenance doses of anti-beta7 antibody. The loading dose is a larger amount of anti-beta7 antibody than the maintenance dose. In certain embodiments, the loading dose is between 400 mg and 450 mg, and the maintenance dose is between 50 mg and 350 mg. In certain embodiments, the loading dose is 400 mg, or 420 mg, or 430 mg, or 450 mg.

Typically, the clinician will administer an antibody of the invention (alone or in combination with a second compound) until a dosage or dosages that provide the desired biological effect is reached.The progress of the therapy of the invention is readily monitored by conventional techniques and assays.

The integrin beta 7 antagonist may be administered by any suitable means, including parenteral, topical, intravenous, subcutaneous, intraperitoneal, intrapulmonary, intranasal and/or intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intrafungal, or subcutaneous administration. Intrathecal administration is also contemplated (see, e.g., U.S. Patent Publication No. 2002/0009444 to Grillo-Lopez). In addition, the integrin beta 7 antagonist may be suitably administered, for example, by pulse infusion of the antibody with gradually decreasing doses. In certain embodiments, administration is intravenous or subcutaneous. Each exposure may be provided by the same or different means of administration. In one embodiment, each exposure of the anti-beta 7 antibody is provided by subcutaneous administration. In one embodiment, the first exposure (e.g., a loading dose) of the anti-beta 7 antibody is provided by intravenous administration, and each subsequent exposure is provided by subcutaneous administration.

In certain embodiments, the anti-beta7 antibody is administered using, for example, a self-injection device, an automatic injection device, or other device designed for self-administration. A variety of self-injection devices, including automatic injection devices, are known in the art and are commercially available. Exemplary devices include, but are not limited to, prefilled syringes (such as BD HYPAK READYFILL™ and STERIFILL SCF™ from Becton Dickinson; CLEARSHOT™ copolymer prefilled syringes from Baxter; and Daikyo Seiko CRYSTAL prefilled syringes available from West Pharmaceutical Service); disposable pen injection devices, such as the BD Pen from Becton Dickinson; ultra-sharp and microneedle devices (such as INJECT-EASE™ and microinfusion sets from Becton Dickinson; H-PATCH™ available from Valeritas); and needle-free injection devices (such as those available from Bioject and patch devices available from Medtronic). In certain embodiments, rhuMAb Beta 7 is a manufactured article comprising a prefilled syringe containing 2 ML (150 mg) of rhuMAb Beta 7. In certain embodiments, rhuMAb Beta 7 is a manufactured article comprising a prefilled syringe containing 1 ML (180 mg) of rhuMAb Beta 7.

As indicated, the integrin beta 7 antagonist can be administered alone or in combination with at least a second therapeutic compound. These second therapeutic compounds are typically used at the same dose and route of administration as previously used, or at about 1% to 99% of the dose previously used. If such a second compound is used, it is used in certain embodiments in a lower amount than when the integrin beta 7 antagonist is not present to eliminate or reduce side effects caused thereby.

As also noted (eg, see below), a variety of second therapeutic compounds suitable for treating IBD (eg, ulcerative colitis and Crohn's disease) are known in the art, as are dosages and methods of administration of such second therapeutic compounds.

Administration of the integrin beta7 antagonist and any second therapeutic compound can be performed simultaneously, for example, as a single composition or as two or more different compositions using the same or different routes of administration. Alternatively, or in addition, administration can be performed sequentially in any order. In certain embodiments, there can be an interval of from a few minutes to a few days, or a few weeks to a few months between the administration of the two or more compositions. For example, the integrin beta7 antagonist can be administered first, followed by the second therapeutic compound. However, simultaneous administration or administration of the second therapeutic compound before the integrin beta7 antagonist is also contemplated.

The standard of care for individuals with active moderately to severely active UC involves treatment with standard doses of aminosialic acid, oral corticosteroids, 6-mercaptopurine (6-MP), and/or azathioprine. Treatment with an integrin beta7 antagonist (such as an anti-beta7 integrin antibody disclosed herein) will result in improved disease remission (rapid control of disease and/or prolonged remission) and/or a clinical response that is superior to the clinical response achieved with the standard of care for such individuals.

In one embodiment, the present invention provides for the treatment of inflammatory bowel disease (IBD) in a human subject suffering from IBD, comprising administering to the subject an effective amount of a therapeutic agent, such as an anti-beta7 integrin antibody, and further comprising administering to the subject an effective amount of a second agent, which is an immunosuppressant, a pain control agent, an anti-diarrhea agent, an antibiotic, or a combination thereof.

In an exemplary embodiment, the second drug is selected from aminosialic acid, oral corticosteroids, 6-mercaptopurine (6-MP) and azathioprine. In another exemplary embodiment, the second drug is another integrin beta7 antagonist, such as another anti-beta7 integrin antibody or an anti-cytokine antibody.

All of these second drugs can be used in combination with each other or themselves in combination with the first drug, so that the expression "second drug" as used herein does not mean that it is the only drug other than the first drug. Therefore, the second drug does not need to be one drug, but can consist of or contain more than one such drug.

Combined administration herein includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein there is typically a period while both (or all) active agents simultaneously exert their biological activities.

The combined administration of the second drug includes co-administration (simultaneous administration) using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein there is generally a period while both (or all) active agents (drugs) simultaneously exert their biological activities.

E. Design a treatment plan

Drug development is a complex and expensive process. The cost of bringing a new drug to market is estimated to be between $800 million and $1 billion. Less than 10% of drugs that undergo Phase I clinical trials reach approval. Two key reasons for late-stage drug failure are a lack of understanding of the relationship between dose-concentration response and unexpected safety events. Given this, it is crucial to have tools that help predict how a drug will behave in vivo and aid in the success of clinical therapeutic candidates (Lakshmi Kamath, Drug Discovery and Development; Modeling Success in PK/PD Testing Drug Discovery & Development (2006)).

Pharmacokinetics (PK) characterizes the absorption, distribution, metabolism, and clearance characteristics of a drug. Pharmacodynamics (PD) defines the physiological and biological responses to an administered drug. PK/PD modeling establishes a mathematical and theoretical link between these two approaches and helps better predict drug action. Integrated PK/PD modeling and computer-aided experimental design (CAD) through simulation are being incorporated into many drug development projects and are having an ever-increasing impact (Lakshmi Kamath, Drug Discovery and Development; Modeling Success in PK/PD Testing Drug Discovery & Development (2006)).

PK/PD testing is typically performed at every stage of the drug development process. As development becomes increasingly complex, time-consuming, and expensive, many companies are seeking to better utilize PK/PD data to eliminate flawed candidates at the outset and identify those with the highest chance of clinical success (Lakshmi Kamath, supra).

PK/PD modeling methods have been shown to be useful in determining the relationship between biomarker responses, drug levels, and dosing regimens. The PK/PD profile of a drug candidate and the ability to predict the patient's response to it are crucial for the success of clinical trials. Recent advances in molecular biology techniques and a better understanding of multiple disease targets have validated biomarkers as good clinical indicators of drug therapy efficacy. Biomarker assays (including those described herein) and the use of such biomarker assays help identify the biological response to a drug candidate. Once a biomarker is clinically validated, it is possible to effectively model the test simulation. Biomarkers have the potential to reach a substitute state, which may one day replace the clinical results in drug development (Lakshmi Kamath, above).

The amount of biomarkers such as those described herein in peripheral blood can be used to identify the biological response to integrin beta7 antagonist treatment and, therefore, can function as a good clinical indicator of the therapeutic efficacy of a candidate treatment.

Traditional PK/PD modeling in drug development defines parameters such as drug dose concentration, drug exposure effect, drug half-life, drug concentration versus time, and drug action versus time. When used more broadly, quantitative techniques such as drug modeling, disease modeling, experimental modeling, and market modeling can support the entire development process, leading to better decision-making through clear consideration of risk and better utilization of knowledge. For drug development researchers, a variety of PK/PD modeling tools are available, such as WinNonlin and the KnowledgebaseServer (PKS) developed by Pharsight, Inc. Mountain View, California.

General biomarker technology

Unless otherwise indicated, the practice of the present invention will utilize conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are fully described in the literature, for example, in "Molecular Cloning: A Laboratory Manual," 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987, and periodically updated); and "PCR: The Polymerase Chain Reaction" (Mullis et al., eds., 1994).

The primers, oligonucleotides, and polynucleotides utilized in the present invention can be generated using annotation techniques known in the art.

Provided herein are gene expression biomarkers that are associated with predicting responsiveness to certain therapeutic agents in IBD patients (including patients with ulcerative colitis or Crohn's disease). These expression levels of mRNA or individual proteins encoded by these genes constitute biomarkers for predicting responsiveness to IBD therapeutics, ulcerative colitis therapeutics, and/or Crohn's disease therapeutics. Thus, the invention disclosed herein finds use in a variety of settings, such as in methods and compositions related to the diagnosis and treatment of inflammatory bowel disease.

Detection of gene expression levels

The nucleic acid of any of the methods described herein can be RNA transcribed from genomic DNA or cDNA produced from RNA or mRNA. The nucleic acid can be derived from a vertebrate, such as a mammal. A nucleic acid is said to be "derived from" a particular source if it is obtained directly from that source, or if it is a copy of a nucleic acid found in that source.

Nucleic acids include copies of nucleic acids, such as copies generated from amplification. In some cases, it may be desirable to amplify, for example, to obtain a desired amount of material for detecting variations. The amplicon can then be subjected to variation detection methods (such as those described below) to determine the expression of certain genes.

The level of mRNA can be measured and quantified by a variety of methods known to those skilled in the art (including the use of commercially available kits and reagents). One such method is polymerase chain reaction (PCR). Another method for quantitative applications is real-time quantitative PCR or qPCR. See, for example, "PCR Protocols, A Guide to Methods and Applications," (M.A.Innis et al., ed., Academic Press, Inc., 1990); "Current Protocols in Molecular Biology" (F.M.Ausubel et al., ed., 1987, and periodically updated); and "PCR: The Polymerase Chain Reaction," (Mullis et al., ed., 1994).

Microarray is a multiplex technology that is usually carried out under high stringency conditions with an array series of tens of thousands of nucleic acid probes, for example, cDNA or cRNA sample hybridization. Detection and quantitative probe-target hybridization are usually carried out by detecting the target of fluorophore, silver or chemiluminescent labeling to measure the relative abundance of nucleic acid sequence in the target. In a typical microarray, the probe is attached to a solid surface by a covalent bond with a chemical matrix (through epoxy-silane, amino-silane, lysine, polyacrylamide or other). The solid surface is, for example, glass, silicon chip or microsphere. A variety of microarrays are available, including, for example, those manufactured by Affymetrix, Inc. and Illumina, Inc.

Biological sample can be obtained with certain method known to those skilled in the art.Biological sample can be obtained from vertebrates, especially mammals.In some cases, biological sample is synovial tissue, serum or peripheral blood mononuclear cells (PBMC).By screening this type of body sample, can reach simple early diagnosis for diseases such as ulcerative colitis and Crohn's disease.In addition, by testing this type of body sample for the variation of target nucleic acid (or encoded polypeptide) expression level, can more easily monitor treatment process.

After determining that an individual or tissue or cell sample contains a gene expression signature or relative levels of certain biomarkers disclosed herein, it is contemplated that an effective amount of an appropriate therapeutic agent can be administered to the individual for the specific disease in the individual, such as UC or Crohn's disease. A skilled practitioner can perform clinical diagnosis of a variety of pathological conditions described herein in mammals. Clinical diagnostic techniques are available in the art that allow, for example, diagnosis or detection of inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease, in mammals.

Reagent test kit

Also provided are test kits or manufactured products for applications described herein or proposed. Such test kits can include compartmentalization to tightly confine one or more container means (such as vials, tubes, etc.), each container means including one of the separate components to be used in the method. For example, one of the container means can include a probe that is detectably labeled or can be detectably labeled. Such probes can be polynucleotides specific to the polynucleotides of one or more genes that comprise a gene expression signature. When the test kit utilizes nucleic acid hybridization to detect a target nucleic acid, the test kit can also include a container comprising one or more nucleic acids for amplifying the target nucleic acid sequence and/or a container comprising a reporter means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzyme, fluorescence, or radioisotope labeling.

The kit will typically comprise the above-mentioned container and one or more other containers comprising materials desired from a commercial and user perspective, including buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use. A label may be present on the container to indicate that the composition is for a specific therapeutic or non-therapeutic application, and may also indicate directions for in vivo or in vitro use, such as those described above. Other optional components in the kit include one or more buffers (e.g., blocking buffer, wash buffer, substrate buffer, etc.), other reagents (e.g., substrates chemically altered by enzyme labeling (e.g., chromogens), epitope retrieval solutions, control samples (positive and/or negative controls), control sections, etc.

Sales Methods

The present invention also encompasses methods for marketing a therapeutic agent or pharmaceutically acceptable composition thereof, comprising promoting, explaining, and/or detailing to a target audience the use of the therapeutic agent or pharmaceutical composition thereof in treating a patient or patient population suffering from a particular disease (e.g., UC or Crohn's disease) from whom a sample has been obtained and which exhibits relative levels of a gene expression signature or serum biomarker disclosed herein.

Marketing is paid coverage, typically through impersonal media, in which the sponsor is identified and the message is controlled. Marketing for the purposes of this document includes publicity, public relations, product placement, sponsorship, underwriting, and sales promotion. The term also includes sponsored information announcements appearing in any printed media designed to appeal to a broad audience to persuade, inform, promote, inspire, or otherwise change behavior toward purchasing, supporting, or endorsing the advantageous model of the invention.

Marketing of the diagnostic methods herein can be achieved by any means. Examples of marketing media used to deliver these messages include television, radio, movies, magazines, newspapers, the Internet, and billboards, including advertisements, which are messages that appear in broadcast media.

The type of marketing used will depend on many factors, such as the nature of the target audience to be influenced, such as hospitals, insurance companies, clinics, doctors, nurses and patients, as well as cost considerations and relevant jurisdictional laws and regulations governing the marketing of pharmaceuticals and diagnostics. Marketing can be individualized or personalized based on user characteristics determined through service interactions and/or other data, such as user demographics and geographic location.

The foregoing written description and the following examples are considered sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the following examples and fall within the scope of the appended claims.

It will be appreciated that application of the teachings of the present invention to a specific problem or situation will be well within the capabilities of one of ordinary skill in the art given the guidance of the teachings contained herein.

Further details of the present invention are illustrated by the following non-limiting examples.The disclosures of all citations in this specification are expressly incorporated herein by reference.

Example

Example 1

A Phase II, randomized, double-blind, placebo-controlled study and open-label extension to evaluate the efficacy and safety of rhuMAb β7 (etrolizumab) in patients with moderate to severe ulcerative colitis

Clinical study description

rhuMAbβ7 (etrolizumab) Description

rhuMAbβ7 (etrolizumab) is a humanized monoclonal antibody based on the human IgG1 subclass III _VH , kappa subclass IV _L consensus sequence, and is specific for the β7 subunit of the integrin heterodimer. See Figures 1A and B. It has been shown to bind with high affinity to α4β7 ( _Kd of approximately 116 pM) and αEβ7 ( _Kd of approximately 1800 pM).

This recombinant antibody has two heavy chains (446 residues) and two light chains (214 residues) covalently linked by interchain and intrachain disulfide bonds typical of IgG1 antibodies. For the studies described herein, it was produced in Chinese hamster ovary (CHO) cells. The molecular weight of the intact, non-glycosylated rhuMAb β7 molecule is approximately 144 kDa. Each heavy chain of rhuMAb β7 has a conserved N-linked glycosylation site at Asn297. The oligosaccharides present at this site are typical of those observed in recombinant antibodies expressed in CHO cells, with the predominant glycoforms being sialic acid-free, bibranched G0 and G1 glycans. The most prevalent form of rhuMAb β7, containing two G0 glycans and no C-terminal lysine residue, has a mass of approximately 147 kDa.

The rhuMAb β7 (etrolizumab) drug product and placebo are manufactured by Genentech. They are clear to slightly opalescent, colorless to slightly yellowish aqueous solutions. Both solutions are sterile and preservative-free liquids intended for IV and SC administration.

Study Design

Study Description

This Phase II study is a randomized, double-blind, placebo-controlled, multicenter study evaluating the efficacy and safety of two rhuMAb β7 dose levels compared to placebo in patients with moderate to severe UC. The primary efficacy endpoint was assessed at Week 10 (2 weeks after the last dose of study drug), and the secondary efficacy endpoint was assessed at Week 6.

Patients were randomized in a 1:1:1 ratio across a dose range of rhuMAb β7 100 mg SC (flat dose) at Weeks 0, 4, and 8, 420 mg SC (flat loading dose) at Week 0, followed by 300 mg SC at Weeks 2, 4, and 8, or matching placebo SC. The study protocol is shown in Figure 2. The study consisted of a 0-35 day screening period, a 10-week double-blind treatment period, an 18-week safety follow-up period, and a 17-month progressive multifocal leukoencephalopathy (PML) follow-up period (2 years after randomization).

The dosage values provided in the previous paragraph are nominal doses. Phase II dose administration uses a vial and syringe with a vial concentration of 150 mg/ml. In order to be able to perform consistent and accurate dose administration, 0.7 ml is selected as the volume of each subcutaneous (SC) injection. Therefore, the actual drug amount of the nominal 100 mg dose arm is 105 mg (1 x 0.7 ml SC injection), and the actual drug amount of the nominal 300 mg dose is 315 mg (3 x 0.7 ml SC injection). The actual loading dose of 420 mg is 420 mg (4 x 0.7 ml SC injection). All SC injections are administered into the abdomen. Therefore, the dosage of "100 mg" and the dosage of "105 mg" are used interchangeably herein. In addition, the dosage of "300 mg" and the dosage of "315 mg" are used interchangeably herein. Finally, in some cases herein, as a shorthand and for convenience, the trial arm in which the patient receives "300 mg + loading dose (LD)" is referred to as the "300 mg dose." Therefore, unless the context clearly indicates otherwise, "300 mg + loading dose" is equivalent to "300 mg dose."

To be eligible, patients must have a duration of at least 12 weeks of UC diagnosed according to the American College of Gastroenterology (ACG) practice guidelines; that is, clinical and endoscopic evidence confirmed by histopathology reports, in some cases evidence of moderate to severe disease confirmed by MCS ≥ 5 or in some cases by MCS ≥ 6, including an endoscopy subscore ≥ 2, a rectal bleeding subscore ≥ 1 (see Table 1), and endoscopic evidence of disease activity at least 25 cm from the anal verge. Other inclusion and exclusion criteria for this study are provided in International Patent Application Publication No. WO/2012/135589.

Table 1. Mayo Clinic Scoring System for Assessing Ulcerative Colitis Activity

^a Each patient served as his or her own control to establish the degree of abnormal stool frequency.

^b Daily bleeding score represents the worst bleeding on that day.

^The physician's global assessment recognized three other criteria: the patient's daily memory of abdominal discomfort and general sense of well-being, and other observations, such as physical examination findings and the patient's performance status.

Prior to randomization, patients must have been on stable doses of concomitant medications for UC. Oral 5-aminosalicylic acid (5-ASA) and immunosuppressants (azathioprine [AZA], 6-mercaptopurine [6-MP], or methotrexate) doses must have remained stable for at least 4 weeks prior to randomization on Day 1. Patients receiving topical 5-ASA or glucocorticoids must have discontinued their medication for 2 weeks prior to randomization on Day 1. Oral glucocorticoid doses must have remained stable for 2 weeks prior to randomization on Day 1. Patients receiving high-dose steroids must have had their dose reduced to ≤20 mg/day for 2 weeks prior to randomization on Day 1. For patients receiving oral glucocorticoids during the study treatment period, steroid tapering must have begun at Week 10 at a rate of 5 mg of prednisone or prednisone equivalents per week for 2 weeks, followed by 2.5 mg of prednisone or prednisone equivalents per week until discontinuation. For patients receiving oral immunosuppressants (other than oral glucocorticoids), tapering of the immunosuppressant must have begun by Week 8, and patients must have completely discontinued the immunosuppressant by Week 10. Patients previously receiving anti-TNF therapy must have discontinued treatment for at least 8 weeks prior to randomization to receive study drug on Day 1. If a patient experiences persistent or increasing disease activity at any time during the study, rescue therapy in the form of increased steroid and/or immunosuppressant doses may be increased or initiated based on the clinical judgment of the investigator. Patients requiring rescue therapy were allowed to remain in the study but discontinued study treatment and were classified as having experienced treatment failure during data analysis.

Patients are evaluated to determine if they have failed to respond to conventional treatment (including at least one anti-TNF agent). As used herein, loss of response and/or tolerance to anti-TNF agents and immunosuppressants refers to the following. For anti-TNF agents, loss of response and/or tolerance refers to symptoms of active disease that persist despite prior treatment with one or more of the following: (a) infliximab: 5 mg/kg IV, 3 doses over 6 weeks, assessed at week 8; (b) adalimumab: one 160 mg SC dose at week 0, then one 80 mg dose at week 2, then 40 mg at weeks 4 and 6, assessed at week 8; or recurrence of active symptoms during regularly scheduled maintenance doses after a previous response (selective treatment interruption is not eligible for patients who have responded and have not lost response); or a history of tolerance to at least one anti-TNF antibody (including but not excluding or limited to infusion-related reactions or injection site reactions, infection, congestive heart failure, demyelination). For immunosuppressive agents, loss of response and/or tolerance refers to the persistence of symptoms of active disease despite prior weekly treatment for at least 8 weeks with one or more azathioprine (≥1.5 mg/kg) or equivalent doses of 6-mercaptopurine mg/kg (≥0.75 mg/kg) or methotrexate 25 mg SC/IM (or as indicated); or a history of intolerance to at least one immunosuppressive agent (including but not excluding pancreatitis, drug fever, rash, nausea/vomiting, elevated liver function tests, genetic mutations in thiopurine S-methyltransferase, infection).

Randomization to study treatment was stratified by concurrent glucocorticoid therapy (yes/no), concurrent immunosuppressant therapy (yes/no), prior anti-TNF exposure (yes/no) (except for patients randomized in the United States), and study site.

UC disease activity was assessed using the MCS at screening (considered to be the baseline MCS), week 6 (2 weeks after the week 4 dose), and week 10 (2 weeks after the last dose of study drug). Biopsies of the colon were obtained during flexible sigmoidoscopy at these same time points. Partial MCS were also collected throughout the study. Patient-reported outcomes (PROs) were also assessed using the Brief Inflammatory Bowel Disease Questionnaire (SIBDQ), which patients completed on day 1 and at weeks 6 and 10. In addition, disease activity, daily symptoms, and UC impact were collected in a patient diary, which patients completed daily from screening (approximately 7 days before day 1 until day 1) and at least 7 days before the week 6 and 10 study visits until the week 6 and 10 study visits. Serum and fecal samples were also obtained for biomarker analysis. Stool was obtained at screening and at weeks 6, 10, and 28 for biomarker measurement. Exemplary biomarkers contemplated for measurement include, but are not limited to, lipocalin, calprotectin, and lactoferrin. Serum and plasma were obtained at screening, day 1, and weeks 4, 6, 10, 16, and 28 for measurement of exploratory biomarkers.

The primary efficacy endpoint of this study was the proportion of patients who achieved clinical remission (defined as an absolute reduction in the MCS to ≤2, with no individual subscore exceeding 1 point) by Week 10. Other secondary endpoints are listed in the study outcome measures described below.

Outcome measures

Main effectiveness outcome measures

The primary efficacy outcome measure was clinical remission at week 10. Clinical remission was defined as an MCS ≤ 2, with no individual subscore exceeding 1 point (see Table 1).

Secondary effectiveness outcome measures

Secondary efficacy outcome measures for this study were: (1) clinical response at Weeks 6 and 10, where clinical response was defined as a decrease in MCS of at least 3 points and a 30% reduction from baseline, a decrease in rectal bleeding subscore of ≥1 point, or an absolute rectal bleeding score of 0 or 1; (2) clinical remission (defined above) at Week 6; and (3) endoscopy score and a pointer to a rectal bleeding score of 0 at Weeks 6 and 10.

Exploratory outcome measures

The exploratory outcome measure used in this study was time to UC flare in patients who achieved response or remission.For this outcome measure, flare was defined as a 2-point reduction in the partial MCS accompanied by 3 consecutive days of rectal bleeding and an endoscopic score of 2 on flexible sigmoidoscopy.

Safety outcome measures

The safety and tolerability of rhuMAbβ7 were assessed using the following measures: (1) the incidence of adverse events and serious adverse events graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0 (NCI CTCAE, version 4); (2) clinically significant changes in vital signs and safety laboratory measurements; (3) discontinuations due to adverse events; (4) the incidence and nature of injection site reactions and hypersensitivity reactions; (5) the incidence of infectious complications; and (6) immunogenicity as measured by the incidence of ATA.

Pharmacokinetic outcome measures

Pharmacokinetic outcome measures included the following: (1) Cmax after the first and last doses; (2) time to maximum concentration (Tmax) after the first and last doses; (3) area under the serum concentration-time curve (AUC) during the dosing interval after the last dose; (4) AUC from time 0 to the time of the last detectable observation (AUClast) or to infinity (AUCinf); (5) apparent clearance (CL/F); (6) apparent volume of distribution (V/F); and (7) elimination half-life (t1/2).

Example 2 - Predictive Biomarker Studies and Analysis

Experimental design for selecting predictive biomarkers of efficacy in etrolizumab-treated patients

To identify novel biomarkers predictive of response to etrolizumab treatment, we first used RNA sequencing to establish baseline gene expression profiles in colon biopsies from all etrolizumab-treated patients. Baseline biopsies from etrolizumab-treated patients who had not received a TNF antagonist were used to identify differential gene expression profiles between etrolizumab-treated patients who experienced remission and those who did not. Differentially expressed genes were further selected based on signal intensity, biological relevance, and expression in other IBD datasets and then evaluated by quantitative polymerase chain reaction in colon biopsies from etrolizumab-treated and placebo-treated patients for subgroup analysis.

Intestinal biopsy collection and processing

Intestinal biopsies were collected from study participants during screening visits (up to 4 weeks before treatment) and flexible sigmoidoscopy/pancolonoscopy at weeks 6 and 10 after starting etrolizumab treatment. Biopsies were collected from the most severely inflamed colon area within 10-40 cm of the anal verge. Biopsies were avoided in necrotic areas of ulcerated mucosa or at suture sites in patients who had previously undergone colectomy. Biopsies were placed in tissue RNA stabilization buffer (RNAlater, Qiagen, catalog number 76104) and shipped frozen, or they were stored in formalin and processed later.

RNA isolation and sequencing

Upon receipt, biopsy tissue samples were thawed, homogenized with 3 mm steel beads using a TissueLyzer (Qiagen, catalog number 69989), and RNA was isolated using the RNeasy Mini kit (Qiagen, catalog number 74106) according to the manufacturer's instructions. RNA integrity was assessed using an Agilent RNA 6000 Pico kit (Agilent Technologies, catalog number 5067-1513) and an Agilent 2100 Bioanalyzer. Samples with low RNA quality (RIN ≤ 5) were excluded from analysis. RNA was quantified using the Quant-iT ^™ RNA Assay kit (Life Technologies Corporation, Carlsbad, CA, USA).

High-quality total RNA (250 ng) was loaded into the Illumina TruSeq RNA Sample Prep Kit v2 workflow and run on the Beckman Coulter Life Sciences Biomek liquid handling platform. RNA libraries were evaluated using Agilent 2200 TapeStation High Sensitivity D1K Tape and qPCR with the KAPA Library Quantification Kit for Illumina Sequencing. 2 nM of the library (12 samples per lane) was loaded for cluster generation and sequenced on the Illumina HiSeq 2000 Sequencing System using 2 x 50 bp read length plus index reads. Passing filter reads were measured, and fastq files were generated using Illumina CASAVA v1.8.

Identification of differentially expressed genes

RNA sequencing data processing and analysis were performed using the R programming language (http://www.r-project.org) along with software packages from the Bioconductor project (http://bioconductor.org). Raw RNA sequencing reads were processed using the HTSeqGenieBioconductor software package. Briefly, reads were aligned to the reference human genome sequence (NCBI build 37) using the GSNAP algorithm (Wu, T.D. et al., Bioinformatics (Oxford, England) 26, 873-881 (2010)). Pairs of uniquely aligned reads that fall within exons were counted to provide estimates of individual gene expression levels.

To calculate differential expression, we used the DESeq2 Bioconductor software package (Anders et al., Genome Biology (2010), vol. 11(10), p. R106), which fits negative binomial generalized linear models to determine log fold changes and p-values for group differences. We used the default library size estimation method from this software package to account for differences in sequencing depth between individuals. Our final analysis included only samples from patients who had not received TNF. The final generalized model included covariates for whether the patient was in remission at week 10, technical aspects of the sequencing response (sequencing plate and lane), and the endoscopic subscore of the Mayo Clinic score. For our statistical analyses, we used nominal (non-multiple testing corrected) p-values to rank candidate genes associated with remission.

Gene expression analysis by quantitative polymerase chain reaction

RNA isolated from the biopsy tissue described above was reverse transcribed into complementary deoxyribonucleic acid using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies Corporation, Carlsbad, CA, USA). Gene expression levels were assessed by real-time polymerase chain reaction (also known as quantitative polymerase chain reaction). Taqman gene expression assays (all from Life Technologies Corporation, Carlsbad, CA, USA) were used for each gene according to the manufacturer's instructions. Real-time polymerase chain reaction was run using TaqMan PreAmp Master Mix (Life Technologies Corporation, Carlsbad, CA, USA) and reagents (Fluidigm) on the BioMark ^™ HD System (Fluidigm Corporation, South San Francisco, CA, USA). Target gene expression was normalized for GAPDH expression using the ΔCt method.

Statistical analysis

Statistical analysis was performed using the Wilcoxon rank sum test or Fisher's exact test as needed. No correction for multiple comparisons was performed. Subgroup analysis using sample median cutoffs was performed for selected genes. The longitudinal stability of gene expression was evaluated in patients receiving placebo, where the concordance rate of biomarker low or biomarker high classification was determined from samples measured from the same patient at baseline, week 6, and week 10. Intra-patient variability was also assessed by calculating the standard deviation of samples from the same patient at baseline, week 6, and week 10.

result

According to the methods and analysis described above, RNA sequence readout results were used to identify differentially expressed genes between patients who experienced remission after etrolizumab treatment and patients who did not experience remission after etrolizumab treatment. The results are summarized in Tables 2 and 3 below. Table 2 shows differentially expressed genes with high baseline expression associated with no remission after etrolizumab treatment. Table 3 shows differentially expressed genes with high baseline expression associated with remission after etrolizumab treatment. As described above, differentially expressed genes were identified in baseline colon biopsy tissue obtained from patients who had not received TNF antagonists. Other baseline variables were controlled using a generalized model that accounted for race/ethnicity, gender, technical batch, and baseline endoscopy score.

Table 2. High Baseline Gene Expression Associated with No Response After Etrolizumab Treatment

Table 3. High Baseline Gene Expression Associated with Response After Etrolizumab Treatment

After identifying differentially expressed candidate genes by RNA sequencing analysis, we selected a subset of these genes (n=46) for further analysis based on signal intensity, biological relevance, and expression patterns in other datasets. For these genes, gene expression in baseline biopsies from all patients (n=106) was quantified by quantitative polymerase chain reaction (qPCR), and enrichment was assessed using the median cutoff method. These analyses were performed in combination with the two different etrolizumab doses described in Example 1. Figure 5 shows the odds ratios for the difference in efficacy across all participants with respect to remission for the indicated genes. In Figure 5, an odds ratio of 1 (no efficacy) is shown as the dashed line in the center of the box; positive efficacy is shown to the right of the median, and negative efficacy is shown to the left of the median. Thus, for example, above-median expression of granzyme A (GZMA) and FoxM1 and below-median tissue expression of TNFSF15 and SLC8A3 were associated with remission after etrolizumab treatment. The qualitative results from Figure 5 and elsewhere are summarized in Tables 4 and 5 below.

Table 4. Genes with expression levels above the median associated with etrolizumab responsiveness among all participants (“highly expressed predictive genes” or “HEPGs”).

Table 5. Genes with expression levels below the median among all participants were associated with etrolizumab responsiveness (“lowly expressed predictive genes” or “LEPGs”).

Gene symbol Clinical endpoint: remission TNFSF15 X SLC8A3 (organization) X MLK7.AS1 X CCL3 X VNN3 X INHBA X VNN2 X IL18RAP X REM2 X TMEM154 X CCL2 X IL1A X LRRC4 X HAMP X LMO4 X MUCL1 X LIF X TM4SF4 X FGF7 X BEST2 X CCL3L3 X MX1 X UROS X SSTR2 X CPA3 X MT1M X

Figure 6 shows the odds ratios for differences in response to treatment for the indicated genes in terms of remission in anti-TNF-naive patients. In Figure 6, an odds ratio of 1 (no response) is shown as the dashed line in the center of the box; positive responses are shown to the right of the median, and negative responses are shown to the left of the median. Thus, for example, expression of granzyme A (GZMA) above the median and expression of SLC8A3 below the median were associated with remission after etrolizumab treatment. The qualitative results from Figure 6 and elsewhere are summarized in Tables 6 and 7 below.

Table 6. Genes expressed above the median in TNF-naive patients are associated with etrolizumab responsiveness ("highly expressed predictive genes" or "HEPGs").

Gene symbol Clinical endpoint: remission FASLG X CCL4 X GZMA X FOXM1 X ECH1 X KLRB1 X TMIGD2 X ITGAE X KCNMA1 X GZMB X PHF14 X CPA2 X DDO X TMEM200A X FAM125B X CXCR6 X SLC8A3 (blood) X

Table 7. Genes with below-median expression levels associated with etrolizumab responsiveness in TNF-naive patients ("lowly expressed predictive genes" or "LEPGs").

Gene symbol Clinical endpoint: remission PMCH X TNFSF15 X SLC8A3 (organization) X MLK7.AS1 X CCL3 X VNN3 X INHBA X VNN2 X IL18RAP X REM2 X TMEM154 X CCL2 X IL1A X LRRC4 X HAMP X LMO4 X MUCL1 X LIF X TM4SF4 X FGF7 X BEST2 X CCL3L1/3 X UROS X SSTR2 X CPA3 X MT1M X

Two-way clustering of baseline gene expression using the selected enriched genes allowed us to identify two distinct patient clusters. As shown in Figure 7A, patients with high expression of T cell-related genes (integrin αE, KLRB1, FOXM1, GZMA, TMEM200A) and low expression of bone marrow/neutrophil genes (IL1A, VNN2, VNN3) were more likely to experience remission. In addition, Figure 7B shows that genes with higher baselines in responders tend to be correlated, while genes with higher baselines in non-responders tend to be correlated.

We then analyzed the percentage of patients who achieved remission, mucosal healing, and response for certain selected genes. Figure 8 shows that gene expression above the median level of the genes shown is enriched for remission in patients treated with etrolizumab in all patients (left half of each figure) and in patients who had not received anti-TNF (right half of the figure). The genes whose high expression is enriched for remission shown in Figure 8 are (A) granzyme A, (B) KLRB1, (C) FOXM1, and (D) integrin αE. For the results shown in Figure 8, we also qualitatively assessed the longitudinal stability of gene expression levels in samples from the placebo arm of the study by measuring the gene expression level of each of granzyme A, KLRB1, FOXM1, and αE in biopsies obtained from individual patients at screening, day 43, and day 71 (i.e., gene expression is above the median, at the median, or below the median). These samples were paired for consistency comparison, and the average consistency was calculated. The average concordance in the placebo samples was 67% for granzyme A, 71% for KLRB1, 70% for FOXM1, and 57% for αE. Without being bound by theory, it is believed that genes with higher average concordance reflect more stable aspects of the underlying biology than genes with lower average concordance, and therefore such concordant genes may ultimately prove more robust as biomarkers enriched for responsiveness to anti-integrin β7 antagonists.

Additional results for each of granzyme A, KLRB1, FOXM1, and αE are shown in Figures 9-12, respectively. As shown in each of Figures 9-12, consistent with the above results, high baseline expression of each of the identified genes was enriched for etrolizumab responsiveness as assessed by remission. In addition, the data in Figures 9-12 show that high baseline expression of each of GZMA, KLRB1, FOXM1, and ITGAE was enriched for etrolizumab responsiveness as assessed by mucosal healing and clinical response, but the degree of enrichment was less significant. Figures 18A-D show data for another gene, ECH1, whose baseline expression was examined in screening biopsies and evaluated for etrolizumab responsiveness by remission, mucosal healing, and clinical response.

We also observed that certain genes were enriched in patients with baseline gene expression below the median level in screening biopsy tissue for responsiveness to etrolizumab treatment. As shown in Figure 13, the gene expression below the median level was enriched in patients treated with etrolizumab in all patients (the left half of each figure) and in patients who had not received anti-TNF (the right half of the figure). The genes whose low expression enrichment was alleviated shown in Figure 13 were SLC8A3 (Figure 13A), TNFSF15 (Figure 13B), CCL2 (Figure 13C) and BEST2 (Figure 13D). For the results shown in Figure 13, we also measured the gene expression level of each of SLC8A3, TNFSF15, CCL2 and BEST2 in the biopsy tissue obtained from individual patients at screening, the 43rd day and the 71st day, and qualitatively (i.e., gene expression was above the median, in the median or below the median) assessed the longitudinal stability of gene expression levels in the samples from the placebo arm of the study. These samples were paired for consistency comparison, and average consistency was calculated. The average concordance in the placebo samples was 66% for SLC8A3, 75% for TNFSF15, 70% for CCL2, and 63% for BEST2. As discussed above, without being bound by theory, it is believed that genes with higher average concordance reflect more stable aspects of the underlying biology than genes with lower average concordance, and therefore such concordant genes may ultimately prove more robust for use as biomarkers enriched for responsiveness to anti-integrin β7 antagonists.

Other results for each of SLC8A3, TNFSF15, CCL2, and BEST2 are shown in Figures 14-17, respectively. As shown in each of Figures 14-17, consistent with the above results, low baseline expression of each of the identified genes was enriched for etrolizumab responsiveness assessed by remission. In addition, the data in Figures 14-17 show that low baseline expression of each of SLC8A3, TNFSF15, CCL2, and BEST2 was enriched for etrolizumab responsiveness assessed by mucosal healing and clinical response, but the degree of enrichment was less significant. Figures 19A-D show data for another gene, VNN2, that was examined for baseline expression in screening biopsy tissue and evaluated for etrolizumab responsiveness by remission, mucosal healing, and clinical response.

In summary, we show that by examining differential gene expression patterns between etrolizumab-treated patients who did not respond to the drug (assessed by clinical endpoints of remission, mucosal healing, or clinical response) and etrolizumab-treated patients who responded to the drug (using the same clinical endpoints), we were able to identify many genes expressed at levels above the median that correlated with etrolizumab response or non-response. Further analysis allowed us to identify many genes with high or low baseline expression that were enriched for etrolizumab responsiveness. Specifically, we determined that high baseline expression of granzyme A, KLRB1, and FOXM1 was enriched for etrolizumab responsiveness as assessed by remission, mucosal healing, and clinical response. Each of these genes demonstrated approximately 70% concordance in three longitudinal biopsies from patients in the placebo arm of the trial. The results reported here confirm and extend our previously reported findings, which showed that high baseline αE expression and certain other genes with high baseline expression were enriched for etrolizumab responsiveness (see, for example, 2014/055824).

In the study reported here, we also determined that low baseline expression of SL8A3 (in biopsies) and TNFSF15 was enriched for etrolizumab responsiveness as assessed by remission, mucosal healing, and clinical response. Each of these genes also demonstrated approximately 70% concordance in three longitudinal biopsies from patients in the placebo arm of the trial.

Peripheral blood gene expression analysis

Untreated patient sample cohort

Cohort 1: Ileal and colon biopsy samples were collected from UC patients (n=30), CD patients (n=67), and non-IBD healthy controls (n=14) during ileocolonic examinations. Biopsies were collected from areas judged to be inflamed or non-inflamed by the endoscopist. RNA was isolated from the biopsy samples as detailed above. Cohort 2: Peripheral blood samples were collected from UC (n=31) or CD (n=32) patients undergoing bowel resection into PAXgene RNA tubes. Additional PAXgene samples were collected from normal healthy controls (n=10). Cohort 3: Colonic biopsies were collected from both inflamed and non-inflamed areas of the colon from both UC patients (n=13) and healthy controls (n=8), placed in RNAlater and then RNA isolated as detailed above, placed in formalin, or immediately processed for cell sorting as detailed above.

Collection and processing of peripheral blood RNA

In the etrolizumab Phase 2 study samples, total RNA was isolated from frozen PAXgene blood tubes by automated separation on a KingFisher ^™ (Thermo Scientific) magnetic particle separator. Briefly, the tubes were thawed at room temperature for 16 hours. After centrifugation and washing to collect the leukocyte pellet, the cells were lysed in a buffer containing guanidine. Organic extraction was performed, and then the binding buffer and magnetic beads in the formulation for KingFisher ^™ operation were added. The method was optimized for the retention of microRNA and included a DNAse treatment step and cleaning before eluting from the magnetic beads. The purity and amount of the total RNA samples were measured by absorbance readings at 260 and 280 nm using a NanoDrop ND-1000UV spectrophotometer. The integrity of the total RNA was assessed by microfluidic electrophoresis on an Agilent Bioanalyzer 2100 using a NanoAssay and CaliperLabChip system. In peripheral blood samples from untreated patient samples (group 2), RNA was isolated using the PAXgene Blood RNA Kit IVD (Qiagen) according to the manufacturer's instructions. RNA was quantified using a Nanodrop spectrophotometer, and RNA integrity was assessed on an Agilent 2100 Bioanalyzer using the RNA 6000 Pico kit (Agilent Technologies).

Isolation of cells from colon biopsy tissue

Colonic biopsies from untreated patient samples (mild to severely active UC patients from cohort 3 (described above)) were processed into single-cell suspensions using previously published methods. Briefly, mononuclear cells were extracted by incubation with 5 mM 1,4-dithiothreitol followed by digestion with 1.5 mg/ml collagenase VIII and 0.05 mg/ml DNase I (all reagents from Sigma, St Louis, MO, USA). Cell suspensions were stained with the following dyes: CD45 PeCy5, TCRαβ PeCy7, CD8α APC Cy7, αE integrin (CD103) FITC, β7 integrin APC (all from Biolegend, London, UK), and CD8β PE (Beckman Coulter, Brea, CA, USA). Based on their expression of αE and β7 integrins, TCRαβ+, CD4+, or CD8+ T cells were sorted directly into RLT buffer containing β-mercaptoethanol (Qiagen, Hilden, Germany), and RNA was isolated from them using the PicoPure RNA isolation kit according to the manufacturer's protocol (Life Technologies Corporation, Carlsbad, CA, USA). The purity of the sorted population was assessed to ensure that the sample purity was ≥85%.

Gene expression analysis by quantitative polymerase chain reaction

The RNA separated from the PAXgene tube and sorted cells described above was reverse transcribed into complementary deoxyribonucleic acid using High-Capacity cDNA Reverse Transcription Kit (Life Technologies Corporation, Carlsbad, CA, USA). Gene expression levels were assessed by real-time polymerase chain reaction (also referred to as quantitative polymerase chain reaction). Taqman gene expression assays (all from Life Technologies Corporation, Carlsbad, CA, USA) were used for each gene according to the manufacturer's instructions. Real-time polymerase chain reaction was run on the BioMark ^™ HD System (Fluidigm Corporation, South San Francisco, CA, USA) using TaqMan PreAmp Master Mix (Life Technologies Corporation, Carlsbad, CA, USA) and reagents (Fluidigm). Target gene expression was normalized for GAPDH expression using the ΔCt method.

Immunohistochemistry and cell quantification

Formalin-fixed tissue samples from the etrolizumab Phase II study were embedded in paraffin blocks and cut into 4 μM sections for staining. Staining was performed with anti-integrin αE antibody (EPR4166; Abcam plc, Cambridge, MA, USA) on a Benchmark XT (Ventana Medical Systems, Inc., Tucson, AZ, USA) automated stainer, developed with 3,3″-diaminobenzidine, and counterstained with hematoxylin. Whole-slide images were acquired at 200× final magnification using an Olympus Nanozoomer automated slide scanning platform (Hamamatsu Photonics KK, Bridgewater, NJ, USA). Scanned slides were analyzed as 24-bit RGB images in a software package (version R2012a; The MathWorks, Inc., Natick, MA, USA). Total cells, αE ⁺ cells, and αE associated with the crypt epithelium were counted. A combination of support vector machines and genetic programming was used to identify crypt epithelial areas in which individual nuclei were segmented and then assessed as immunohistochemically positive when ≥25% of their total area colocalized with ^3,3 "-diaminobenzidine.

Double immunofluorescence studies in untreated patient samples (cohort 3 (described above)) were performed on formalin-fixed paraffin-embedded tissue samples using the anti-integrin αE antibody described above and anti-granzyme A polyclonal goat IgG (R&D Systems, Minneapolis, MN, USA) incubated for 60 minutes at 37° C. Anti-goat Omnimap-HRP was used as detection (antibody), and sections were stained with DAPI and hematoxylin-II (Ventana Medical Systems, Inc., Tucson, AZ, USA).

Statistical analysis

Statistical analysis was performed with the Wilcoxon rank-sum test or Fisher's exact test, as appropriate. No correction for multiple comparisons was performed. Subgroup analyses using sample median cutoffs were performed for selected genes. Longitudinal stability of gene expression was assessed in patients randomized to the placebo arm of the study.

result

Baseline gene expression in peripheral blood RNA samples was evaluated for prediction of response to etrolizumab using the methods and analyses described above. We analyzed baseline and day 1 peripheral blood samples from all patients (n=107). Gene expression was quantified by quantitative polymerase chain reaction (qPCR) and enrichment was assessed using the median cutoff method. These analyses were performed in combination with the two different etrolizumab doses described in Example 1.

We analyzed the percentage of patients who achieved remission, mucosal healing, and response in patients stratified by baseline peripheral blood gene expression levels (low (below the median) vs. high (at or above the median)). Figures 20A-D show that in patients treated with etrolizumab, remission was enriched for ITGAE gene expression at and above the median level in all patients. As shown in Figures 21A-D, we also observed enrichment for remission in patients with high (at or above the median) baseline peripheral blood ECH1 expression. Peripheral blood gene expression of FOXM1 (Figures 22A-D), GZMA (Figures 23A-D), and KLRB1 (Figures 24A-D) was also evaluated. As shown in Figures 25A-D, unlike the results observed when screening biopsy tissue gene expression (Figures 14A-D), patients with high levels of SLC8A3 peripheral blood gene expression had improved remission in response to etrolizumab treatment.

The SLC8A3 gene encodes the sodium/calcium exchanger NCX3, which acts as a membrane transporter for bidirectional sodium and calcium exchange to maintain calcium homeostasis in cells. NCX3 is expressed on human macrophages and myofibroblasts. Although the relationship between tissue cells and circulating cells of the monocyte/macrophage lineage is not fully understood, without being limited by theory, it may be that low monocyte/macrophage cell counts in biopsy tissue indicate higher levels of circulating monocytes/macrophages. In this case, patients with low tissue monocyte/macrophage content indicated by low SLC8A3 gene expression can roughly overlap with patients with high circulating monocyte/macrophage content indicated by high SLC8A3 gene expression. Therefore, low levels of SLC8A3 gene expression in tissues are correlated with high levels of SLC8A3 gene expression in peripheral blood, and this relationship explains why low levels of expression in tissues (Figures 14A-D) and high levels of expression in peripheral blood (Figures 25A-D) can each predict responsiveness to etrolizumab treatment.

We also observed that baseline peripheral blood gene expression of certain genes below the median level was enriched for patients' responsiveness to etrolizumab treatment. As shown in Figures 26A-D, below-median expression of the TNFSF15 gene was enriched for remission in patients treated with etrolizumab. We also found that below-median expression of the VNN2 gene was enriched for remission in response to etrolizumab, as shown in Figures 27A-D.

We then evaluated the effect of inflammation on gene expression in mucosal biopsies obtained from untreated patients as described above. Ileal and colon biopsy samples were collected from CD patients, UC patients, or non-IBD healthy controls. Biopsies were collected from the sigmoid colon of non-IBD controls and UC patients, and from the colon and ileum of non-IBD controls and CD patients. Non-inflamed biopsies were collected from normal mucosa as judged by the endoscopist; if inflammatory disease was documented, additional biopsies were collected from inflamed areas. As shown in Figure 28A, ITGAE gene expression was higher in ileal biopsies compared to colon biopsies in both non-IBD and IBD patients. However, in IBD patients, there were no significant differences between inflamed and non-inflamed colon biopsies or inflamed and non-inflamed ileal biopsies. Similarly, GZMA (Figure 28B), KLRB1 (Figure 28E), and TNFSF15 (Figure 28G) were more highly expressed in ileal biopsies compared to colon biopsies, with no difference between inflamed and non-inflamed colon or ileal biopsies. VNN2 (Figure 28C) was higher in the ileum than in the colon and was also elevated in the inflamed ileum compared to the non-inflamed ileum of CD patients. Finally, ECH1 (Figure 28D) was lower in the inflamed colon compared to the non-inflamed colon biopsies from CD patients.

Peripheral blood gene expression was evaluated in untreated patient sample cohort 2. As shown in Figure 29A, lower levels of ITGAE peripheral blood gene expression were observed in UC patients compared to healthy controls. Higher levels of GZMA (Figure 29B) and ECH1 (Figure 29D) peripheral blood gene expression were found in CD patients compared to healthy controls. VNN2 (Figure 29C) and FOXM1 (Figure 29H) gene expression levels were higher in both UC and CD patients compared to healthy controls.

At baseline, the number of αE+ cells in the crypt epithelium was significantly higher in both granzyme A ^{high and αE high patients compared to granzyme A low and αE low} patients (Figures 30A-B, respectively). As shown in Figures 30C-D, etrolizumab treatment significantly reduced the number of αE+ cells in the crypt epithelium at week 10 in granzyme A ^high and αE ^high patients, whereas in granzyme A ^low and αE ^low patients, etrolizumab treatment did not significantly reduce the number of αE+ cells in the crypt epithelium.

In flow cytometric sorted CD4+αE+T cells isolated from colon biopsies of untreated UC patients in cohort 3, granzyme A gene expression was significantly upregulated compared to CD4+αE-T cells; the level of granzyme A in CD4+αE+ cells was significantly higher in UC patients compared to non-IBD controls (Figure 31A). In sorted CD4+αE+T cells from UC patients, but not CD8+αE+ cells, granzyme A gene expression was positively correlated with αE expression (Figure 31B). Consistent with the data from untreated patient sample cohort 3, in baseline colon biopsies from the etrolizumab Phase II study population, gene expression of granzyme A and αE was significantly positively correlated (Figure 31C). Consistent with these observations, in colon biopsies from patients recruited in the etrolizumab Phase II study, colon biopsy gene expression of granzyme A was significantly correlated with the number of αE+ cells in the crypt epithelium and lamina propria (Figure 31D). Double immunofluorescence staining analysis showed that granzyme A staining was limited to the αE+ cell subtype in colonic tissue (Figure 31E). These data suggest that αE+ cells can be the source of granzyme A in the colon of UC patients.

In summary, we have shown that high baseline peripheral blood expression of ITGAE, ECH1, and SLC8A3 is enriched for etrolizumab responsiveness as assessed by remission, mucosal healing, and clinical response. The results reported here confirm and extend our previously reported findings showing that high baseline αE expression and certain other genes with high baseline expression are enriched for etrolizumab responsiveness. We also specifically determined that low baseline peripheral blood expression of VNN2 and TNFSF15 is enriched for etrolizumab responsiveness as assessed by remission, mucosal healing, and clinical response. We evaluated peripheral blood gene expression of these genes in an independent cohort and found that they overlap with healthy controls, with some variation in samples from IBD patients.

We also evaluated biopsy gene expression of the genes described herein in untreated patient cohorts and found that gene expression was elevated in the ileum in some cases (ITGAE, GZMA, KLRB1, TNFSF15), but not in inflamed samples from patients with CD or UC. VNN2 had elevated gene expression in the ileum and was also elevated in inflamed versus non-inflamed biopsies. ECH1 was lower in inflamed colon compared to non-inflamed colon. Further investigation of the overlap between ITGAE and GZMA showed that ITGAE and GZMA were correlated in biopsies, with GZMA expression being higher in sorted αE+ CD4+ T cells compared to αE- CD4+ T cells, and immunofluorescence showed that the αE+ cell subset co-expressed granzyme A.

In summary, the expression levels of the genes described herein (alone or in combination) therefore show potential for use as predictive biomarkers for identifying IBD patients (such as UC and Crohn's disease patients) who are most likely to benefit from therapeutics targeting the β7 integrin subunit, including etrolizumab. Biomarkers can be measured by a number of methods (e.g., by qPCR) and can be measured using a variety of tissue samples (e.g., intestinal biopsy tissue or peripheral blood).

Claims (42)

1. The use of reagents for measuring the mRNA expression level of VNN2, a predictive gene with low expression in a sample, in the preparation of kits for predicting and/or identifying the response of patients with gastrointestinal inflammatory disorders to treatment containing an integrin β7 antagonist, wherein the prediction and/or identification includes: Obtain biological samples from patients; Measure the mRNA expression level of VNN2 in the sample; The mRNA expression level of VNN2 measured in the sample will be compared with the measured mRNA level of VNN2 in the reference sample; and When the measured VNN2 mRNA expression level is lower than the measured VNN2 mRNA level in the reference sample, it predicts that the patient will respond to integrin β7 antagonist treatment and/or identifies a patient who may respond to integrin β7 antagonist treatment. The integrin β7 antagonist is an anti-β7 antibody, which comprises three heavy chain hypervariable regions (HVR-H1-H3) and three light chain hypervariable regions (HVR-L1-L3), wherein: (i)HVR-L1 is SEQ ID NO:9; (ii) HVR-L2 is SEQ ID NO:2; (iii) HVR-L3 is SEQ ID NO:3; (iv)HVR-H1 is SEQ ID NO:4; (v)HVR-H2 is SEQ ID NO:5; and (vi)HVR-H3 is SEQ ID NO:19. 2. The use of reagents for measuring the mRNA expression level of VNN2 in samples and reagents for measuring the mRNA expression level of at least one, at least two, or at least three low-expression predictive genes selected from tissue SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, and VNN3 in the preparation of a kit for predicting and/or identifying the responsiveness of patients with gastrointestinal inflammatory disorders to integrin β7 antagonist therapy, wherein the prediction and/or identification includes measuring the mRNA expression level of biological samples from patients. The mRNA expression level of VNN2 and the mRNA expression levels of at least one, at least two, or at least three low-expression predictive genes selected from tissues SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, and VNN3, wherein the reduced mRNA expression level of each low-expression predictive gene, compared to the measured mRNA level of each low-expression predictive gene in a reference sample, would predict and/or identify the patient as a potential responder to integrin β7 antagonist therapy. The integrin β7 antagonist is an anti-β7 antibody, which comprises three heavy chain hypervariable regions (HVR-H1-H3) and three light chain hypervariable regions (HVR-L1-L3), wherein: (i)HVR-L1 is SEQ ID NO:9; (ii) HVR-L2 is SEQ ID NO:2; (iii) HVR-L3 is SEQ ID NO:3; (iv)HVR-H1 is SEQ ID NO:4; (v)HVR-H2 is SEQ ID NO:5; and (vi)HVR-H3 is SEQ ID NO:19. 3. The use of reagents for measuring the mRNA expression level of VNN2 in samples and reagents for measuring the mRNA expression level of at least one, at least two, at least three, or at least four highly expressed predictive genes selected from GZMA, KLRB1, FOXM1, CCDC90A, CCL4, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and peripheral blood SLC8A3 in the preparation of a kit for predicting and/or identifying the response of patients with gastrointestinal inflammatory disorders to treatment containing an integrin β7 antagonist, wherein the prediction and/or identification includes measuring the mRNA expression level of VNN2 in biological samples from patients and selecting The mRNA expression levels of at least one, at least two, at least three, or at least four highly expressed predictive genes from GZMA, KLRB1, FOXM1, CCDC90A, CCL4, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and peripheral blood SLC8A3, wherein a decreased mRNA expression level of VNN2 and an increased mRNA expression level of each highly expressed predictive gene in the reference sample, compared with the measured mRNA level of VNN2 and the measured mRNA level of each highly expressed predictive gene in the reference sample, would predict and/or identify the patient as a patient who may respond to integrin β7 antagonist therapy. The integrin β7 antagonist is an anti-β7 antibody, which comprises three heavy chain hypervariable regions (HVR-H1-H3) and three light chain hypervariable regions (HVR-L1-L3), wherein: (i)HVR-L1 is SEQ ID NO:9; (ii) HVR-L2 is SEQ ID NO:2; (iii) HVR-L3 is SEQ ID NO:3; (iv)HVR-H1 is SEQ ID NO:4; (v)HVR-H2 is SEQ ID NO:5; and (vi)HVR-H3 is SEQ ID NO:19. 4. Reagents for measuring the mRNA expression level of VNN2 in the sample; reagents for measuring the mRNA expression level of at least one, at least two, or at least three low-expression predicted genes selected from tissue SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, and VNN3 in the sample; and reagents for measuring the mRNA expression level of at least one, at least two, or at least three low-expression predicted genes in the sample. The use of reagents selected from at least one, at least two, at least three, or at least four highly expressed mRNA expression levels of predictive genes, including GZMA, KLRB1, FOXM1, CCDC90A, CCL4, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and peripheral blood SLC8A3, in the preparation of kits for predicting and/or identifying the response of patients with gastrointestinal inflammatory disorders to treatment containing an integrin β7 antagonist, wherein the prediction and/or identification includes: (a) Obtaining biological samples from patients; (b) Measure the mRNA expression level of VNN2 in the sample, the mRNA expression levels of at least one, at least two, or at least three low-expression predicted genes selected from tissue SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, and VNN3, and the mRNA expression levels of GZMA, KLRB1, and other genes selected from the same gene pool. The mRNA expression levels of at least one, at least two, at least three, or at least four highly expressed predicted genes, including FOXM1, CCDC90A, CCL4, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and peripheral blood SLC8A3, were measured and compared with the mRNA levels of each low-expression predicted gene and each highly expressed predicted gene in the reference sample. (c) When the mRNA expression levels of the low-expression predicted genes measured in (b) are lower than the mRNA levels of the low-expression predicted genes in the reference sample, and the mRNA expression levels of the high-expression predicted genes are higher than the mRNA levels of the high-expression predicted genes in the reference sample, predict and/or identify patient response to integrin β7 antagonist treatment. The integrin β7 antagonist is an anti-β7 antibody, which comprises three heavy chain hypervariable regions (HVR-H1-H3) and three light chain hypervariable regions (HVR-L1-L3), wherein: (i)HVR-L1 is SEQ ID NO:9; (ii) HVR-L2 is SEQ ID NO:2; (iii) HVR-L3 is SEQ ID NO:3; (iv)HVR-H1 is SEQ ID NO:4; (v)HVR-H2 is SEQ ID NO:5; and (vi)HVR-H3 is SEQ ID NO:19. 5. The use of any one of claims 2-4, further comprising measuring the mRNA expression level of an additional high-expression predictive gene, wherein the additional high-expression predictive gene is ITGAE. 6. The use of any one of claims 2-4, wherein at least one, at least two, at least three, or at least four highly expressed predictive genes are selected from GZMA, KLRB1, FOXM1, peripheral blood SLC8A3, and ECH1. 7. The use of claim 6, further comprising measuring the mRNA expression level of an additional high-expression predictive gene, wherein the additional high-expression predictive gene is ITGAE. 8. The use of claim 2 or 4, wherein at least one, at least two, or at least three low-expression predictive genes are selected from tissue SLC8A3, TNFSF15, BEST2, and CCL2. 9. The use of claim 8, wherein at least one or at least two low-expression predictive genes are selected from tissue SLC8A3 and TNFSF15. 10. The use of claim 4, wherein at least one, at least two, at least three, or at least four highly expressed predictive genes are selected from GZMA, KLRB1, FOXM1, peripheral blood SLC8A3, and ECH1, and wherein at least one, at least two, or at least three low-expressed predictive genes are selected from tissue SLC8A3, TNFSF15, BEST2, and CCL2. 11. The use of claim 10, further comprising measuring the mRNA expression level of an additional high-expression predictive gene, wherein the additional high-expression predictive gene is ITGAE. 12. The use of claim 10, wherein at least one or at least two low-expression predictive genes are selected from tissue SLC8A3 and TNFSF15. 13. The use of any one of claims 1-4, wherein 105 mg of integrin β7 antagonist is administered subcutaneously once every four weeks. 14. The use according to any one of claims 1-4, wherein an initial dose of 210 mg integrin β7 antagonist is administered subcutaneously, followed by a first follow-up dose of 210 mg integrin β7 antagonist administered subcutaneously 2 weeks after the initial dose, a second follow-up dose of 210 mg integrin β7 antagonist administered subcutaneously 4 weeks after the initial dose, a third follow-up dose of 210 mg integrin β7 antagonist administered subcutaneously 8 weeks after the initial dose, and a fourth follow-up dose of 210 mg integrin β7 antagonist administered subcutaneously 12 weeks after the initial dose. 15. The use of any one of claims 1-4, wherein the gastrointestinal inflammatory disorder is inflammatory bowel disease. 16. The use of claim 15, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease. 17. The use of claim 16, wherein the inflammatory bowel disease is ulcerative colitis, and the response is selected from clinical response, mucosal healing, and remission. 18. Use according to any one of claims 1-4, wherein the biological sample is selected from intestinal tissue and peripheral whole blood. 19. The use of claim 18, wherein the mRNA expression level is measured by RNA sequencing, microarray or PCR. 20. The use of claim 19, wherein the PCR method includes qPCR. 21. The use of claim 19, wherein the measurement comprises amplifying VNN2 and one or more of GZMA, KLRB1, FOXM1, CCDC90A, CCL4, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and peripheral blood SLC8A3 mRNA, and detecting the amplified mRNA, thereby measuring the level of the amplified mRNA. 22. The use of claim 21, wherein the measurement further comprises amplifying ITGAE mRNA and detecting the amplified mRNA, thereby measuring the level of the amplified mRNA. 23. The use of claim 19, wherein the measurement comprises amplifying one or more of the following mRNAs: VNN2 and amplified tissue SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, and VNN3, and detecting the amplified mRNAs, thereby measuring the level of the amplified mRNAs. 24. Use according to any one of claims 1-4, wherein the mRNA levels in each reference sample are median. 25. Use according to any one of claims 1-4, wherein the patient has not previously been treated with an anti-TNF agent. 26. The use of claim 14, wherein the administration of the integrin β7 antagonist results in one or more of the following: (1) a reduction of 3 points in the Mayo Clinical Scoring System as shown in Table 1 and a decrease of 30% from baseline, and a reduction of ≥1 point in the rectal bleeding sub-score or an absolute rectal bleeding score of 0 or 1; (2) an endoscopic sub-score of 0 or 1; (3) a Mayo Clinical Scoring System score ≤2 as shown in Table 1, with no single sub-score >1. 27. Use according to any one of claims 1-4 for stratifying patients into potential responders and non-responders to integrin β7 antagonist treatment. 28. The use of claim 27, wherein the kit is used to determine the mRNA level of VNN2 and the mRNA level of at least one, at least two, at least three, or at least four markers selected from GZMA, KLRB1, FOXM1, peripheral blood SLC8A3, and ECH1. 29. The use of claim 28, wherein the kit is used for further determination of ITGAE mRNA levels. 30. The use of claim 27, wherein the kit is used to determine the mRNA level of VNN2 and the mRNA level of at least one, at least two, or at least three markers selected from tissue SLC8A3, TNFSF15, BEST2, and CCL2. 31. The use of claim 30, wherein the kit is used for further determination of ITGAE mRNA levels. 32. A kit for stratifying patients with inflammatory gastrointestinal disorders, wherein the kit comprises: (a) A reagent for measuring the mRNA levels of VNN2 in biological samples obtained from patients and the mRNA levels of at least one, at least two, at least three, or at least four markers selected from GZMA, KLRB1, FOXM1, CCDC90A, CCL4L1.2, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and peripheral blood SLC8A3; and (b) Regarding the following: (i) Measure the mRNA level of VNN2 and the mRNA levels of at least one, at least two, at least three, or at least four markers selected from GZMA, KLRB1, FOXM1, CCDC90A, CCL4L1.2, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and peripheral blood SLC8A3; (ii) Measure the mRNA level of VNN2 and the mRNA levels of markers selected from GZMA, The levels of at least one, at least two, at least three, or at least four markers of KLRB1, FOXM1, CCDC90A, CCL4L1.2, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and peripheral blood SLC8A3 were compared with the mRNA levels in a reference sample; and (iii) based on the comparison, patients were stratified into categories of integrin β7 antagonist responders or integrin β7 antagonist non-responders. The integrin β7 antagonist is an anti-β7 antibody, which comprises three heavy chain hypervariable regions (HVR-H1-H3) and three light chain hypervariable regions (HVR-L1-L3), wherein: (i)HVR-L1 is SEQ ID NO:9; (ii) HVR-L2 is SEQ ID NO:2; (iii) HVR-L3 is SEQ ID NO:3; (iv)HVR-H1 is SEQ ID NO:4; (v)HVR-H2 is SEQ ID NO:5; and (vi)HVR-H3 is SEQ ID NO:19. 33. The kit of claim 32, wherein the kit comprises reagents for measuring the mRNA levels of VNN2 and the mRNA levels of at least one, at least two, at least three, or at least four markers selected from GZMA, KLRB1, FOXM1, peripheral blood SLC8A3, and ECH1 in a sample obtained from a patient. 34. The kit of claim 32 or 33, wherein the kit further comprises reagents for measuring the mRNA level of ITGAE in a sample obtained from a patient. 35. A kit for stratifying patients with inflammatory gastrointestinal disorders, wherein the kit comprises: (a) A reagent for measuring the mRNA level of VNN2 in biological samples obtained from patients and the mRNA levels of at least one, at least two, or at least three markers selected from tissue SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, and VNN3; and (b) Regarding the following: (i) Measure the mRNA level of VNN2 and the mRNA levels of at least one, at least two, or at least three markers selected from tissue SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, and VNN3; (ii) Measure the mRNA level of VNN2 and the mRNA levels of at least one, at least two, or at least three markers selected from tissue SLC8A3, TNFSF15, BEST2, CCL2, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, and VNN3; The levels of at least one, at least two, or at least three of the markers 8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, and VNN3 were compared with the mRNA levels in a reference sample; and (iii) based on the comparison, patients were stratified into categories of integrin β7 antagonist responders or integrin β7 antagonist non-responders. The integrin β7 antagonist is an anti-β7 antibody, which comprises three heavy chain hypervariable regions (HVR-H1-H3) and three light chain hypervariable regions (HVR-L1-L3), wherein: (i)HVR-L1 is SEQ ID NO:9; (ii) HVR-L2 is SEQ ID NO:2; (iii) HVR-L3 is SEQ ID NO:3; (iv)HVR-H1 is SEQ ID NO:4; (v)HVR-H2 is SEQ ID NO:5; and (vi)HVR-H3 is SEQ ID NO:19. 36. The kit of claim 35, wherein the kit comprises reagents for measuring the mRNA level of VNN2 and the mRNA level of at least one, at least two, or at least three markers selected from tissue SLC8A3, TNFSF15, BEST2, and CCL2 in a sample obtained from a patient. 37. A kit for stratifying patients with inflammatory gastrointestinal disorders, wherein the kit comprises: (a) Used to measure the mRNA level of VNN2 in biological samples obtained from patients and the levels of tissue-derived SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, and VNN3. A reagent containing mRNA levels of at least one, at least two, or at least three markers, and mRNA levels of at least one, at least two, at least three, or at least four markers selected from GZMA, KLRB1, FOXM1, CCDC90A, CCL4L1.2, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and peripheral blood SLC8A3; and (b) Regarding the following: (i) Measuring the mRNA level of VNN2 and at least two or more of the following: tissue SLC8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, and VNN3. (ii) The mRNA levels of three markers, and the mRNA levels of at least one, at least two, at least three, or at least four markers selected from GZMA, KLRB1, FOXM1, CCDC90A, CCL4L1.2, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and peripheral blood SLC8A3; The levels of at least one, at least two, or at least three of the following markers: 8A3, TNFSF15, BEST2, CCL2, CCL3, CCL3L1/3, CPA3, FGF7, HAMP, IL1A, IL18RAP, INHBA, LIF, LMO4, LRRC4, MLK7.AS1, MT1M, MUCL1, MX1, PMCH, REM2, SSTR2, TM4SF4, TMEM154, UROS, and VNN3, and the levels of GZMA, KLRB1, FOXM1, and CCDC90. A) The mRNA levels of at least one, at least two, at least three, or at least four markers of CCL4L1.2, CPA2, CXCR6, DDO, ECH1, FAM125B, FASLG, FGF9, GPR15, GZMB, KCNMA1, PHF14, TIFAB, TMEM200A, TMIGD2, and peripheral blood SLC8A3 were compared with the mRNA levels in a reference sample; and (iii) Based on the comparison, patients were stratified into categories of integrin β7 antagonist responders or integrin β7 antagonist non-responders. The integrin β7 antagonist is an anti-β7 antibody, which comprises three heavy chain hypervariable regions (HVR-H1-H3) and three light chain hypervariable regions (HVR-L1-L3), wherein: (i)HVR-L1 is SEQ ID NO:9; (ii) HVR-L2 is SEQ ID NO:2; (iii) HVR-L3 is SEQ ID NO:3; (iv)HVR-H1 is SEQ ID NO:4; (v)HVR-H2 is SEQ ID NO:5; and (vi)HVR-H3 is SEQ ID NO:19. 38. The kit of any one of claims 35-37, wherein the kit further comprises a reagent for measuring the mRNA level of ITGAE in a sample obtained from a patient. 39. The use of claims 1-4 or the kit of claim 37, wherein the anti-β7 antibody is selected from chimeric antibodies, human antibodies and humanized antibodies. 40. The use or kit of claim 39, wherein the anti-β7 antibody is an antibody fragment. 41. The use of claims 1-4 or the kit of claim 37, wherein the anti-β7 antibody comprises a variable light chain of amino acid sequence SEQ ID NO:31 and a variable heavy chain of amino acid sequence SEQ ID NO:32. 42. The use of claims 1-4 or the kit of claim 37, wherein the anti-β7 antibody is etrolizumab.