EP3329276A2 - Méthodes de diagnostic et de traitement de la maladie intestinale inflammatoire - Google Patents

Méthodes de diagnostic et de traitement de la maladie intestinale inflammatoire

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Publication number
EP3329276A2
EP3329276A2 EP16750314.3A EP16750314A EP3329276A2 EP 3329276 A2 EP3329276 A2 EP 3329276A2 EP 16750314 A EP16750314 A EP 16750314A EP 3329276 A2 EP3329276 A2 EP 3329276A2
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EP
European Patent Office
Prior art keywords
cells
level
patient
seq
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP16750314.3A
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German (de)
English (en)
Inventor
Teresa Ramirez MONTAGUT
Akiko CHAI
Franklin FUH
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F Hoffmann La Roche AG
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F Hoffmann La Roche AG
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Publication date
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Publication of EP3329276A2 publication Critical patent/EP3329276A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Biomarkers predictive of responsiveness to integrin beta7 antagonists including anti-beta7 integrin subunit antibodies, and methods of using such biomarkers are provided.
  • methods of treating gastrointestinal inflammatory disorders such as inflammatory bowel diseases including ulcerative colitis and Crohn's disease are provided.
  • methods of using such predictive biomarkers for the treatment of inflammatory bowel diseases including ulcerative colitis and Crohn's disease.
  • IBD Inflammatory bowel disease
  • GI gastrointestinal
  • CD ulcerative colitis
  • CD Crohn's disease
  • UC ulcerative colitis
  • CD Crohn's disease
  • Both conditions are characterized clinically by frequent bowel motions, malnutrition, and dehydration, with disruption in the activities of daily living.
  • CD is frequently complicated by the development of malabsorption, strictures, and fistulae and may require repeated surgery.
  • UC less frequently, may be complicated by severe bloody diarrhea and toxic megacolon, also requiring surgery.
  • Both IBD conditions are associated with an increased risk for malignancy of the GI tract.
  • the etiology of IBD is complex, and many aspects of the pathogenesis remain unclear.
  • TNF-a tumor necrosis factor alpha
  • infliximab a chimeric antibody
  • adalimumab a fully human antibody
  • AEs adverse events associated with anti TNFs
  • Other adverse events (AEs) associated with anti TNFs include elevated rates of bacterial infection, including tuberculosis, and, more rarely, lymphoma and demyelination (Chang et al, Nat Clin Pract Gastroenterol Hepatology 3:220 (2006); Hoentjen et al, World J. Gastroenterol. 15(17):2067 (2009)).
  • No currently 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)).
  • the integrins are alpha/beta heterodimeric cell surface glycoprotein receptors that play a role in numerous 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 a and ⁇ transmembrane subunits that bind specifically to distinct cell adhesion molecules (CAMs) on endothelia, epithelia, and extracellular matrix proteins.
  • CAMs cell adhesion molecules
  • integrins can function as tissue-specific cell adhesion receptors aiding in the recruitment of leukocytes from blood into nearly all tissue sites in a highly regulated manner, playing a role in the homing of leukocytes to normal tissue and to sites of inflammation (von Andrian et al, N Engl J Med 343: 1020-34 (2000)).
  • integrins are involved in leukocyte trafficking, adhesion and infiltration during inflammatory processes (Nakajima, H. et al, J. Exp. Med., 1994, 179: 1145-1154). Differential expression of integrins regulates the adhesive properties of cells and different integrins are involved in different inflammatory responses. (Butcher, E. C.
  • beta7 containing integrins ⁇ i.e., alpha4beta7 and alphaEbeta7
  • alpha4beta7 and alphaEbeta7 are expressed primarily on monocytes, lymphocytes, eosinophils, basophils, and macrophages but not on neutrophils (Elices, M. J. et al, Cell, 1990, 60:577-584).
  • the ⁇ 4 ⁇ 7 integrin is a leukocyte-homing receptor that is important in the migration of cells to the intestinal mucosa and associated lymphoid tissues, such as Peyer's patches in the small intestine, lymphoid follicles in the large intestine, and mesenteric lymph nodes.
  • lymphoid tissues such as Peyer's patches in the small intestine, lymphoid follicles in the large intestine, and mesenteric lymph nodes.
  • chemokines In the gut, leukocyte rolling and firm adhesion to the mucosal endothelium is initiated by signals from chemokines and is mediated via mucosal addressin cell adhesion molecule (MAdCAM)- 1 -associated sialyl Lewis X.
  • MAdCAM mucosal addressin cell adhesion molecule
  • Chemokine signaling induces the ⁇ 4 ⁇ 7 integrin to undergo a change from low to high MAdCAM-1 binding affinity.
  • the leukocyte then arrests and begins the process of extravasation through the vascular endothelium to underlying tissue. This extravasation process is believed to occur in both the normal immune cell recirculation state and in inflammatory conditions (von Andrian et al., supra).
  • the numbers of ⁇ 4 ⁇ 7 + cells in infiltrates and the expression of the ligand MAdCAM-1 are higher at sites of chronic inflammation such as in the intestinal tract of patients with UC or CD (Briskin et al, Am J Pathol 151 :97-110 (1997); Souza et al, Gut 45:856-63 (1999)).
  • ⁇ 4 ⁇ 7 binds preferentially to high endothelial venules expressing MAdCAM-1 and vascular cell adhesion molecule
  • VCAM extracellular matrix molecule fibronectin fragment CS-1
  • MAdCAM-1 extracellular matrix molecule fibronectin fragment CS-1
  • the ⁇ 4 ⁇ 7 integrin plays a selective role in leukocyte gut tropism but does not seem to contribute to homing of leukocytes to the peripheral tissue or the CNS. Instead, peripheral lymphoid trafficking has been associated with ⁇ 4 ⁇ 1 interaction with VCAM-1 (Yednock et al., Nature 356:63-6 (1992); Rice et al, Neurology 64: 1336-42 (2005)).
  • ⁇ 7 integrin Another member of the ⁇ 7 integrin family, expressed exclusively on T lymphocytes and associated with mucosal tissues, is the ⁇ 7 integrin, otherwise known as CD103.
  • the ⁇ 7 integrin binds selectively to E-cadherin on epithelial cells and has been proposed to play a role in the retention of T cells in the mucosal tissue in the intraepithelial lymphocyte compartment (Cepek et al., J Immunol 150:3459-70 (1993); Karecla et al. Eur J Immunol 25:852-6 (1995)).
  • ⁇ 7 + cells in the lamina propria have been reported to exhibit cytotoxicity against stressed or infected epithelial cells (Hadley et al, J Immunol 159:3748-56 (1997); Buri et al, J Pathol 206: 178-85 (2005)).
  • ⁇ 7 The expression of ⁇ 7 is increased 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-a ⁇ 7 antibody treatment has been reported to attenuate experimental colitis in mice, implicating a role for ⁇ 7 + lymphocytes in experimental models of IBD (Ludviksson et al, J Immunol 162:4975-82 (1999)).
  • An anti-a4 antibody (natalizumab) reportedly has efficacy in treatment of patients with CD (Sandborn et al, N Engl J Med 2005;353: 1912-25) and an anti-a4p7 antibody (MLN-02, MLN0002, vedolizumab) reportedly is effective in patients with UC (Feagan et al, N Engl J Med 2005;352:2499-507).
  • a second anti-alpha4/beta7 antibody (AMG 181) is also in development and clinical trials have recently begun (clinicaltrials(dot)gov identifier,
  • rhuMAb Beta7 is derived from the rat anti-mouse/human monoclonal antibody FIB504 (Andrew et al. 1994). It was engineered to include human IgGl -heavy chain and Kl-light chain frameworks. Intn'l Patent Pub. No. WO2006/026759. Administration of etrolizumab to human patients according to certain dosing regimens has been described previously. See, e.g., Intn'l Patent Pub. No. WO/2012/135589.
  • RhuMAb Beta7 (etrolizumab) binds ⁇ 4 ⁇ 7 (Holzmann et al, Cell 56:37-46 (1989); Hu et al, Proc Natl Acad Sci USA 89:8254-8 (1992)) and ⁇ 7 (Cepek et al., J Immunol 150:3459-70 (1993)), which regulate trafficking and retention of lymphocyte subsets, respectively, in the intestinal mucosa.
  • rhuMAb Beta7 binds specifically to the ⁇ 7 subunit of ⁇ 4 ⁇ 7 and ⁇ 7 and does not bind to a4 or ⁇ integrin individual subunits. This was demonstrated by the inability of the antibody to inhibit adhesion of ⁇ 4 ⁇ 1+ ⁇ 4 ⁇ 7- Ramos cells to vascular cell adhesion molecule 1 (VCAM 1) at concentrations as high as 100 nM. Importantly, this characteristic of rhuMAb Beta7 indicates selectivity: T cell subsets expressing ⁇ 4 ⁇ 1 but not ⁇ 7 should not be directly affected by rhuMAb Beta7.
  • rhuMAb Beta7 Support for the gut-specific effects of rhuMAb Beta7 on leukocyte homing comes from several in vivo nonclinical studies. In severe combined immunodeficient (SCID) mice reconstituted with CD45RB hi 8 h CD4+ T cells (an animal model of colitis), rhuMAb Beta7 blocked radiolabeled lymphocyte homing to the inflamed colon but did not block homing to the spleen, a peripheral lymphoid organ. See, e.g., Intn'l Patent Pub. No. WO2006/026759.
  • rat-mouse chimeric anti-murine ⁇ 7 (anti ⁇ 7, muFIB504) was unable to reduce the histologic degree of central nervous system (CNS) inflammation or improve disease survival in myelin basic protein T cell receptor (MBP-TCR) transgenic mice with experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis. Id.
  • rhuMAb Beta7 induced a moderate increase in peripheral blood lymphocyte numbers that was largely due to a marked (approximately three- to sixfold) increase in CD45RA ⁇ 7 1 ⁇ 1 ⁇ 11 peripheral blood T cells, a subset that is phenotypically similar to gut-homing memory/effector T cells in humans. See, e.g., Intn'l Patent Pub. No. WO2009/140684; Stefanich et al, Br. J. Pharmacol. 162: 1855-1870 (2011).
  • rhuMAb Beta7 had minimal to no effect on the number of CD45RA+ ⁇ 7intermediate peripheral blood T cells, a subset that is phenotypically similar to naive T cells in humans, and no effect on the number of CD45RA ⁇ 7 1 ⁇ peripheral blood T cells, a subset that is phenotypically similar to peripheral homing memory /effector T cells in humans, confirming the specificity of rhuMAb Beta7 for the gut homing lymphocyte subpopulation.
  • natalizumab treatment has been associated with confirmed cases of progressive multifocal leukoencephalopathy (PML) in patients with Crohn's disease (and separately, multiple sclerosis) who received concomitant treatment with natalizumab and immunosupressives.
  • PML is a potentially fatal neurological condition linked to reactivation of a polyomavirus (JC virus) and active viral replication in the brain. No known interventions can reliably prevent PML or adequately treat PML, if it occurs.
  • vedolizumab treatment is that it is administered intravenously which can be inconvenient for the patient and can also be associated with undesirable or adverse events, e.g., infusion site reactions. Accordingly, there is a need for improved therapeutic approaches to the treatment of gastrointestinal inflammatory disorders such as IBD, e.g., ulcerative colitis and Crohn's disease, as well as more desirable dosing regimens.
  • IBD gastrointestinal inflammatory disorders
  • Crohn's disease e.g., ulcerative colitis and Crohn's disease
  • the methods of the invention are based, at least in part, on the discovery that levels of certain cells, such as T cells, Treg cells, and/or Thl7 cells in biological samples obtained from patients, e.g., blood, are predictive of responsiveness of patients suffering from a gastrointestinal inflammatory disorder to treatment with integrin beta7 antagonists.
  • methods of predicting the response of a patient suffering from a gastrointestinal inflammatory disorder to a therapy comprising an integrin beta7 antagonist are provided.
  • the method comprises:
  • methods of predicting responsiveness of a gastrointestinal inflammatory disorder patient to an integrin beta7 antagonist treatment comprises determining the level of Treg cells in a biological sample from the patient, wherein an elevated level of Treg cells compared to a reference level of Treg cells identifies the patient as one who is likely to respond to the integrin beta7 antagonist treatment.
  • an elevated level of Treg cells is a level that is greater than a particular percentage or proportion of Treg cells in the sample.
  • an elevated level of Treg cells is a level that is equal to or greater than about 1.2% or about 1.25% or about 1.28% or about 1.3% or about 1.32% of cells in the sample.
  • the sample is a peripheral blood sample.
  • methods of identifying a patient suffering from a gastrointestinal inflammatory disorder as likely to respond to a therapy comprising an integrin beta7 antagonist are provided.
  • the method comprises:
  • methods of treating a patient having a gastrointestinal inflammatory disorder are provided.
  • the method comprises:
  • the patient is TNF-nai ' ve.
  • the reference level is the level of Treg cells in a population of patients suffering from a gastrointestinal inflammatory disorder. In some embodiments, the reference level is the level of Treg cells in a TNF-nai ' ve population of patients suffering from a gastrointestinal inflammatory disorder. In some embodiments, the level of Treg cells is determined as a proportion of Treg cells in the biological sample. In some embodiments, the proportion of Treg cells is determined by dividing the number of copies of demethylated FOXP3 gene in the biological sample by the number of copies of a control gene in the biological sample. In some embodiments, the method comprises determining the number of copies of demethylated FOXP3 Treg-specific demethylated region (TSDR). In some embodiments, the control gene is selected from GAPDH, ACTB, and COQ3.
  • methods of monitoring the response of a patient suffering from a gastrointestinal inflammatory disorder to a therapy comprising an integrin beta7 antagonist are provided.
  • the method comprises:
  • a method of monitoring responsiveness of a gastrointestinal inflammatory disorder patient to an integrin beta7 antagonist treatment comprises determining a first level of cells selected from CD3+ T cells, Treg cells, and Thl7 in a first biological sample from the patient taken at a first time point and determining a second level of the respective cells in a second biological sample from the patient taken at a second time point, wherein a higher level of the cells in the second biological sample compared to the first biological sample identifies the patient as one who is likely to experience clinical remission.
  • methods of identifying a patient suffering from a gastrointestinal inflammatory disorder and receiving a therapy comprising an integrin beta7 antagonist as likely to experience clinical remission comprise: a) measuring a first level of cells selected from CD3+ T cells, Treg cells, and Thl7 in a first biological sample from the patient taken at a first time point;
  • a method of treating a patient having a gastrointestinal inflammatory disorder comrprises:
  • the cells are CD3+ T cells. In some embodiments, the cells are Treg cells. In some embodiments, the cells are Thl7 cells.
  • the second time point is after the first time point.
  • the patient began treatment with an integrin beta7 antagonist after the first time point but prior to the second time point.
  • the patient began treatment with an integrin beta7 antagonist prior to the first time point, and continues treatment until at least the second time point.
  • the second time point is between 2 and 10 weeks after beginning treatment, or between 2 and 9 weeks, or between 2 and 8 weeks, or between 2 and 7 weeks, or between 2 and 6 weeks, or between 2 and 5 weeks, or between 2 and 4 weeks after beginning treatment.
  • the second time point is between 10 and 30 weeks after beginning treatment, or between 10 and 26 weeks after beginning treatment, or between 10 and 22 weeks after beginning treatment, or between 10 and 18 weeks after beginning treatment, or between 10 and 14 weeks after beginning treatment.
  • the first time point is before treatment is begun. In some embodiments, the first time point is within 4 weeks before treatment is begun, or within 3 weeks, or within 2 weeks, or within 1 week, or within 5 days, or within 4 days, or within 3 days, or within 2 days, or within 1 day before treatment is begun.
  • the patient is TNF-nai ' ve. In some embodiments, the patient is a TNF inadequate responder.
  • the level of cells is determined as a proportion of the cells in the biological sample.
  • the proportion of Treg cells is determined by dividing the number of copies of demethylated FOXP3 gene in the biological sample by the number of copies of a control gene in the biological sample.
  • the method comprises determining the number of copies of demethylated FOXP3 Treg-specific demethylated region (TSDR).
  • the proportion of T cells is determined by dividing the number of copies of demethylated CD3D/G gene in the biological sample by the number of copies of a control gene in the biological sample.
  • the proportion of Thl7 cells is determined by dividing the number of copies of demethylated IL17A gene in the biological sample by the number of copies of a control gene in the biological sample.
  • the control gene is selected from GAPDH, ACTB, and COQ3.
  • determining the number of copies of a demethylated gene comprises bisulfite conversion of genomic DNA. In some embodiments, determining the number of copies of a demethylated gene comprises quantitative PCR.
  • the response is clinical remission. In some embodiments, the response is mucosal healing. In some embodiments, the response is clinical response. In certain embodiments, remission in the patient is determined to be induced when the absolute Mayo Clinic Score ⁇ 2 and no individual subscore >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, patients who experience mucosal healing are determined to have an endoscopy subscore of 0. In certain embodiments, clinical response is determined to have occurred when the patient experiences a 3-point decrease and 30% reduction from baseline in MCS and > 1 -point decrease in rectal bleeding subscore or absolute rectal bleeding score of 0 or 1.
  • the patient is a human. In some embodiments, the patient is not previously treated with an anti-TNF therapeutic agent. In some embodiments, the gastrointestinal inflammatory disorder is an inflammatory bowel disease. In some embodiments,
  • the inflammatory bowel disease is ulcerative colitis or Crohn's disease. In some embodiments, the inflammatory bowel disease is ulcerative colitis and the response is selected from clinical response, mucosal healing and remission. In some embodiments, remission in the patient is determined to be induced when the absolute Mayo Clinic Score ⁇ 2 and no individual subscore >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, patients who experience mucosal healing are determined to have an endoscopy subscore of 0. In certain embodiments, clinical response is determined to have occurred when the patient experiences a 3-point decrease and 30% reduction from baseline in MCS and > 1 -point decrease in rectal bleeding subscore or absolute rectal bleeding score of 0 or 1.
  • 105 mg of the integrin beta7 antagonist is administered subcutaneously once every four weeks.
  • an initial dose of 210 mg of the integrin beta7 antagonist is administered subcutaneously followed by subsequent doses, each subsequent dose of 210 mg of the integrin beta7 antagonist administered subcutaneously, administered at each of weeks 2, 4, 8 and 12 after the initial dose.
  • the patient is a human.
  • the patient is not previously treated with an anti- TNF therapeutic agent.
  • the gastrointestinal inflammatory disorder is an inflammatory bowel disease.
  • the inflammatory bowel disease is ulcerative colitis or Crohn's disease.
  • the inflammatory bowel disease is ulcerative colitis and the response is selected from clinical response, mucosal healing and remission.
  • remission in the patient is determined to be induced when the absolute Mayo Clinic Score ⁇ 2 and no individual subscore >1, which is also referred to as clinical remission.
  • 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.
  • patients who experience mucosal healing are determined to have an endoscopy subscore of 0.
  • clinical response is determined to have occurred when the patient experiences a 3-point decrease and 30% reduction from baseline in MCS and > 1 -point decrease in rectal bleeding subscore or absolute rectal bleeding score of 0 or 1.
  • the biological sample is peripheral whole blood. In some embodiments, the biological sample is peripheral blood mononucleocytes. In some
  • the biological sample is intestinal tissue.
  • the intestinal tissue is intestinal biopsy tissue, for example, obtained from an area of the intestine showing signs of disease or an area of the intestine suspected of having disease.
  • the response is clinical remission.
  • the response is mucosal healing.
  • the response is clinical response.
  • remission in the patient is determined to be induced when the absolute Mayo Clinic Score ⁇ 2 and no individual subscore >1 , which is also referred to as clinical remission.
  • 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.
  • patients who experience mucosal healing are determined to have an endoscopy subscore of 0.
  • clinical response is determined to have occurred when the patient experiences a 3-point decrease and 30% reduction from baseline in MCS and > 1 -point decrease in rectal bleeding subscore or absolute rectal bleeding score of 0 or 1.
  • kits for determining the level of Treg cells in a biological sample obtained from a patient suffering from a gastrointestinal inflammatory disorder is provided.
  • the use is for stratifying patients into likely responders and non-responders for therapeutic treatment with an integrin beta7 antagonist.
  • the kit comprises reagents for determining the number of copies of demethylated FOXP3 gene in the biological sample.
  • the kit further comprises reagents for determining the number of copies of a control gene in the biological sample.
  • the control gene is selected from GAPDH, ACTB, and COQ3.
  • the kit comprises instructions for (i) determining the level of Treg cells in the biological sample; (ii) comparing the level to a reference level; and (iii) stratifying the patient into the category of integrin beta7 antagonist responder or integrin beta7 antagonist non- responder based on the comparison.
  • kits for determining the level of a cells selected from CD3+ T cells, Treg cells, and Thl7 cells in a biological sample obtained from a patient suffering from a gastrointestinal inflammatory disorder for monitoring the response of the patient to therapeutic treatment with an integrin beta7 antagonist.
  • the cells are CD3+ T cells.
  • the cells are Treg cells.
  • the cells are Thl7 cells.
  • the kit comprises reagents for determining the number of copies of a demethylated gene in the biological sample.
  • the demethylated gene is selected from CD3D/G, FOXP3, and IL17A.
  • the kit further comprises reagents for determining the number of copies of a control gene in the biological sample.
  • the control gene is selected from GAPDH, ACTB, and COQ3.
  • the kit comprises instructions for (i) determining a first level of the cells in a biological sample taken at a first time point; (ii) determining a second level of the cells in a biological sample taken at a second time point; (iii) comparing the first level to the second level; and (iv) stratifying the patient into the category of integrin beta7 antagonist remitter or integrin beta7 antagonist non-remitter based on the comparison.
  • the integrin beta7 antagonist is for use in treating the patient wherein 105 mg is administered subcutaneously once every four weeks.
  • an initial dose of 210 mg of the integrin beta7 antagonist is administered subcutaneously followed by subsequent doses, each of 210 mg of the integrin beta7 antagonist administered subcutaneously, administered at each of weeks 2, 4, 8 and 12 after the initial dose.
  • administration of the integrin beta7 antagonist results in one or more of the following: (1) a 3-point decrease and 30% reduction from baseline in MCS and > 1 -point decrease in rectal bleeding subscore or absolute rectal bleeding score of 0 or 1, (2) an endoscopic subscore of 0 or 1, (3) MCS ⁇ 2 with no individual subscore > 1.
  • 105 mg of the integrin beta7 antagonist is administered subcutaneously once every four weeks.
  • an initial dose of 210 mg of the integrin beta7 antagonist is administered subcutaneously followed by subsequent doses, each of 210 mg of the integrin beta7 antagonist administered subcutaneously, administered at each of weeks 2, 4, 8 and 12 after the initial dose.
  • administering results in one or more of the following: (1) a 3-point decrease and 30% reduction from baseline in MCS and > 1-point decrease in rectal bleeding subscore or absolute rectal bleeding score of 0 or 1, (2) an endoscopic subscore of 0 or 1, (3) MCS ⁇ 2 with no individual subscore > 1.
  • 105 mg of the integrin beta7 antagonist is administered subcutaneously once every four weeks.
  • an initial dose of 210 mg of the integrin beta7 antagonist is administered subcutaneously followed by subsequent doses, each of 210 mg of the integrin beta7 antagonist administered subcutaneously, administered at each of weeks 2, 4, 8 and 12 after the initial dose.
  • the gastrointestinal inflammatory disorder is an inflammatory bowel disease, and in certain such embodiments, the inflammatory bowel disease is ulcerative colitis (UC) or Crohn's disease (CD), and in certain such embodiments, the integrin beta7 antagonist is a monoclonal anti-beta7 antibody.
  • UC ulcerative colitis
  • CD Crohn's disease
  • the integrin beta7 antagonist is a monoclonal anti-beta7 antibody.
  • 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 Al-Al l, wherein Al-Al l is RASESVDTYLH (SEQ ID NO: l); RASESVDSLLH (SEQ ID NO:7), RASESVDTLLH (SEQ ID NO: 8), or RASES VDDLLH (SEQ ID NO: 9) or a variant of SEQ ID NOs: 1, 7, 8 or 9 (SEQ ID 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 A4 is selected from the
  • HVR-L2 comprises amino acid sequence B1-B8, wherein B1-B8 is KYASQSIS (SEQ ID NO:2), RYASQSIS (SEQ ID NO:20), or XaaYASOSIS (SEQ ID NO:21, where Xaa represents any amino acid) or a variant of SEQ ID NOs: 2, 20 or 21 (SEQ ID NO: 27) wherein amino acid Bl is selected from the group consisting of K, R, N, V, A, F, Q, H, P, I, L, Y and Xaa (where 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;
  • 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, I L, and M;
  • HVR-H1 comprises amino acid sequence D1-D10 wherein D1-D10 is GFFITNNYWG (SEQ ID NO:4);
  • 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 El 2 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 El 7 is selected from the group consisting of S and G; and (vi) HVR-H3 comprises amino acid sequence F2-F11 wherein F2 -Fl l is MTGSSGYFDF (SEQ ID NO:6) or RT
  • ARTGSSGYFDF SEQ ID NO: 17
  • AQTGSSGYFDF SEQ ID NO: 18
  • a variant of SEQ ID NOs:6, 16, 17, 18, or 19 SEQ ID NO:30
  • amino acid F2 is R, M, A, E, G, Q, S, and/or amino acid Fl 1 is selected from the group consisting of F and Y.
  • 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:
  • HVR-L1 comprises SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9;
  • HVR-L2 comprises SEQ ID NO:2;
  • HVR-L3 comprises SEQ ID NO:3;
  • HVR-H1 comprises SEQ ID NO:4;
  • HVR-H2 comprises SEQ ID NO:5;
  • HVR-H3 comprises SEQ ID NO:6 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 19.
  • 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:
  • HVR-L1 comprises SEQ ID NO:9;
  • HVR-L2 comprises SEQ ID NO:2;
  • HVR-L3 comprises SEQ ID NO: 3;
  • HVR-H1 comprises SEQ ID NO:4;
  • HVR-H2 comprises SEQ ID NO:5;
  • HVR-H3 comprises SEQ ID NO: 19.
  • 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.
  • the anti-beta7 antibody is etrolizumab, also referred to as rhuMAb Beta7.
  • Figures 1A and IB show alignment of sequences of the variable light and heavy chains for the following consensus sequences and anti-beta7 subunit antibody sequences: light chain human subgroup kappa I consensus sequence (FIG. 1A, SEQ ID NO: 12), heavy chain human subgroup III consensus sequence (FIG. IB, SEQ ID NO: 13), rat anti-mouse beta7 antibody (Fib504) variable light chain (FIG. 1A, SEQ ID NO: 10), rat anti-mouse beta7 antibody (Fib504) variable heavy chain (FIG. IB, SEQ ID NO: 11), and humanized antibody variants: Humanized hu504Kgraft variable light chain (FIG.
  • FIG. 1A SEQ ID NO: 14
  • humanized hu504K graft variable heavy chain FIG. IB
  • variants hu504-5, hu504-16, and hu504-32 amino acid variations from humanized hu504K graft are indicated in FIG. 1 A
  • FIG. IB dasheavy chain for variants hu504-5, hu504-16, and hu504-32 (SEQ ID NO:25).
  • Figure 2 shows the variable light chain region (Fig. 2 A) (SEQ ID NO:31) and the variable heavy chain region (Fig. 2B) (SEQ ID NO: 32) of etrolizumab.
  • Figure 3 shows the study schema for the Phase II clinical study as described in
  • Figure 4 shows the study schema for the Phase II open label extension clinical study as described in Example 1.
  • Figure 5A-C show changes from baseline levels of T cells (Fig. 5 A), Treg cells (Fig. 5B), and Thl7 cells (Fig. 5C) over time in UC patients admininstered 100 mg rhuMAb Beta7 (etrolizumab, "ab7"), 300 mg rhuMAb Beta7 (etrolizumab, "ab7"), or placebo.
  • Figure 6A-C show changes from baseline levels of T cells (Fig. 6A), Treg cells (Fig. 6B), and Thl7 cells (Fig. 6C) over time in UC patients administered rhuMAb Beta7 (etrolizumab) that experienced clinical remission, UC patients administered rhuMAb Beta7 (etrolizumab) that did not experience remission, and UC patients that received placebo.
  • Figure 7A-C show changes from baseline levels of T cells (Fig. 7A), Treg cells (Fig. 7B), and Thl7 cells (Fig. 7C) over time in TNF-nai ' ve and TNF-inadequate responder UC patients administered rhuMAb Beta7 (etrolizumab) that experienced clinical remission, TNF- nai ' ve and TNF-inadequate responder UC patients administered rhuMAb Beta7 (etrolizumab) that did not experience remission, and UC patients that received placebo.
  • Figures 8A-B show the percentage of Treg cells at baseline in blood of TNF-nai ' ve UC patients administered rhuMAb Beta7 (etrolizumab) that experienced clinical remission and TNF-nai ' ve UC patients administered rhuMAb Beta7 (etrolizumab) that did not experience clinical remission (patients treated with placebo were excluded) (Fig.
  • Figures 9A-C show percentage of all patients in remission in biomarker high vs biomarker low patients.
  • Figures 10A-B show percentage of TNF -naive patients in remission in biomarker high vs biomarker low patients.
  • Ranges provided in the specification and appended claims include both end points and all points between the end points.
  • a range of 2.0 to 3.0 includes 2.0, 3.0, and all points between 2.0 and 3.0.
  • Treatment refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • Treatment regimen refers to a combination of dosage, frequency of
  • Effective treatment regimen refers to a treatment regimen that will offer beneficial response to a patient receiving the treatment.
  • Modifying a treatment refers to changing the treatment regimen including, changing dosage, frequency of administration, or duration of treatment, and/or addition of a second medication.
  • Patient response or “patient responsiveness” can be assessed using any endpoint indicating a benefit to the patient, including, without limitation, (1) inhibition, to some extent, of disease progression, including slowing down and complete arrest; (2) reduction in the number of disease episodes and/or symptoms; (3) reduction in lesional size; (4) inhibition (i.e., reduction, slowing down or complete stopping) of disease cell infiltration into adjacent peripheral organs and/or tissues; (5) inhibition (i.e., reduction, slowing down or complete stopping) of disease spread; (6) decrease of auto-immune, immune, or inflammatory response, which may, but does not have to, result in the regression or ablation of the disease lesion; (7) relief, to some extent, of one or more symptoms associated with the disorder; (8) increase in the length of disease-free presentation following treatment; and/or (9) decreased mortality at a given point of time following treatment.
  • responsiveness refers to a measurable response, including complete response (CR) and partial response (PR).
  • Partial response refers to a decrease of at least 50% in the severity of inflammation, in response to treatment.
  • a "beneficial response" of a patient to treatment with an integrin beta7 antagonist and similar wording refers to the clinical or therapeutic benefit imparted to a patient at risk for or suffering from a gastrointestinal inflammatory disorder from or as a result of the treatment with the antagonist, such as an anti-beta7 integrin antibody.
  • Such benefit includes cellular or biological responses, a complete response, a partial response, a stable disease (without progression or relapse), or a response with a later relapse of the patient from or as a result of the treatment with the antagonist.
  • non-response or “lack of response” or similar wording means an absence of a complete response, a partial response, or a beneficial response to treatment with an integrin beta7 antagonist.
  • sample refers to a composition that is obtained or derived from a subject of interest that contains a cellular and/or other molecular entity that is to be characterized and/or identified, for example based on physical, biochemical, chemical and/or physiological characteristics.
  • disease sample and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and/or molecular entity that is to be characterized.
  • the sample can be obtained from a tissue for the subject of interest or from peripheral blood of the subject.
  • the sample may be obtained from blood and other liquid samples of biological origin and tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom.
  • the source of the tissue sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids; and cells from any time in gestation or development of the subject or plasma.
  • sample or “test sample” includes biological samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides, or embedding in a semi-solid or solid matrix for sectioning purposes.
  • a "section" of a tissue sample is meant a single part or piece of a tissue sample, e.g. 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, lypmph fluid, synovial fluid, cellular extracts, and combinations thereof.
  • a "reference sample,” as used herein, refers to any sample, standard, or level that is used for comparison purposes.
  • a reference sample is obtained from a healthy and/or non-diseased part of the body (e.g., tissue or cells) of the same subject or patient.
  • a reference sample is obtained from an untreated tissue and/or cell of the body of the same subject or patient.
  • a reference sample is obtained from a healthy and/or non-diseased part of the body (e.g., tissues or cells) of an individual who is not the subject or patient.
  • a reference sample is obtained from an untreated tissue and/or cell part of the body of an individual who is not the subject or patient.
  • a beta7 integrin antagonist or “beta7 antagonist” refers to any molecule that inhibits one or more biological activities or blocking binding of beta7 integrin with one or more of its associated molecules.
  • Antagonists of the invention can be used to modulate one or more aspects of beta7 associated effects, including but not limited to association with alpha4 integrin subunit, association with alphaE integrin subunit, binding of alpha4beta7 integrin to MAdCAM, VCAM-1 or fibronectin and binding of alphaEbeta7 integrin to E-cadherin.
  • beta7 antagonist is an anti-beta7 integrin antibody (or anti-beta7 antibody).
  • the anti-beta7 integrin antibody is a humanized anti-beta7 integrin antibody and more particularly a recombinant humanized monoclonal anti-beta7 antibody, for example, rhuMAb beta7, also referred to as etrolizumab.
  • the anti-beta7 antibodies of the present invention are anti-integrin beta7 antagonistic antibodies that inhibit or block the binding of beta7 subunit with alpha4 integrin subunit, association with alphaE integrin subunit, binding of alpha4beta7 integrin to MAdCAM, VCAM-1 or fibronectin and binding of alphaEbeta7 integrin to E-cadherin.
  • beta7 subunit or " ⁇ 7 subunit” is meant the human ⁇ 7 integrin subunit (Erie et al, (1991) J. Biol. Chem. 266: 11009-11016).
  • the beta7 subunit associates with alpha4 integrin subunit, such as the human .alpha.4 subunit (Kilger and Holzmann (1995) J. Mol. Biol. 73:347-354).
  • alpha4beta7 integrin is reportedly expressed on a majority of mature lymphocytes, as well as a small population of thymocytes, bone marrow cells and mast cells. (Kilshaw and Murant (1991) Eur. J. Immunol.
  • the beta7 subunit also associates with the alphaE subunit, such as the human alphaE integrin subunit (Cepek, K. L, et al. (1993) J. Immunol. 150:3459).
  • the alphaEbeta7 integrin is expressed on intra- intestinal epithelial lymphocytes (ilELs) (Cepek, K. L. (1993) supra).
  • alphaE subunit or "alphaE integrin subunit” or “ocE subunit” or “ocE integrin subunit” or “CD 103” is meant an integrin subunit found to be associated with beta7 integrin on intra-epithelial lymphocytes, which alphaEbeta7 integrin mediates binding of the iELs to intestinal epithelium expressing E-cadherin (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 composed of three immunoglobulin-like domains.
  • the cDNAs for murine, human and macaque MAdCAM-1 have been cloned (Briskin, et al, (1993) Nature, 363:461-464; Shyjan ⁇ a/., (1996) J. Immunol. 156:2851-2857).
  • VCAM-1 or "vascular cell adhesion molecule-1”
  • CD 106 refers to a ligand of alpha4beta7 and alpha4betal, expressed on activated endothelium and important in
  • 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 contains an extracellular domain, a single transmembrane segment and two tandem intracytoplasmic catalytic domains, and thus belongs to receptor type PTP. This gene is specifically expressed in hematopoietic cells. This PTP has been shown to be an essential regulator of T- and B-cell antigen receptor signaling.
  • CD45RA CD45RA, CD45RB, CD45RC, CD45RAB, CD45RAC, CD45RBC, CD45RO, CD45R (ABC).
  • CD45 is also highly glycosylated.
  • CD45R is the longest protein and migrates at 200 kDa when isolated from T cells.
  • B cells also express CD45R with heavier glycosylation, bringing the molecular weight to 220 kDa, hence the name B220;
  • B220 expression is not restricted to B cells and can also be expressed on activated T cells, on 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 exon 4 CD45 polymorphism in HIV seropositive Kenyans.”
  • "Gut-homing lymphocytes” refer to a subgroup of lymphocytes having the characteristic of selectively homing to intestinal lymph nodes and tissues but not homing to peripheral lymph nodes and tissues. This subgroup of lymphocytes are characterized by an unique expression partem of a combination of multiples cell surface molecules, including, but not limited to, the combination of CD4, CD45RA and Beta7. Typically, at least two subsets of peripheral blood CD4 + lymphocytes can be subdivided based on the markers of CD45RA and Beta7, CD45RA ⁇ 7 hi ⁇ h , and CD45RA ⁇ 7 low CD4 + cells.
  • CD45RA ⁇ 7 hi ⁇ h CD4 + cells home preferentially to intestinal lymph nodes and tissues
  • CD45RA ⁇ 7 low CD4 + cells home preferentially to peripheral lymph nodes and tissues
  • Gut- homing lymphocytes are therefore a distinctive subgroup of lymphocytes identified as CD45RA ⁇ 7 hl h CD4 + in a flow cytometry assay. The methods of identifying this group of lymphocytes are well-known in the art.
  • CD4 + lymphocytes are a group of lymphocytes having CD4 expressed on their cell surfaces.
  • CD45RA lymphocytes are a group of lymphocytes having no CD45RA expressed on their cell surfaces.
  • An “amount” or “level” of biomarker can be determined using methods known in the art and disclosed herein, such as flow cytometry analysis or qPCR.
  • a "change in the amount or level of a biomarker" is as compared to a
  • a reference or comparator amount of the biomarker can be the amount of a biomarker before treatment and more particularly, can be the baseline or pre-dose amount.
  • Gastrointestinal inflammatory disorders are a group of chronic disorders that cause inflammation and/or ulceration in the mucous membrane. 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 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 necrotizing enterocolitis and esophagitis.
  • IBD Inflammatory Bowel Disease
  • IBD ulcerative colitis
  • Crohn's disease (CD) and “ulcerative colitis (UC)” are chronic inflammatory bowel diseases of unknown etiology. Crohn's disease, unlike ulcerative colitis, can affect any part of the bowel. The most prominent feature Crohn's disease is the granular, reddish-purple edematous thickening of the bowel wall. With the development of inflammation, these granulomas often lose their circumscribed borders and integrate with the surrounding tissue. Diarrhea and obstruction of the bowel are the predominant clinical features.
  • Crohn's disease As with ulcerative colitis, the course of Crohn's disease may be continuous or relapsing, mild or severe, but unlike ulcerative colitis, Crohn's disease is not curable by resection of the involved segment of bowel. Most patients with Crohn's disease require surgery at some point, but subsequent relapse is common and continuous medical treatment is usual.
  • Crohn's disease may involve any part of the alimentary tract from the mouth to the anus, although typically it appears in the ileocolic, small-intestinal or colonic-anorectal regions. Histopathologically, the disease manifests by discontinuous granulomatomas, crypt abscesses, fissures and aphthous ulcers.
  • the inflammatory infiltrate is mixed, consisting of lymphocytes (both T and B cells), plasma cells, macrophages, and neutrophils. There is a disproportionate increase in IgM- and IgG-secreting plasma cells, macrophages and neutrophils.
  • Anti-inflammatory drugs sulfasalazine and 5-aminosalisylic acid (5-ASA) are used for treating mildly active colonic Crohn's disease and are commonly prescribed in an attempt to maintain remission of the disease.
  • Metroidazole and ciprofloxacin are similar in efficacy to sulfasalazine and are particularly prescribed for treating perianal disease.
  • corticosteroids are prescribed to treat active exacerbations and can sometimes maintain remission.
  • Azathioprine and 6-mercaptopurine have also been used in patients who require chronic administration of corticosteroids. It has been suggested that these drugs may play a role in the long-term prophylaxis.
  • Antidiarrheal drugs can also provide symptomatic relief in some patients.
  • Nutritional therapy or elemental diet can improve the nutritional status of patients and induce symptomatic improvement of acute disease, but it does not induce sustained clinical remissions.
  • Antibiotics are used in treating secondary small bowel bacterial overgrowth and in treatment of pyogenic complications.
  • Ulcerative colitis afflicts the large intestine.
  • the course of the disease may be continuous or relapsing, mild or severe.
  • the earliest lesion is an inflammatory infiltration with abscess formation at the base of the crypts of Lieberkuhn. Coalescence of these distended and ruptured crypts tends to separate the overlying mucosa from its blood supply, leading to ulceration.
  • Symptoms of the disease include cramping, lower abdominal pain, rectal bleeding, and frequent, loose discharges consisting mainly of blood, pus and mucus with scanty fecal particles.
  • a total colectomy may be required for acute, severe or chronic, unremitting ulcerative colitis.
  • UC ulcerative colitis
  • onset may be insidious or abrupt, and may include diarrhea, tenesmus and relapsing rectal bleeding.
  • fulminant involvement of the entire colon toxic megacolon, a life-threatening emergency, may occur.
  • Extraintestinal manifestations include arthritis, pyoderma gangrenoum, uveitis, and erythema nodosum.
  • Treatment for UC includes sulfasalazine and related salicylate-containing drugs for mild cases and corticosteroid drugs in severe cases.
  • Topical administration of either salicylates or corticosteroids is sometimes effective, particularly when the disease is limited to the distal bowel, and is associated with decreased side effects compared with systemic use.
  • Supportive measures such as administration of iron and antidiarrheal agents are sometimes indicated.
  • Azathioprine, 6-mercaptopurine and methotrexate are sometimes also prescribed for use in refractory corticosteroid-dependent cases.
  • an “effective dosage” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • the term "patient” refers to any single subject for which treatment is desired.
  • the patient herein is a human.
  • a "subject" herein is typically a human.
  • a subject is a non-human mammal.
  • exemplary non-human mammals include laboratory, domestic, pet, sport, and stock animals, e.g., mice, cats, dogs, horses, and cows.
  • the subject is eligible for treatment, e.g., treatment of a gastrointestinal inflammatory disorder.
  • antibody and “immunoglobulin” are used interchangeably in the broadest sense and include monoclonal antibodies (for example, 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 greater detail herein).
  • An 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, and typically most or all, of the functions normally associated with that portion when present in an intact antibody.
  • an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen.
  • an antibody fragment for example one that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half life modulation, ADCC function and complement binding.
  • an antibody fragment is a monovalent antibody that has an in vivo half life substantially similar to an intact antibody.
  • such an antibody fragment may comprise on antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that 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 with or homologous to
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which 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 lo sequence.
  • the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • a "human antibody” is one which comprises an amino acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as 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 for the production of human monoclonal antibodies (see, e.g., Kozbor J.
  • 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: 4
  • An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or
  • the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and often more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS- PAGE under reducing or nonreducing conditions using Coomassie blue or silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • hypervariable region when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops.
  • antibodies comprise six hypervariable regions; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3).
  • a number of hypervariable region delineations are in use and are encompassed herein.
  • the Kabat Complementarity Determining Regions are based on sequence variability and are the most commonly used (Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
  • the AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.
  • the "contact" hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.
  • Hypervariable regions may comprise "extended hypervariable regions” as follows: 24-36 or 24-34 (LI), 46-56 or 49-56 or 50-56 or 52-56 (L2) and 89-97 (L3) in the VL and 26- 35 (HI), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in the VH.
  • the variable domain residues are numbered according to Kabat et al, supra for each of these definitions.
  • a "human consensus framework” is a framework which represents the most commonly occurring amino acid residue in a selection of human immunoglobulin VL or VH framework sequences.
  • the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
  • the subgroup of sequences is a subgroup as in Kabat et al.
  • the subgroup is subgroup kappa I as in Kabat et al.
  • the subgroup III as in Kabat et al.
  • affinity matured antibody is one with one or more alterations in one or more CDRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s).
  • affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen.
  • Affinity matured antibodies are produced by procedures known in the art. Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by: Barbas et al. Proc Nat. Acad.
  • Binding affinity generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.
  • Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.
  • a variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention.
  • variable in connection with antibodies or immunoglobulins refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs).
  • the variable domains of native heavy and light chains each comprise four FRs, largely adopting a ⁇ -sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit 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 yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.
  • Fv is the minimum antibody fragment which contains a complete antigen- recognition and antigen-binding site. This region consists of a dimer of one heavy chain 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 an antigen- binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising 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 (CHI) of the heavy chain.
  • Fab fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear at least one free thiol group.
  • F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have 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 can be assigned to different classes.
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG2, IgG3, IgG4, IgAi, and IgA2.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • An antibody may be part of a larger fusion molecule, formed by covalent or non- covalent association of the antibody with one or more other proteins or peptides.
  • full-length antibody “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below.
  • a "naked antibody” for the purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.
  • Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • the numbering of the residues in an immunoglobulin heavy chain 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), expressly incorporated herein by reference.
  • the "EU index as in Kabat” refers to the residue numbering of the human IgGl EU antibody.
  • a "functional Fc region” possesses an "effector function" of a native sequence
  • effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • phagocytosis down regulation 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 using various assays as herein disclosed, for example.
  • 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 a native sequence human IgGl Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof.
  • a “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification.
  • the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, and in certain embodiments from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide.
  • the variant Fc region herein will possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, or at least about 90% homology therewith, or at least about 95% homology therewith.
  • intact antibodies can be assigned to different "classes.” There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy-chain constant domains that correspond to the different classes of antibodies are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • Antibody-dependent cell-mediated cytotoxicity and “ADCC” refer to a cell- mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
  • FcRs Fc receptors
  • FcR expression on hematopoietic cells in summarized is Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).
  • ADCC activity of a molecule of interest may be assessed in vitro, such as that described in U.S. Patent No. 5,500,362 or 5,821,337.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
  • Human effector cells are leukocytes which express one or more FcRs and perform effector functions. In certain embodiments, the cells express at least FCYRIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer
  • monocytes cytotoxic T cells and neutrophils.
  • the effector cells may be isolated from a native source thereof, e.g., from blood or PBMCs as described herein.
  • Fc receptor or “FcR” are used to describe a receptor that binds to the Fc region of an antibody.
  • FcR is a native sequence human FcR.
  • FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • FcyRII receptors include FcyRIIA (an “activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (IT AM) in its cytoplasmic domain.
  • Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (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).
  • FcR FcR
  • the term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al, J. Immunol. 117:587 (1976) and Kim et al, J. Immunol. 24:249 (1994)), and regulates homeostasis of immunoglobulins.
  • Antibodies with improved binding to the neonatal Fc receptor (FcRn), and increased half-lives, are described in WO00/42072 (Presta, L.) and US2005/0014934A1 (Hinton et al).
  • these antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn.
  • the Fc region may have substitutions 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).
  • the Fc region- comprising antibody variant with improved FcRn binding comprises amino acid substitutions at one, two or three of positions 307, 380 and 434 of the Fc region thereof (Eu numbering of residues).
  • Single-chain Fv or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • HER2 antibody scFv fragments are described in W093/16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458.
  • diabodies refers to small antibody fragments with two antigen- binding sites, which fragments comprise a variable heavy domain (VH) connected to a variable light domain (VL) in the same polypeptide chain (VH - VL).
  • VH variable heavy domain
  • VL variable light domain
  • linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11 161 ; and Hollinger et ctl, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
  • affinity matured antibody is one with one or more alterations in one or more hypervariable regions thereof which result an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s).
  • affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen.
  • Affinity matured antibodies are produced by procedures known in the art. Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by: Barbas et al. Proc Nat. Acad.
  • amino acid sequence variant antibody herein is an antibody with an amino acid sequence which differs from a main species antibody.
  • amino acid sequence variants will possess at least about 70% homology with the main species antibody, or they will be at least about 80%, or at least about 90% homologous with the main species antibody.
  • the amino acid sequence variants possess substitutions, deletions, and/or additions at certain positions within or adjacent to the amino acid sequence of the main species antibody.
  • amino acid sequence variants herein include an acidic variant (e.g., deamidated antibody variant), a basic variant, an antibody with an amino-terminal leader extension (e.g.
  • VHS- on one or two light chains thereof, an antibody with a C-terminal lysine residue on one or two heavy chains thereof, etc, and includes combinations of variations to the amino acid sequences of heavy and/or light chains.
  • the antibody variant of particular interest herein is the antibody comprising an amino-terminal leader extension on one or two light chains thereof, optionally further comprising other amino acid sequence and/or glycosylation differences relative to the main species antibody.
  • a "glycosylation variant” antibody herein is an antibody with one or more carbohydrate moieties attached thereto which differ from one or more carbohydrate moieties attached to a main species antibody.
  • glycosylation variants herein include antibody with a Gl or G2 oligosaccharide structure, instead a GO oligosaccharide structure, attached to an Fc region thereof, antibody with one or two carbohydrate moieties attached to one or two light chains thereof, antibody with no carbohydrate attached to one or two heavy chains of the antibody, etc, and combinations of glycosylation alterations. Where the antibody has an Fc region, an oligosaccharide structure may be attached to one or two heavy chains of the antibody, e.g.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g. At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes 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.
  • radioactive isotopes e.g. At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu
  • chemotherapeutic agents e.g. At 211 , 1 131 , 1 125 , Y 90 , Re 186
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.
  • cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone 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); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-a and - ⁇ ; mullerian- inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF- ⁇ ; platelet
  • ILs interleukins
  • IL-1 interleukins
  • IL-la interleukins
  • IL-2 interleukins
  • IL-3 interleukins
  • IL-4 interleukins
  • IL-5 IL-6
  • IL-7 tumor necrosis factor
  • IL-8 IL-9
  • IL-10 IL- 11, IL-12
  • KL kit ligand
  • cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
  • immunosuppressive agent refers to substances that act to suppress or mask the immune system of the subject being treated herein. This would include substances that suppress cytokine production, down-regulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Patent No.
  • non-steroidal antiinflammatory drugs NSAIDs
  • ganciclovir tacrolimus
  • glucocorticoids such as Cortisol or aldosterone
  • anti-inflammatory agents such as a cyclooxygenase inhibitor; a 5-lipoxygenase inhibitor; or a leukotriene receptor antagonist
  • purine antagonists such as azathioprine or mycophenolate mofetil (MMF)
  • alkylating agents such as cyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde (which masks the MHC antigens, as described in U.S. Patent No. 4,120,649)
  • anti-idiotypic antibodies for MHC antigens and MHC fragments NSAIDs
  • tacrolimus such as Cortisol or aldosterone
  • anti-inflammatory agents such as a cyclooxygenase inhibitor; a 5-lipoxygenase inhibitor; or a leukot
  • cyclosporine 6 mercaptopurine; steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogs, e.g., prednisone, methylprednisolone, including SOLU- MEDROL.RTM.
  • steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogs, e.g., prednisone, methylprednisolone, including SOLU- MEDROL.RTM.
  • methylprednisolone sodium succinate, and dexamethasone dihydrofolate reductase inhibitors such as methotrexate (oral or subcutaneous); anti-malarial agents such as chloroquine and hydroxychloroquine; sulfasalazine; leflunomide; cytokine or cytokine receptor antibodies or antagonists including anti-interferon-alpha, -beta, or -gamma antibodies, antitumor necrosis factor(TNF)-alpha antibodies (infliximab (REMICADE.RTM.) or
  • adalimumab anti-TNF-alpha immunoadhesin (etanercept), anti-TNF-beta 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-CDl la and anti- CD 18 antibodies; anti-L3T4 antibodies; heterologous anti -lymphocyte globulin; pan-T antibodies, anti-CD3 or anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3 binding domain (WO 90/08187 published Jul. 26, 1990); streptokinase; transforming growth factor-beta (TGF-beta); streptodomase; RNA or DNA from the host; FK506; RS-61443;
  • 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) and see also definition below
  • biologic agents that interfere with T cell helper signals 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 CTLA
  • ameliorates or “amelioration” as used herein refers to a decrease, reduction or elimination of a condition, disease, disorder, or phenotype, including an abnormality or symptom.
  • a "symptom" of a disease or disorder is any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by a subject and indicative of disease.
  • a “therapeutically effective amount” refers to an amount that is effective for preventing, ameliorating, or treating a disease or disorder (e.g., inflammatory bowel disease, e.g., ulcerative colitis or Crohn's disease).
  • a “therapeutically effective amount” of an antibody refers to an amount of the antibody that is effective for preventing, ameliorating, or treating the specified disease or disorder.
  • 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, in
  • combination is effective for preventing, ameliorating, or treating the specified disease or disorder.
  • a combination of two compounds does not mean that the compounds have to be administered in admixture with each other.
  • treatment with or use of such a combination encompasses a mixture of the compounds or separate administration of the compounds, and includes administration on the same day or different days.
  • the terminology “combination” means two or more compounds are used for the treatment, either individually or in admixture with each other.
  • an antibody and a second compound for example, are administered in combination to a subject, the antibody is present in the subject at a time when the second compound is also present in the subject, whether the antibody and second compound are administered individually or in admixture to the subject.
  • a compound other than the antibody is administered prior to the antibody.
  • a compound other than the antibody is administered after the antibody.
  • TNF-alpha tumor necrosis factor-alpha
  • TNF-alpha refers to a human TNF-alpha molecule comprising the amino acid sequence as described in Pennica et al, Nature, 312:721 (1984) or Aggarwal et al, JBC, 260:2345 (1985).
  • TNF-alpha inhibitor is used interchangeably herein with "anti-TNF therapeutic agent” and refers to an agent that inhibits, to some extent, a biological function of TNF-alpha, generally through binding to TNF-alpha and neutralizing its activity.
  • TNF inhibitors specifically contemplated herein are etanercept (ENBREL®), infliximab (REMICADE®), adalimumab (HUMIRA®), golimumab (SIMPONITM), and certolizumab pegol (CIMZIA®).
  • TNF -naive or "a patient not previously treated with an anti-TNF therapeutic agent” refers to a patient who has not previously been administred an anti-TNF therapeutic agent.
  • Corticosteroid refers to any one of several synthetic or naturally occurring substances with the general chemical structure of steroids that mimic or augment the effects of the naturally occurring corticosteroids.
  • synthetic corticosteroids include prednisone, prednisolone (including methylprednisolone), dexamethasone triamcinolone, and betamethasone.
  • Antagonist refers to a molecule capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with the activities of a particular or specified protein, including its binding to one or more receptors in the case of a ligand or binding to one or more ligands in case of a receptor.
  • Antagonists include antibodies and antigen-binding fragments thereof, proteins, peptides, glycoproteins, glycopeptides, glycolipids,
  • Antagonists also include small molecule inhibitors of the protein, and fusion proteins, receptor molecules and derivatives which bind specifically to the protein thereby sequestering its binding to its target, antagonist variants of the protein, antisense molecules directed to the protein, RNA aptamers, and ribozymes against the protein.
  • a "self-inject device” refers to a medical device for self-administration, e.g., by a patient or in-home caregiver, of a therapeutic agent.
  • Self-inject devices include autoinjector devices and other devices designed for self-administration.
  • Oligonucleotide refers to short, single stranded
  • Oligonucleotides that are at least about seven nucleotides in length and less than about 250 nucleotides in length. Oligonucleotides may be synthetic. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.
  • primer refers to a single stranded polynucleotide that is capable of hybridizing to a nucleic acid and allowing the polymerization of a complementary nucleic acid, generally by providing a free 3'-OH group.
  • Amplification refers to the process of producing one or more copies of a reference nucleic acid sequence or its complement. Amplification may be linear or exponential (e.g., PCR). A “copy” does not necessarily mean perfect sequence
  • copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable, but not fully complementary, to the template), and/or sequence errors that occur during amplification.
  • nucleotide analogs such as deoxyinosine
  • intentional sequence alterations such as sequence alterations introduced through a primer comprising a sequence that is hybridizable, but not fully complementary, to the template
  • sequence errors that occur during amplification.
  • detection includes any means of detecting, including direct and indirect detection.
  • Elevated levels or “higher levels” refers to an increased amount or proportion of a cell type in a patient relative to a control or to a reference level, such as an individual or individuals who are not suffering from an autoimmune disease, e.g., IBD, or relative to a pre- established threshold or cut-off value, or relative to the median for a population of patients and/or subjects.
  • a control or to a reference level such as an individual or individuals who are not suffering from an autoimmune disease, e.g., IBD, or relative to a pre- established threshold or cut-off value, or relative to the median for a population of patients and/or subjects.
  • Reduced levels refers to a decreased amount or proportion of a cell type in a patient relative to a control or to a reference level, such as an individual or individuals who are not suffering from an autoimmune disease, e.g., IBD, or relative to a pre- established threshold or cut-off value, or relative to the median for a population of patients and/or subjects.
  • a control or to a reference level such as an individual or individuals who are not suffering from an autoimmune disease, e.g., IBD, or relative to a pre- established threshold or cut-off value, or relative to the median for a population of patients and/or subjects.
  • multiplex-PCR refers to a single PCR reaction carried out on nucleic acid obtained from a single source (e.g., a patient) using more than one primer set for the purpose of amplifying two or more DNA sequences in a single reaction.
  • Biomarker refers to an indicator of a phenotype of a patient, e.g., a pathological state or likely responsiveness to a therapeutic agent, which can be detected in a biological sample of the patient.
  • Biomarkers include, but are not limited to, particular cell typies, such as immune cells, T cells, and T cell subsets.
  • diagnosis is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition.
  • diagnosis may refer to identification of a particular type of gastrointestinal inflammatory disorder, or the classification of a particular sub-type of gastrointestinal inflammatory disorder, by tissue/organ involvement (e.g., inflammatory bowel disease), or by other features (e.g., a patient subpopulation characterized by responsiveness to a treatment, such as to a treatment with an integrin beta7 antagonist), or by molecular features (e.g., a subtype characterized by expression of one or a combination of particular genes or proteins encoded by said genes).
  • tissue/organ involvement e.g., inflammatory bowel disease
  • other features e.g., a patient subpopulation characterized by responsiveness to a treatment, such as to a treatment with an integrin beta7 antagonist
  • molecular features e.g., a subtype characterized by expression of one or a combination of particular genes or proteins encoded by said genes.
  • a method of aiding diagnosis of IBD can comprise measuring the level of one or more biomarkers in a biological sample from an individual.
  • prognosis is used herein to refer to the prediction of the likelihood of disease symptoms, including, for example, recurrence, flaring, and drug resistance, of a gastrointestinal inflammatory disorder.
  • prediction is used herein to refer to the likelihood that a patient will respond either favorably or unfavorably to a drug (therapeutic agent) or set of drugs or a therapeutic regimen. In one embodiment, the prediction relates to the extent of those responses. In one embodiment, the prediction relates to whether and/or the probability that a patient will survive or improve following treatment, for example treatment with a particular therapeutic agent, or for a certain period of time without disease recurrence.
  • the predictive methods of the invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient.
  • the predictive methods of the present invention are valuable tools in predicting if a patient is likely to respond favorably to a treatment regimen, such as a given therapeutic regimen, including for example, administration of a given therapeutic agent or combination, surgical intervention, steroid treatment, etc., or whether long-term survival of the patient or remission or sustained remission, following a therapeutic regimen is likely.
  • a treatment regimen such as a given therapeutic regimen, including for example, administration of a given therapeutic agent or combination, surgical intervention, steroid treatment, etc., or whether long-term survival of the patient or remission or sustained remission, following a therapeutic regimen is likely.
  • the predictive methods described herein are used to determine whether a patient who is being treated with a particular therapeutic regimen will have a particular outcome, for example, before the outcome may be otherwise known.
  • control subject refers to a healthy subject who has not been diagnosed as having a particular disease, e.g., IBD, and who does not suffer from any sign or symptom associated with that disease.
  • correlate or “correlating” is meant comparing, in any way, the
  • a first analysis or protocol may be used in carrying out a second protocols and/or one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed.
  • biomarker level analysis or protocol one may use the results of the biomarker level analysis or protocol to determine whether a specific therapeutic regimen should be performed.
  • comparing refers to comparing the level of the biomarker in the sample from the individual or patient with the reference level of the biomarker specified elsewhere in this description. It is to be understood that comparing as used herein usually refers to a comparison of corresponding parameters or values, e.g., an absolute amount is compared to an absolute reference amount while a concentration is compared to a reference concentration or an intensity signal obtained from the biomarker in a sample is compared to the same type of intensity signal obtained from a reference sample.
  • the comparison may be carried out manually or computer assisted. Thus, the comparison may be carried out by a computing device (e.g., of a system disclosed herein).
  • the value of the measured or detected level of the biomarker in the sample from the individual or patient and the reference level can be, e.g., compared to each other and the said comparison can be automatically carried out by a computer program executing an algorithm for the comparison.
  • the computer program carrying out the said evaluation will provide the desired assessment in a suitable output format.
  • the value of the determined amount may be compared to values corresponding to suitable references which are stored in a database by a computer program.
  • the computer program may further evaluate the result of the comparison, i.e. automatically provide the desired assessment in a suitable output format.
  • the value of the determined amount may be compared to values corresponding to suitable references which are stored in a database by a computer program.
  • the computer program may further evaluate the result of the comparison, i.e. automatically provides the desired assessment in a suitable output format.
  • the phrase "recommending a treatment” as used herein refers to using the information or data generated relating to the level of the biomarkers (such as level of T cells, Treg cells, and/or Thl7 cells) in a biological sample from a patient, to identify the patient as suitably treated or not suitably treated with a therapy.
  • the therapy comprises an integrin beta7 antagonist, including an anti-integrin beta7 antibody such as etrolizumab.
  • the phrase "recommending a treatment/therapy” includes the identification of a patient who requires adaptation of an effective amount of the integrin beta7 antagonist being administered.
  • recommending a treatment includes recommending that the amount of integrin beta7 antagonist being administered is adapted.
  • the phrase "recommending a treatment” as used herein also may refer to using the information or data generated for proposing or selecting a therapy comprising an integrin beta7 antagonist for a patient identified or selected as more or less likely to respond to the therapy comprising an integrin beta7 antagonist.
  • the information or data used or generated may be in any form, written, oral or electronic.
  • using the information or data generated includes communicating, presenting, reporting, storing, sending, transferring, supplying, transmitting, dispensing, or combinations thereof.
  • communicating, presenting, reporting, storing, sending, transferring, supplying, transmitting, dispensing, or combinations thereof are performed by a computing device, analyzer unit or combination thereof.
  • communicating, presenting, reporting, storing, sending, transferring, supplying, transmitting, dispensing, or combinations thereof are performed by a laboratory or medical professional.
  • the information or data includes a comparison of the level of the biomarker(s) to a reference level.
  • the information or data includes an indication that one or more of the biomarkers identified is present at elevated or reduced levels in the sample.
  • the information or data includes an indication that the patient is suitably treated or not suitably treated with a therapy comprising an integrin beta7 antagonist, including an anti-integrin beta7 antibody such as etrolizumab.
  • a "package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products or medicaments, 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 medicaments and the like.
  • a "kit” is any manufacture (e.g. a package or container) comprising at least one reagent, e.g. , a medicament for treatment of an IBD, e.g., UC or Crohn's disease, or a probe for specifically detecting a biomarker gene or protein of the invention.
  • the manufacture is promoted, distributed, or sold as a unit for performing the methods of the present invention.
  • a "target audience” is a group of people or an institution to whom or to which a particular medicament is being promoted or intended to be promoted, as by marketing or advertising, especially for particular uses, treatments, or indications, such as individual patients, patient populations, readers of newspapers, medical literature, and magazines, television or internet viewers, radio or internet listeners, physicians, drug companies, etc.
  • whole blood refers to any whole blood sample obtained from an individual. Typically, whole blood contains all of the blood components, e.g., cellular components and plasma. Methods for obtaining whole blood from mammals are well known in the art.
  • the phrase "not responsive" includes a description of those subjects who are resistant and/or refractory to the previously administered medication(s), and includes the situations in which a subject or patient has progressed while receiving the medicament(s) that he or she is being given, and in which a subject or patient has progressed within 12 months (for example, within six months) after completing a regimen involving the medicament(s) to which he or she is no longer responsive.
  • the non-responsiveness to one or more medicaments thus includes subjects who continue to have active disease following previous or current treatment therewith. For instance, a patient may have active disease activity after about one to three months, or three to six months, or six to 12 months, of therapy with the medicament(s) to which they are non-responsive.
  • Such responsiveness may be assessed by a clinician skilled in treating the disorder in question.
  • a subject who experiences "a clinically unacceptably high level of toxicity" from previous or current treatment with one or more medicaments experiences one or more negative side-effects or adverse events associated therewith that are considered by an experienced clinician to be significant, such as, for example, serious infections, congestive heart failure, demyelination (leading to multiple sclerosis), significant hypersensitivity, neuropathological events, high degrees of
  • a cancer such as endometrial cancer, non-Hodgkin's lymphoma, breast cancer, prostate cancer, lung cancer, ovarian cancer, or melanoma, tuberculosis (TB), and the like.
  • the "amount” or "level” of a biomarker associated with an increased clinical benefit to a patient suffering from a certain disease or disorder, or predictive of response to a particular therapeutic agent or treatment regimen, is a detectable level in a biological sample. These can be measured by methods known to one skilled in the art and also disclosed herein. The level of a biomarker can be used to determine the response or the predicted response to a treatment or therapeutic agent.
  • beta7 integrin antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with beta7 integrin, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments.
  • a potential antagonist may be a closely related protein, for example, a mutated form of the beta7 integrin that recognizes the ligand but imparts no effect, thereby competitively inhibiting the action of the beta7 integrin.
  • beta7 integrin antagonist is an antisense RNA or DNA construct prepared using antisense technology, where, e.g., an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation.
  • Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
  • the 5' coding portion of the polynucleotide sequence, which encodes the beta7 integrin herein is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a 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 the beta7 integrin.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into beta7 integrin protein (antisense—Okano, Neurochem, 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, Fla., 1988).
  • the oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the PRO polypeptide.
  • antisense DNA is used, oligodeoxyribonucleotides derived from the translation-initiation site, e.g., 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, the ligand or binding molecule binding site, thereby blocking the normal biological activity of the beta7 integrin.
  • 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 capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential RNA target can-be identified by known techniques. For further details see, e.g., Rossi, Current Biology, 4:469-471 (1994), and PCT Publication No. WO 97/33551 (published Sep. 18, 1997).
  • Nucleic acid molecules in triple-helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides.
  • the base composition of these oligonucleotides is designed such that it promotes triple-helix formation via Hoogsteen base- pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex.
  • PCT Publication No. WO 97/33551 See, e.g., PCT Publication No. WO 97/33551.
  • These small molecules can be identified by any one or more of the screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art.
  • Screening assays for antagonists are designed to identify compounds that bind or complex with the beta7 integrin encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins.
  • Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.
  • the 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.
  • the beta7 integrin antagonists are anti-beta7 antibodies.
  • Exemplary antibodies include polyclonal, monoclonal, humanized, human, bispecific, and heteroconjugate antibodies, etc., as described below.
  • SC subcutaneous
  • IP intraperitoneal
  • a protein that is immunogenic in the species to be immunized e.g., keyhole limpet hemocyanin, serum albumin
  • Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 ⁇ g or 5 ⁇ g of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
  • the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
  • Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
  • the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent.
  • Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.
  • Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al, Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567).
  • a mouse or other appropriate host animal such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
  • lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).
  • a suitable fusing agent such as polyethylene glycol
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium which medium may contain one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner).
  • a suitable culture medium which medium may contain one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner).
  • the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT)
  • HGPRT or HPRT the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody- producing cells, and are sensitive to a selective medium that selects against the unfused parental cells.
  • myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Va., USA.
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al, Anal. Biochem, 107:220 (1980). Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59- 103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D- MEM or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal e.g., by i.p. injection of the cells into mice.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion-exchange
  • DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells serve as a source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein.
  • Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al, Curr. Opinion in Immunol, 5:256-262 (1993) and
  • monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using e.g., the techniques 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, respectively, using phage libraries.
  • the DNA that encodes the antibody may be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy chain and light chain constant domain (CH and CL) sequences for the homologous murine 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 for a non- immunoglobulin polypeptide (heterologous polypeptide).
  • CH and CL constant domain
  • non-immunoglobulin polypeptide sequences can substitute for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.
  • Exemplary anti-beta7 antibodies are Fib504, Fib 21, 22, 27, 30 (Tidswell, M. J
  • the anti-beta7 integrin antibodies of the 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 (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • humanized antibodies are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally 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)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al, Nature, 321 :522-525 (1986); Riechmann et al, Nature, 332:323-327 (1988); Verhoeyen et al, Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized” antibodies are chimeric antibodies (U.S.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity and HAMA response (human anti-mouse antibody) when the antibody is intended for human therapeutic use.
  • the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences.
  • the human V domain sequence which is closest to that of the rodent is identified and the human framework region (FR) within it accepted for the humanized antibody (Sims et al, J. Immunol. 151 :2296 (1993); C o a. et al, J. Mol. Biol, 196:901 (1987)).
  • Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci.
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three- dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three- dimensional conformational structures of selected candidate immunoglobulin sequences.
  • the humanized antibody may be an antibody fragment, such as a Fab, which is optionally conjugated with one or more cytotoxic agent(s) in order to generate an
  • humanized anti-beta7 antibodies include, but are not limited to rhuMAb Beta7, which is a humanized monoclonal antibody against the integrin subunit ⁇ 7 and was derived from the rat anti-mouse/human monoclonal antibody FIB504 (Andrew et al, 1994 J Immunol 1994;153:3847-61). It has been engineered to include human immunoglobulin IgGl heavy chain and ⁇ light chain frameworks and is produced by Chinese hamster ovary cells. This antibody binds to two integrins, ⁇ 4 ⁇ 7 (Holzmann et al.
  • IBD inflammatory bowel diseases
  • UC ulcerative colitis
  • CD Crohn's disease
  • rhuMAb Beta7 is a potent in vitro blocker of the cellular interaction between ⁇ 4 ⁇ 7 and its ligands (mucosal addressin cell adhesion molecule-1 [MAdCAM]-l, vascular cell adhesion molecule [VCAM]-1, and fibronectin) as well as the interaction between ⁇ 7 and its ligand (E-cadherin).
  • rhuMAb Beta7 binds reversibly, with similar high affinity, to ⁇ 7 on lymphocytes from rabbits, cynomolgus monkeys, and humans. It also binds to mouse ⁇ 7 with high affinity.
  • the amino acid sequence as well as the making and using of rhuMAb Beta7 and its variants are disclosed in detail in e.g., U.S. Patent Application Publication No. 20060093601 (issued as U.S. Patent No.
  • FIGS. 1 A and IB depict alignment of sequences of the variable light and heavy chains for the following: light chain human subgroup kappa I consensus sequence (FIG. 1 A, SEQ ID NO: 12), heavy chain human subgroup III consensus sequence (FIG. IB, SEQ ID NO: 13), rat anti-mouse beta7 antibody (Fib504) variable light chain (FIG. 1A, SEQ ID NO: 10), rat anti-mouse beta7 antibody (Fib504) variable heavy chain (FIG. IB, SEQ ID NO: 11), and humanized antibody variants: Humanized hu504Kgraft variable light chain (FIG. 1A, SEQ ID NO: 14), humanized hu504K graft variable heavy chain (FIG.
  • human antibodies can be generated.
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • JH antibody heavy-chain joining region
  • transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc. Natl. Acad. Sci.
  • V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as Ml 3 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell.
  • a filamentous bacteriophage such as Ml 3 or fd
  • Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3 :564-571 (1993).
  • 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.
  • a repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al, J. Mol. Biol. 222:581-597 (1991), or Griffith et al, EMBO J.
  • human antibodies may also be generated by in vitro activated B cells (see U. S. Patent Nos. 5,567,610 and 5,229,275).
  • F(ab')2 fragments can be isolated directly from recombinant host cell culture.
  • the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; US Patent No. 5,571,894; and US Patent No. 5,587,458.
  • the antibody fragment may also be a "linear antibody," e.g., as described in US Patent No. 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific.
  • Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes.
  • Exemplary bispecific antibodies may bind to two different epitopes of beta7 integrin as described herein.
  • Other such antibodies may combine a TAT binding site with a binding site for another protein.
  • an anti-Beta7 integrin arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g., CD3), or Fc receptors for IgG (Fc.y.R), such as FcyRI (CD64),
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express TAT. These antibodies possess a TAT-binding arm and an arm which binds the cytotoxic agent (e.g., saporin, anti-interferon-. alpha., vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).
  • immunoglobulin heavy chain-light chain pairs where the two chains have different specificities (Millstein et al, Nature 305 :537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, Cm, and CH3 regions.
  • the first heavy-chain constant region (CHI) containing the site necessary for light chain bonding present in at least one of the fusions.
  • the bispecific antibodies are 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, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al, Methods in Enzymology 121 :210 (1986).
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the interface comprises at least a part of the Cro domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).
  • Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
  • Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089).
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Patent No. 4,676,980, along with a number of cross-linking techniques.
  • bispecific antibodies can be prepared using chemical linkage.
  • Brennan et al, Science 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab').sub.2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
  • the Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives:
  • TAB thionitrobenzoate
  • One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the "diabody” technology described by Hollinger et al, Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.
  • the fragments comprise a V.sub.H connected to a V.sub.L by a linker which is too short to allow pairing between the two domains on the same chain.
  • V.sub.H and V.sub.L domains of one fragment are forced to pair with the complementary V.sub.L and V.sub.H domains of another fragment, thereby forming two antigen-binding sites.
  • Another strategy for making bispecific antibody fragments by the use of 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.
  • trispecific antibodies can be prepared. Tutt et al, J. Immunol. 147:60 (1991).
  • Heteroconjugate antibodies are also within the scope of the present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No.
  • a multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind.
  • the antibodies of the present invention can be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody.
  • the multivalent antibody can comprise a dimerization domain and three or more antigen binding sites.
  • the dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region.
  • the multivalent antibody herein comprises (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 polypeptide chain(s) comprise two or more variable domains.
  • the polypeptide chain(s) may comprise VDl-(Xl).sub.n-VD2-(X2).sub.n-Fc, wherein VDl is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, XI and X2 represent an amino acid or polypeptide, and n is 0 or 1.
  • the polypeptide chain(s) may comprise: VH- CHl-flexible linker-VH-CHl-Fc region chain; or VH-CHl -VH-CHl-Fc region chain.
  • the multivalent antibody herein may further comprise at least two (and typically four) light chain variable domain polypeptides.
  • the multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides.
  • the light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.
  • ADCC antigen-dependent cell-mediated cytotoxicity
  • CDC complement dependent cytotoxicity
  • cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have improved internalization capability and/or increased complement- mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, J. Exp Med. 176: 1191 -1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992).
  • Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al, Cancer Research 53 :2560-2565 (1993).
  • an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al, Anti- Cancer Drug Design 3:219-230 (1989).
  • a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Patent No. 5,739,277, for example.
  • the term "salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgGi, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
  • the antagonist or antibody used in the methods herein is optionally conjugated to another agent, such as a cytotoxic agent, or cytokine.
  • Conjugation will ordinarily be achieved through a covalent linkage, the precise nature of which will be determined by the targeting molecule and the linking site on the integrin beta7 antagonist or antibody polypeptide.
  • a non-peptidic agent is modified by the addition of a linker that allows conjugation to anti-beta7 integrin antibody through its amino acid side chains, carbohydrate chains, or reactive groups introduced on antibody by chemical modification.
  • a drug may be attached through the .epsilon.
  • Protein modifying agents include amine-reactive reagents (e.g., reactive esters, isothiocyanates, aldehydes, and sulfonyl halides), thiol-reactive reagents (e.g., haloacetyl derivatives and maleimides), and carboxylic acid- and aldehyde- reactive reagents.
  • Integrin beta7 antagonist or antibody polypeptides can be covalently joined to peptidic agents through the use of bifunctional cross-linking reagents.
  • Heterobifunctional reagents are more commonly used and permit the controlled coupling of two different proteins through the use of two different reactive moieties (e.g., amine-reactive plus thiol,
  • an anti-beta7 integrin antibody polypeptide can be linked to a peptidic moiety through preparation of a fusion polypeptide.
  • bifunctional protein coupling agents include N- succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene).
  • SPDP N- succinimid
  • the anti-beta7 integrin antibodies disclosed herein may also be formulated as immunoliposomes.
  • a "liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al, Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al, Proc. Natl Acad. Sci. USA 77:4030 (1980); U.S.
  • Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al, J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange reaction.
  • a chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al, J. National Cancer Inst. 81(19): 1484 (1989).
  • nucleic acids encoding the anti-beta7 antibodies or polypeptide agents described herein, vectors and host cells comprising the nucleic acids and recombinant techniques for the production of the antibodies.
  • the nucleic acid encoding it may be isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression.
  • the antibody may be produced by homologous recombination, e.g., as described in U.S. Patent No. 5,204,244, specifically incorporated herein by reference.
  • DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, e.g., as described in U.S. Patent No. 5,534,615 issued Jul. 9, 1996 and specifically incorporated herein by reference.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above.
  • Suitable prokaryotes 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, and Shigella, as well as Bacilli such as B. subtilis and B.
  • Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus
  • Salmonella e.g., Salmonella typhimurium
  • Serratia e.g.,
  • E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli XI 776 (ATCC 31,537), and £. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-beta7 integrin antibody-encoding vectors.
  • Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.
  • Candida Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
  • Suitable host cells for the expression of glycosylated anti-Beta7 antibody are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified.
  • a variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
  • vertebrate cells have been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure.
  • useful mammalian host cell lines are monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney 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 (CVl ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2);
  • kidney cells MDCK, ATCC CCL 34
  • buffalo rat liver cells BBL 3A, ATCC CRL 1442
  • human lung cells W138, ATCC CCL 75
  • human liver cells Hep G2, HB 8065
  • 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 appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the host cells used to produce the anti-beta7 integrin antibody of this invention may be cultured in a variety of media.
  • Commercially available 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 the host cells.
  • 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Patent Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, 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 GENT AMYCIN.TM. drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the antibody When using recombinant techniques, the antibody can be produced
  • the particulate debris either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al,
  • Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a
  • the antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the typical purification technique.
  • affinity chromatography is the typical purification technique.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human .
  • gamma.1 , .gamma.2, or .gamma.4 heavy chains (Lindmark et al, J. Immunol. Meth. 62: 1 -13 (1983)). Protein G is recommended for all mouse isotypes and for human .gamma.3 (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 available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the Bakerbond ABX.TM.resin J. T. Baker, Phillipsburg, N.J.
  • Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE.TM. chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
  • 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 2.5-4.5, typically performed at low salt concentrations (e.g., from about 0-0.25 M salt).
  • Therapeutic formulations comprising the therapeutic agents, antagonists or antibodies of the invention are prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of aqueous solutions, lyophilized or other dried formulations.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, 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 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 immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, hist
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, typically 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 purpose intended.
  • the active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin
  • microspheres microspheres, microemulsions, nano-particles and nanocapsules
  • macroemulsions Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
  • formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the immunoglobulin of the invention, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and .gamma.
  • sustained-release preparations include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and .gamma.
  • ethyl-L- glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-gly colic acid copolymers such as the LUPRON DEPOT.TM. (injectable microspheres composed of lactic acid-gly colic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-gly colic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • encapsulated immunoglobulins When encapsulated immunoglobulins remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S--S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • the physician administering treatment will be able to determine the appropriate dose for the individual subject for weight-based dosing or, for flat dosing, will follow the instructions on the label.
  • Preparation and dosing schedules for commercially available second therapeutic and other compounds administered in combination with the integrin beta7 antagonists may be used according to manufacturers' instructions or determined empirically by the skilled practitioner.
  • the appropriate dosage of the integrin beta7 antagonist and any second therapeutic or other compound administered in combination with the non-depleting 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 administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the integrin beta7 antagonist or combination, and the discretion of the attending physician.
  • the integrin beta7 antagonist or combination is suitably administered to the patient at one time or more typically over a series of treatments.
  • the integrin beta7 antagonist is administered once every week, or once every two weeks, or once every four weeks, or once every six weeks, or once every eight weeks for a period of one month (4 weeks), or two months, three months, or six months, or 12 months, or 18 months, or 24 months, or chronically for the lifetime of the patient.
  • the treatment is self-administered by the patient.
  • anti-beta7 antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • the treatment is sustained until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful.
  • a flat dose of anti-beta7 antibody is administered to the patient.
  • a flat dose is a particular amount of anti-beta7 antibody that is administered to every patient regardless of weight.
  • a flat dose of between about 50 mg and 450 mg of anti-beta7 antibody is administered to the patient, which may be one or more separate injections or infusions or administrations.
  • Such flat dose can be administered intravenously or subcutaneously or by other routes as described herein.
  • the flat 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.
  • an initial flat loading dose of anti-beta7 antibody is followed by one or more flat maintenance doses of anti-beta7 antibody.
  • the loading dose is a larger quantity of anti-beta7 antibody than the maintenance dose.
  • the loading dose is between about 400 mg and 450 mg and the maintenance dose is between about 50 mg and 350 mg.
  • the loading dose is 400 mg, or 420 mg, or 430 mg, or 450 mg.
  • the maintenance 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 350 mg.
  • the clinician will administer an antibody (alone or in combination with a second compound) of the invention until a dosage(s) is reached that provides the required biological effect.
  • an antibody alone or in combination with a second compound of the invention.
  • the progress of the therapy of the invention is easily monitored by conventional techniques and assays.
  • the integrin beta7 antagonist can be administered by any suitable means, including parenteral, topical, intravenous, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and/or intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • Intrathecal administration is also contemplated (see, e.g., U. S. Patent Publication No. 2002/0009444 by Grillo-Lopez).
  • the integrin beta7 antagonist may suitably be administered by pulse infusion, e.g., with declining doses of the antibody.
  • the dosing is given intravenously or subcutaneously.
  • each exposure may be provided using the same or a different administration means.
  • each exposure to anti-beta7 antibody is by subcutaneous administration.
  • the first exposure to anti-beta7 antibody, e.g., the loading dose is by intravenous administration and each subsequent exposure is by subcutaneous administration.
  • an anti-beta7 antibody is administered using, for example, a self-inject device, autoinjector device, or other device designed for self- administration.
  • a self-inject device for example, a self-inject device, autoinjector device, or other device designed for self- administration.
  • autoinjector device for example, a self-inject device, autoinjector device, or other device designed for self- administration.
  • Various self-inject devices, including autoinjector devices are known in the art and are commercially available.
  • Exemplary devices include, but are not limited to, prefilled syringes (such as BD HYPAK SCF®, READYFILLTM, and STERIFILL SCFTM from Becton Dickinson; CLEARSHOTTM copolymer prefilled syringes from Baxter; and Daikyo Seiko CRYSTAL ZENITH® prefilled syringes available from West Pharmaceutical Services); disposable pen injection devices such as BD Pen from Becton Dickinson; ultra-sharp and microneedle devices (such as INJECT-EASETM and microinfuser devices from Becton Dickinson; and H-PATCHTM available from Valeritas) as well as needle-free injection devices (such as BIOJECTOR® and IJECT® available from Bioject; and SOF-SERTER® and patch devices available from Medtronic).
  • prefilled syringes such as BD HYPAK SCF®, READYFILLTM, and STERIFILL SCFTM
  • rhuMAb Beta7 is an article of manufacture comprising a prefilled syringe comprising 2 ML (150 mg) rhuMAb Beta7. In certain embodiments, rhuMAb Beta7 is an article of manufacture comprising a prefilled syringe comprising 1 ML (180 mg) rhuMAb Beta7.
  • the integrin beta7 antagonist can be administered alone or in combination with at least a second therapeutic compound.
  • second therapeutic compounds are generally used in the same dosages and with administration routes as used heretofore, or about from 1 to 99% of the heretofore-employed dosages. If such second compounds are used, they are used in certain embodiments in lower amounts than if the integrin beta7 antagonist were not present, so as to eliminate or reduce side effects caused thereby.
  • a variety of suitable second therapeutic compounds for the treatment of IBD e.g., ulcerative colitis and Crohn's disease are known in the art, and dosages and administration methods for such second therapeutic compounds have likewise been described.
  • Administration of the integrin beta7 antagonist and any second therapeutic compound can be done simultaneously, e.g., as a single composition or as two or more distinct compositions using the same or different administration routes. Alternatively, or additionally, the administration can be done sequentially, in any order. In certain embodiments, intervals ranging from minutes to days, to weeks to months, can be present between the administrations of the two or more compositions.
  • the integrin beta7 antagonist may be administered first, followed by the second therapeutic compound.
  • simultaneous administration or administration of the second therapeutic compound prior to the integrin beta7 antagonist is also contemplated.
  • the standard of care for subjects with active moderate-severe active UC involves therapy with standard doses of: an aminosalicylate, an oral corticosteroid, 6- mercaptopurine (6-MP) and/or azathioprine.
  • an integrin beta7 antagonist such as an anti-beta7 integrin antibody as disclosed herein will result in an improvement in disease remission (rapid control of disease and/or prolonged remission), and/or clinical response, superior to that achieved with the standard of care for such subjects.
  • the treatment of the present invention for inflammatory bowel disease (IBD) in a human subject with IBD comprises administering to the subject an effective amount of an therapeutic agent, such as an anti-beta7 integrin antibody, and further comprising administering to the subject an effective amount of a second medicament, that is an immunosuppressant, a pain-control agent, an antidiarrheal agent, an antibiotic, or a
  • an therapeutic agent such as an anti-beta7 integrin antibody
  • a second medicament that is an immunosuppressant, a pain-control agent, an antidiarrheal agent, an antibiotic, or a
  • said secondary medicine is selected from the group consisting of an aminosalicylate, an oral corticosteroid, 6-mercaptopurine (6-MP) and azathioprine.
  • said secondary medicine is another integrin beta7 antagonist, such as another anti-beta7 integrin antibody or an antibody against a cytokine.
  • second medicaments may be used in combination with each other or by themselves with the first medicament, so that the expression "second medicament” as used herein does not mean it is the only medicament besides the first medicament, respectively.
  • the second medicament need not be one medicament, but may constitute or comprise 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 generally there is a time period while both (or all) active agents simultaneously exert their biological activities.
  • the combined administration of a second medicament includes coadministration (concurrent administration), using separate formulations or a single
  • PK Pharmacokinetics
  • PD Pharmacodynamics
  • PK/PD modeling establishes a mathematical and theoretical link between these two processes and helps better predict drug action.
  • Integrated PK/PD modeling and computer-assisted trial design via simulation are being incorporated into many drug development programs and are having a growing impact (Lakshmi Kamath, Drug Discovery and Development; Modeling Success in PK/PD Testing Drug Discovery &
  • PK/PD testing is typically performed at every stage of the drug development process. Because development is becoming increasingly complex, time consuming, and cost intensive, companies are looking to make better use of PK/PD data to eliminate flawed candidates at the beginning and identify those with the best chance of clinical success.
  • PK/PD modeling approaches are proving useful in determining relationships between biomarker response, drug levels, and dosing regimens.
  • the PK/PD profile of a drug candidate and the ability to predict a patient's response to it are critical to the success of clinical trials.
  • Recent advances in molecular biology techniques and a better understanding of targets for various diseases have validated biomarkers as a good clinical indicator of a drug's therapeutic efficacy.
  • Biomarker assays (including those described herein) and use of such biomarker assays help identify a biological response to a drug candidate. Once a biomarker is clinically validated, trial simulations can be effectively modeled. Biomarkers have the potential to achieve surrogate status that may someday substitute for clinical outcomes in drug development. (Lakshmi Kamath, supra).
  • biomarkers in blood such as those described herein can be used in identifying the biological response to a treatment with integrin beta7 antagonists and can therefore function as a good clinical indicator for the therapeutic efficacy of a candidate treatment.
  • PK/PD modeling in drug development defines parameters such as drug dose concentration, drug exposure effects, drug half-life, drug concentrations against time, and drug effects against time.
  • quantitative techniques such as drug modeling, disease modeling, trial modeling, and market modeling can support the entire development process, which results in better decisions through explicit consideration of risk and better utilization of knowledge.
  • PKS Knowledgebase Server
  • Primers, oligonucleotides and polynucleotides employed in the present invention can be generated using standard techniques known in the art.
  • Biomarkers associated with predicting responsiveness of IBD patients including patient suffering from UC or Crohn's disease to certain therapeutic agents are provided herein. Accordingly, the invention disclosed herein is useful in a variety of settings, e.g., in methods and compositions related to diagnosis and therapy of inflammatory bowel diseases.
  • the biomarkers provided herein including T cells, Treg cells, and Thl7 cells
  • T cells may be detected using methods in the art.
  • the T cells, Treg cells, and/or Thl7 cells are detecting using epigenetic analysis, which detects the methylation state of genomic DNA.
  • T cells are identified as cells in blood that have a demethylated region in the CD3D/G gene.
  • Treg cells are identified as cells in blood that have a demethylated region in the FOXP3 gene.
  • the demethylated region in the FOXP3 gene is the Treg specific demethylated region (TSDR).
  • the Thl7 cells are identified as cells in blood that have a demethylated region in the IL17 A gene.
  • Nonlimiting exemplary methods of identifying and quantifying T cells, Treg cells, and/or Thl7 cells using epigenetic analysis are described, e.g., in Wieczorek et al., 2009, Cancer Res. 69: 599-608; Sehouli et al, 2011, Epigenetics 6: 236-246; Turbachova et al , 2013, Epigenetics, 8: 11, 1226-1235; PCT Publication No. WO 2013/057202; and European Publication No. EP 1 826 279.
  • genomic DNA may be isolated from a sample, for example, whole blood or peripheral blood mononucleocytes (PBMCs).
  • PBMCs peripheral blood mononucleocytes
  • the genomic DNA is subject to bisulfite conversion, which converts unmethylated C to U (which is typically replaced by T during amplification), but leaves methylated C as C.
  • Kits for bisulfite conversion of genomic DNA are commercially available from various suppliers, and bisulfite conversion is described, e.g., in Olek et al, 1996, Nulc. Acids Res. , 54: 5064-5066.
  • sequence of the unmethylated genomic DNA is altered relative to the sequence of the methylated DNA.
  • Sequence-specific quantitative PCR may be used to quantify the number of copies of unmethylated genomic DNA. Since most genes are present at two copies per cell (for example, if located on an autosome), the number of cells having the demethylated gene will be 1 ⁇ 2 the number of copies of the gene detected. In some embodiments, if the gene is located on the X chromosome, it will be present at two copies per cell in women and one copy per cell in men. Accordingly, for an X-linked gene, the correlation between the number of cells having the demethylated gene and the number of copies of the demethylated gene detected will be 1 : 1 for men and 1 :2 for women.
  • the number of copies is determined by comparison to a standard curve.
  • Quantitative PCR is described, e.g., in “PCR Protocols, A Guide to Methods and Applications,” (M.A. Innis et al, eds., Academic Press, Inc., 1990); “Current Protocols in Molecular Biology” (F. M. Ausubel et al, eds., 1987, and periodic updates); and “PCR: The Polymerase Chain Reaction", (Mullis et al, eds., 1994).
  • quantitative PCR is also used to quantify the total number of cells in the sample, for example, by quantifying the number of copies of a control gene, independent of methylation state.
  • the total number of ceils is determined by quantifying the number of copies of a housekeeping gene.
  • housekeeping genes that may be used to quantity the total number of cells in a sample include, but are not limited to, GAPDH, ACTB, and UBC.
  • primers and probes for detecting bisulfite-converted demethylated genes are discussed below. It is understood that the sequences of the primers and/or probes may differ from the genomic such that they are specific for the bisulfite-converted sequence.
  • Nonlimiting exemplary primers and probe for detecting bisulfite-converted demethylated FOXP3 gene include a forward primer corresponding to a region within chromosomal region X:49004163-49004191 : 1 and a reverse primer corresponding to a region within chromosomal region NCBI36:X:49004227 -49004251 : 1.
  • a nonlimiting exemplar ⁇ 7 probe corresponds to a region within chromosomal region X: 49004200-49004222: 1. See, e.g., Wieczorek et al. , 2009, Cancer Res. 69: 599-608.
  • Nonlimiting exemplar ⁇ ' primers and probe for detecting bisulfite-converted demethylated FOXP3 gene include a forward primer corresponding to a region within chromosomal region X: 491 17219-49117246: 1 and a reverse primer corresponding to a region within chromosomal region X:491 17283-49117307: 1.
  • a nonlimiting exemplar ⁇ ' probe corresponds to a region within chromosomal region X:49117256-
  • Nonlimiting exemplar ⁇ " primers and probe for detecting bisulfite-converted demethylated ILI 7A gene include:
  • IL17A forward primer A 5 ' - ATTCTTCTATAACCTC ATTAAAAGC A-3 ' (SEQ ID NO: 33);
  • IL17A forward primer IL17A-B 5 ' -TTCTTCTATAACCTCATTAAAAGCAA-3 ' (SEQ ID NO: 34);
  • IL17A reverse primer A 5 ' -GAGATGGATAAAATGTAGTGTTATT-3 ' (SEQ ID NO: 35);
  • IL17A reverse primer B 5 ' -GATGGATAAAATGTAGTGTTATTG-3 ' (SEQ ID NO: 36);
  • 1L17A probe 5'-CCACTACAACACACCACATAAAT-3' (SEQ ID NO: 37).
  • detecting bisulfite-converted demethylated IL17A gene comprises IL17A forward primer A, IL17A reverse primer B, and IL17A probe. See, e.g., PCT
  • Nonlimiting exemplar ⁇ ' primers and probe for detecting bisulfite-converted demethylated CD3D/G gene include a forward primer corresponding to a region within chromosomal region 11 : 1 18213632- 1 J 8213653: 1 and a reverse primer corresponding to a region within chromosomal region 11 : 118213686-118213709: 1.
  • a nonlimiting exemplar ⁇ ' probe corresponds to a region within chromosomal region 1 1 : 118213664-1 18213690: 1. See, e.g., Sehouli et al, 201 1 , Epigenetics, 6: 236-246.
  • Nonlimiting exemplary primers and probe for detecting GAPDH include a forward primer corresponding to a region within chromosomal region 12:6644378-6644399: 1 and a reverse primer corresponding to a region within chromosomal region 12:6644456-6644476: 1.
  • a nonlimiting exemplary probe corresponds to a region within chromosomal region
  • a biological sample may be obtained using certain methods known to those skilled in the art.
  • Biological samples may be obtained from vertebrate animals, and in particular, mammals.
  • a biological sample is whole blood or peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • a blood sample may contain a preservative and/or anti-coagulant, including, but not limited to, EDTA, citrate, and/or heparin.
  • the blood sample may also be a fresh sample or a previously frozen sample.
  • about 1 ml of whole blood is used for analysis.
  • about 500 ⁇ , or about 400 ⁇ , or about 300 ⁇ , or about 200 ⁇ , or about 100 ⁇ , or about 50 ⁇ of whole blood is used for analysis.
  • an effective amount of an appropriate therapeutic agent may be administered to the subject to treat the particular disease in the subject, e.g., UC or Crohn's disease.
  • Clinical diagnosis in mammals of the various pathological conditions described herein can be made by the skilled practitioner.
  • Clinical diagnostic techniques are available in the art which allow, e.g., for the diagnosis or detection of inflammatory bowel diseases in a mammal, e.g., ulcerative colitis and Crohn's disease.
  • kits or articles of manufacture are also provided.
  • Such kits may comprise a carrier means being
  • one of the container means may comprise a probe that is or can be detectably labeled.
  • probe may be a polynucleotide specific for a demethylated region of one or more genes that is indicative of a cell type.
  • probe may be a
  • kits may also have containers containing nucleotide(s) for amplification of 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 enzymatic, florescent, or radioisotope label.
  • a reporter means such as a biotin- binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label.
  • Kits will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • a label may be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic application, and may also indicate directions for either in vivo or in vitro use, such as those described above.
  • Other optional components in the kit include one or more buffers (e.g.
  • substrate e.g., chromogen
  • control samples positive and/or negative controls
  • control slide(s) etc.
  • the invention herein also encompasses a method for marketing a therapeutic agent or a pharmaceutically acceptable composition thereof comprising promoting to, instructing, and/or specifying to a target audience, the use of the agent or pharmaceutical composition thereof for treating a patient or patient population with a particular disease, e.g., UC or Crohn's disease, from which a sample has been obtained showing levels of biomarkers disclosed herein.
  • a particular disease e.g., UC or Crohn's disease
  • Marketing is generally paid communication through a non-personal medium in which the sponsor is identified and the message is controlled.
  • Marketing for purposes herein includes publicity, public relations, product placement, sponsorship, underwriting, and sales promotion. This term also includes sponsored informational public notices appearing in any of the print communications media designed to appeal to a mass audience to persuade, inform, promote, motivate, or otherwise modify behavior toward a favorable pattern of purchasing, supporting, or approving the invention herein.
  • the marketing of the diagnostic method herein may be accomplished by any means.
  • marketing media used to deliver these messages include television, radio, movies, magazines, newspapers, the intemet, and billboards, including commercials, which are messages appearing in the broadcast media.
  • the type of marketing used will depend on many factors, for example, on the nature of the target audience to be reached, e.g., hospitals, insurance companies, clinics, doctors, nurses, and patients, as well as cost considerations and the relevant jurisdictional laws and regulations governing marketing of medicaments and diagnostics.
  • the marketing may be individualized or customized based on user characterizations defined by service interaction and/or other data such as user demographics and geographical location.
  • RhuMAb Beta7 (etrolizumab) is a humanized monoclonal antibody based on the human IgGl subgroup III VH, K subgroup-I VL consensus sequences and is directed specifically against the ⁇ 7 subunit of the integrin heterodimer. See Figs. 1 A and B. It has been shown to bind with high affinity to ⁇ 4 ⁇ 7 (Kd of about 116 pM) and ⁇ 7 (Kd of about 1800 pM).
  • This recombinant antibody has two heavy chains (446 residues) and two light chains (214 residues) that are covalently linked by interchain and intrachain disulfide bonds typical of IgGl antibodies.
  • it was produced in Chinese hamster ovary (CHO) cells.
  • the molecular mass of the intact, non glycosylated rhuMAb Beta7 molecule was approximately 144 kDa.
  • Each heavy chain of rhuMAb Beta7 has one conserved N linked glycosylation site at Asn297.
  • the oligosaccharides present at this site were typical of those observed in recombinant antibodies expressed in CHO cells, with the predominant glycoforms being the asialo, biantennary GO, and Gl glycans.
  • the mass of the most prevalent rhuMAb Beta7 form containing two GO glycans and no C terminal lysine residues was approximately 147 kDa.
  • RhuMAb Beta7 drug product and placebo were prepared by Genentech. They were clear to slightly opalescent, colorless to slightly yellow aqueous solutions. Both solutions were sterile and preservative-free liquid intended for IV and SC administration. Study Design
  • This Phase II study was a randomized, double-blind, placebo-controlled multicenter study to evaluate the efficacy and safety across two rhuMAb Beta7 dose levels compared with placebo in patients with moderate to severe UC.
  • the primary efficacy endpoint was evaluated at Week 10 (2 weeks after the final dose of study drug was administered) with a secondary efficacy endpoint at Week 6.
  • the dose values provided in the preceding paragraph are nominal doses.
  • the phase II dose administration used a vial and a syringe with a vial concentration of 150 mg/ml.
  • 0.7 ml was the selected volume per subcutaneous (SC) injection.
  • the actual drug amount therefore, for the nominal 100 mg dose arm was 105 mg (1 x 0.7 ml SC injection) and for the nominal 300 mg dose was 315 mg (3 x 0.7 ml SC injections).
  • the actual loading dose of 420 mg was 420 mg (4 x 0.7 ml SC injections). All SC injections were administered into the abdomen. Accordingly, a dose of "100 mg" and a dose of "105 mg” are used interchangeably herein.
  • the daily bleeding score represents the most severe bleeding of the day.
  • the physician's global assessment acknowledges the three other criteria, the patient's daily recollection of abdominal discomfort and general sense of well-being, and other observations, such as physical findings and the patient's performance status.
  • azathioprine [AZA], 6-mercaptopurine [6-MP], or methotrexate) doses must have been kept stable for at least 4 weeks prior to randomization on Day 1. Patients who were receiving topical 5-ASA or corticosteroids must have discontinued 2 weeks before randomization on Day 1. Oral corticosteroid doses must have been kept stable for 2 weeks prior to randomization on Day 1. Patients receiving high-dose steroids must have had the dose reduced to ⁇ 20 mg/day for 2 weeks prior to randomization on Day 1.
  • immunosuppressants must have been commenced at Week 8, and patients must have completely discontinued immunosuppressants by Week 10. Patients who have previously received anti-TNF therapy must have discontinued therapy for a minimum of 8 weeks prior to randomization to receive study drug on Day 1. If patients experienced persisting or increasing disease activity at any time during the study, rescue therapy in the form of an increase in steroids and or immunosuppressant dose may be increased or initiated according to the investigator's clinical judgment. Patients who required rescue therapy were permitted to remain in the study but discontinued study treatment and, during data analysis, were classified as having experienced treatment failure.
  • TNF-IR loss of response and/or intolerance and/or inadequate response to anti-TNF agents
  • immunosuppressants means the following. With respect to anti-TNF agents, loss of response and/or intolerance and/or inadequate response means that symptoms of active disease persist despite previous treatment with one or more of (a) infliximab: 5 mg/kg IV, 3 doses over 6 weeks with assessment at 8 weeks; (b) adalimumab: one 160-mg SC does at week 0, followed by one 80-mg dose at week 2 and then 40 mg at 4 and 6 weeks, with assessment at 8 weeks; or recurrent active symptoms during regularly scheduled maintenance dosing following a previous response (elective discontinuation of treatment by patient who has responded and did not lose response does not qualify); or history of intolerance to at least one anti-TNF antibody (including but not exclusive of or limited to infusion-related reaction or injection-site reaction, infection, congestive heart failure, demyelination).
  • infliximab 5 mg/kg IV, 3 doses over 6 weeks with assessment at 8 weeks
  • adalimumab one 160-m
  • loss of response and/or intolerance means that symptoms of active disease persist despite previous treatment with one or more of azathioprine (> 1.5 mg/kg) or equivalent dose of 6- mercaptopurine mg/kg (> 0.75 mg/kg) or methotrexate, 25 mg SC/intramuscular (or as indicated) per week for at least 8 weeks; or history of intolerance of at least one
  • immunosuppressive including, but not exclusive of pancreatitis, drug fever, rash,
  • Randomization to study treatment were stratified by concomitant treatment with corticosteroids (yes/no), concomitant treatment with immunosuppressants (yes/no), previous anti-TNF exposure (yes/no) (except for patients randomized in the United States), and study site. Patients who had previous anti-TNF exposure were considered "TNF experienced,” regardless of whether or not they were TNF-IR.
  • UC disease activity was assessed using the MCS at Screening (and this was considered the baseline MCS), Week 6 (2 weeks after dosing at Week 4), and Week 10 (2 weeks after the final dose of study drug). Biopsies of the colon were obtained during the flexible sigmoidoscopy conducted at these same time points. Partial MCS was also collected throughout the study. Patient Reported Outcomes (PROs) were also assessed by using a Short Inflammatory Bowel Disease Questionnaire (SIBDQ) and MCS, which were to be completed by patients at Day 1 and at Weeks 6 and 10.
  • SIBDQ Short Inflammatory Bowel Disease Questionnaire
  • disease activity, daily symptoms, and impact of UC were collected in a patient diary, to be completed daily by patients from screening (approximately 7 days prior to and up to Day 1) and at least 7 days prior to and up to the study visits at Weeks 6 and 10.
  • Serum and fecal samples were also obtained for biomarker analysis. Stool was obtained at screening and Weeks 6, 10, and 28 for measurement of biomarkers. Exemplary biomarkers that were considered for measurement include, but are not limited to, lipocalin, calprotectin, and lactorferrin.
  • Serum and plasma were obtained at screening, at Day 1, and at Weeks 4, 6, 10, 16, and 28 for measurement of exploratory biomarkers.
  • the primary efficacy endpoint for this study was the proportion of patients who achieved clinical remission, defined as an absolute reduction in MCS to ⁇ 2 with no individual subscore exceeding 1 point, by Week 10. Additional secondary endpoints are listed in the study outcome measures as described below.
  • the primary efficacy outcome measure was clinical remission at Week 10.
  • Clinical remission is defined by an MCS ⁇ 2 with no individual subscore exceeding 1 point (see Table 1).
  • the secondary efficacy outcome measures for this study were (1) Clinical response at Week 6 and Week 10 where clinical response was defined by at least a 3-point decrease and 30% reduction from baseline in MCS and a > 1-point decrease in rectal bleeding subscore or absolute rectal bleeding score of 0 or 1 ; (2) Clinical remission (defined above) at Week 6; and (3) An indicator for endoscopic score and rectal bleeding score of 0 at Week 6 and Week 10.
  • the exploratory outcome measure for this study were the time to flare of UC in patients who achieved response or remission.
  • a flare is defined as a 2 point increase in partial MCS accompanied by 3 days of continuous rectal bleeding and an endoscopy score of 2 on flexible sigmoidoscopy.
  • rhuMAb Beta7 The safety and tolerability of rhuMAb Beta7 were assessed using the following measures: (1) Incidence of adverse events and serious adverse events graded according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) Version 4.0; (2) Clinically significant changes in vital signs and safety laboratory measures; (3) Discontinuation due to adverse event(s); (4) Incidence and nature of injection-site reactions and hypersensitivity; (5) Incidence of infectious complications; and (6) Immunogenicity as measured by the incidence of AT As.
  • NCI CTCAE National Cancer Institute Common Terminology Criteria for Adverse Events
  • the pharmacokinetic outcome measures included the following: (1) Cmax after the first and final doses; (2) Time to maximum concentration (Tmax) after the first and final doses; (3) Area under the serum concentration-time curve (AUC) within a dose interval after the final dose; (4) AUC from time 0 to time of the last detectable observation (AUCiast) or to infinity (AUCinf); (5) Apparent clearance (CL/F); (6) Apparent volume of distribution (V/F); and (7) Elimination half-life (ti/ 2 ).
  • Biomarker data was analyzed in 42 anti-TNF treatment nai e (TNF-nai ' ve) and
  • T cells have demethylated CD3D/G
  • Treg cells have demethylated FOXP3 TSDR (Treg specific demethylated region)
  • Thl 7 cells have demethylated ill 7 A. See, e.g., Sing et al., 2014, Allergy, Asthma & Clinical Immunology 10:32; Wieczorek et al, 2009, Cancer Res. 69: 599-608; and Sehouli et al, 2011, Epigenetics 6: 236-246. Quantitation of each cell type through detection of demethylation is performed substantially as follows.
  • Genomic DNA is isolated from whole blood using the DNeasy blood and tissue kit (Qiagen) according to the manfacturer's protocol. Bisulfite conversion of genomic DNA resulting in either CpG variants (if DNA is methyled) or TpG variants (if DNA is unmethylated) is performed, e.g., as described in Olek et al., 1996, Nucl. Acids Res., 1524: 5064-5066; or using EpiTect
  • BisulfiteKit (Qiagen). Briefly, sodium bisulfite treatment of genomic DNA results in the conversion of unmethylated cytosine to uracil, which is replaced with thymine in subsequent PCT amplification. Methylated cytosine remains unchanged, and is therefore still cytosine after PGR amplification.
  • Quantitative PGR (qPCR) assays specific for demethyiated FOXl ⁇ 3 TSDR (Treg ceils), demethyiated CD3D/G (T cells), demethyiated III 7 A (Thl 7 cells), and GAPDH control are performed substantially as described previously. See, e.g., Wieczorek et al., 2009, Cancer Res. 69: .599-608; Sehouli et al, 201 1 , Epigenetics 6: 236-246; Turbachova et al., 2013, Epigenetics, 8: 11, 1226-1235; PCT Publication No. WO 2013/057202; and European
  • EP 1 826 279 each of which is incorporated by reference herein in its entirety for any purpose. Because the assays are specific for the demethyiated loci (e.g., using primers specific for bisulfite-converted sequences comprising T in place of methylated C in the native genomic sequence), methylated loci (e.g., in other cell types) will not be amplified in the qPCR assay.
  • the GAPDH assay serves as a control to determine the total number of cells in the blood sample. The percentage of each ceil type in whole blood may then be determined.
  • Figure 5 shows the change from baseline (study day 0) in the percentage of T cells (Fig. 5A), Treg cells (Fig. 5B), and Thl7 cells (Fig. 5C) in blood of patients administered placebo, rhuMAb Beta7 ("ab7") at 100 mg dose and rhuMAb Beta7 at 300 mg dose.
  • ab7 rhuMAb Beta7
  • No differences at baseline for T cell (CD3+), Treg (FoxP3), and Thl7 (IL-17) content were observed for the three treatment arms (placebo, 100 mg, and 300 mg).
  • patients with UC showed generally reduced T cell and Treg values.
  • peripheral levels of all three cell types were relatively stable over time in the placebo group, whereas etrolizumab-treated patients showed a trend towards an increase for all three cell types.
  • the median increase for T cell, Treg, and Thl7 cells for the 300 mg dose group compared to placebo was significant at day 29 (P ⁇ 0.05).
  • Figure 6 shows the change from baseline (study day 0) in the percentage of T cells (Fig.
  • Treg cells Fig. 6B
  • Thl7 cells Fig. 6C
  • induction treatment with rhuMAb Beta7 trends toward increased T cells, Treg cells, and Thl7 cells were associated with clinical remission, however statistical significance was observed only at day 29 for the 300 mg dose group (P ⁇ 0.05 for CD3+ and FoxP3).
  • Figure 7 shows the change from baseline (study day 0) in the percentage of T cells (Fig. 7A), Treg cells (Fig. 7B), and Thl7 cells (Fig. 7C) in blood of patients who experienced clinical remission and patients who did not, divided into TNF naive and TNF inadequate responders, as well as in patients admininstered placebo.
  • the median increase in CD3+ T cells appeared more profound in remitters that were TNF-experienced than in those that were TNF- naive (day 71 CD3+ T cells increased 2.9-fold and 1.27-fold, respectively).
  • Figure 8A shows the percentage of Treg cells at baseline in blood of TNF -naive patients who experienced clinical remission (left bar) and patients who did not (right bar). Increased levels of Tregs at baseline was associated with an increased likelihood of remission in TNF -naive patients, compared to nonremitters. Using a median of baseline Treg percentage of 1.28 as a cutoff, 28% of patients that were Treg high (> 1.28) at baseline experienced clinical remission, compared to 13% of patients that were Treg low ( ⁇ 1.28) at baseline. See Figure 8B.
  • UC patients have lower peripheral blood T cell and Treg levels compared to healthy donors, particularly for the subgroup of TNF-experienced patients.
  • rhuMAb Beta7 Upon treatment with rhuMAb Beta7, all tested T cell types in peripheral blood increased, with more pronounced increases seen in patients who achieved clinical remission. Finally, higher levels of Treg at baseline may be associated with clinical remission upon rhuMAb Beta7 treatment.
  • the level of Treg cells shows potential for use as predictive biomarkers to identify IBD patients, such as UC and Crohn's disease patients, most likely to benefit from treatment with therapeutic agents that target the beta7 integrin subunit, including etrolizumab.
  • an elevated level of Treg cells in TNF -naive IBD patients may be used to select or predict which patients are more likely to be remitters upon treatment with therapeutic agents that target the beta7 integrin subunit, including etrolizumab.
  • a greater increase in Treg cells in IBD patients after beginning treatment with therapeutic agents that target the beta7 integrin subunit, including etrolizumab indicates that the patient is morelikely to go into remission.
  • EXAMPLE 3 Predictive Biomarker Analysis in Intestinal Biopsy Tissue
  • Intestinal biopsies were collected from study participants during flexible sigmoidoscopy/full colonoscopy at the screening visit (up to 4 weeks prior to treatment). Biopsies were taken from the most inflamed area of the colon within 10-40 cm of the anal verge. Biopsies within necrotic areas of ulcerated mucosa or at suture sites in patients with prior colonic resection were avoided. Biopsies were placed into a tissue RNA stabilizing buffer (RNAlater, Qiagen, Cat. No. 76104) and frozen for shipment. Upon receipt, genomic DNA is isolated from intestinal biopsy tissue using the DNeasy blood and tissue kit (Qiagen) according to the manufacturer's protocol.
  • RNAlater tissue RNA stabilizing buffer
  • Phase II clinial study was determined by measuring the presence of epigenetic activation at the respective loci in relation to epigenetic activation of the GAPDH locus which determines the total cell number.
  • Demethylation or epigenetic activation of a particular DNA CpG motif can be detected through a demethylation-specific qPCR analysis that quantifies the presence of epigenetically active CD3, FoxP3, or IL-17, as described herein.
  • Differences were calculated by high biomarker group minus low biomarker group based on the median values of all available patient samples at screen. High biomarker group consists with patients with values greater than or equal to the median value. Low biomarker group consists with patients with values less than the median value.
  • the horizontal line represents 90% confidence limits of the difference in proportions.
  • Figure 9B at 95% confidence intervals, the data indicates increased levels of CD3 T-cells in biopsy tissue are associated with increased likelihood and a similar trend for increased levels of Treg in biopsy tissue.
  • Figure 9C shows that increased percentage of patients in remission is observed in patients in the Treg and CD3 T-cell high group.
  • the error bar represents 90% upper confidence limits of the difference in proportions.
  • Al is R
  • A2 is selected from the group consisting of A, G, antibody S, T, and V
  • A3 is selected from the group consisting of S, G, I, K, N, P, Q,
  • HVR-Ll.vA R, and T; A4 is selected from the group consisting of E, V, Q, A, D, G, H, I,
  • A5 is selected from the group consisting of S, Y, A, D, G, H, I, K, N, P, R, T, and V;
  • A6 is selected from the group consisting of V, R, I, A, G, K, L, M, and Q;
  • A7 is selected from the group consisting of D, V, S, A, E, G, H, I, K, L, N, P, S, and T;
  • A8 is selected from the group consisting of D, G, N, E, T, P and S;
  • A9 is selected from the group consisting of L, Y, I and M;
  • A10 is selected from the group consisting of L, A, I, M, and V; and
  • Al 1 is selected from the group consisting of H, Y, F, and S
  • Bl is selected from the group consisting of K, R, N, V, A, F, antibody Q, H, P, I, L, Y and Xaa (where Xaa represents any amino acid); B2 is Y; B3
  • HVR.-L2.vB is A; B4 is selected from the group consisting of S and D; B5 is selected from the group consisting of Q and S; B6 is selected from the group consisting of S, D, L, and R; B7 is selected from the group consisting of I, V, E, and K; and B8 is S
  • C7 are QQGNSLP
  • C8 is selected from the group antibody consisting of N, V, W, Y, R, S, T, A, F, H, I L, and M
  • C9 is T
  • E12 is selected from the group consisting of N, T, A, and D
  • E13 is selected from the group consisting of P, H, D, and A
  • E14 is S
  • E15 is selected from the group consisting of L and V
  • E16 is K
  • amino acid E17 is selected from the group consisting of S and G
  • Fl anti-beta7 F2-F11 or Fl-Fl l
  • F2 is R, M, A, E, G, Q, S
  • F3 to F10 is antibody TGSSGYFD
  • Fl 1 is selected from the group consisting of F and Y

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Abstract

L'invention porte sur des biomarqueurs prédictifs de la réactivité à des antagonistes de l'intégrine bêta 7, y compris à des anticorps de sous-unités de l'intégrine anti-bêta 7, et sur des procédés d'utilisation de tels biomarqueurs. De plus, l'invention concerne des méthodes de traitement de troubles inflammatoires gastro-intestinaux, tels que les maladies inflammatoires de l'intestin, y compris la colite ulcéreuse et la maladie de Crohn. L'invention concerne également des méthodes d'utilisation de tels biomarqueurs prédictifs pour le traitement de maladies inflammatoires de l'intestin, y compris de la colite ulcéreuse et de la maladie de Crohn.
EP16750314.3A 2015-07-27 2016-07-26 Méthodes de diagnostic et de traitement de la maladie intestinale inflammatoire Withdrawn EP3329276A2 (fr)

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RU2678450C2 (ru) * 2011-09-15 2019-01-29 Ногра Фарма Лимитед Способы контроля восприимчивости анти-smad7 терапии
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