WO2023086910A1 - Methods of treating crohn's disease using integrin beta7 antagonists - Google Patents

Methods of treating crohn's disease using integrin beta7 antagonists Download PDF

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WO2023086910A1
WO2023086910A1 PCT/US2022/079676 US2022079676W WO2023086910A1 WO 2023086910 A1 WO2023086910 A1 WO 2023086910A1 US 2022079676 W US2022079676 W US 2022079676W WO 2023086910 A1 WO2023086910 A1 WO 2023086910A1
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patients
patient
score
antibody
therapy
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PCT/US2022/079676
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French (fr)
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Azra HASSANALI
Rhian JACOB-MOFFATT
Zaineb Huzefa SHARAFALI
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Genentech, Inc.
F. Hoffmann-La Roche Ag
Hoffmann-La Roche Inc.
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Publication of WO2023086910A1 publication Critical patent/WO2023086910A1/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily

Definitions

  • Methods of treating Crohn’s disease are provided. Also provided are methods of administering and dosing integrin beta7 antagonists, such as anti-integrin beta7 antibodies. In addition, methods of administrating and dosing such integrin beta7 antagonists to maintain improvement of Crohn’s disease, including clinical remission and/or endoscopic improvement, are provided.
  • IBD Inflammatory bowel disease
  • GI gastrointestinal
  • CD ulcerative colitis
  • CD Crohn’s disease
  • UC ulcerative colitis
  • CD is a chronic, relapsing form of IBD that can affect any portion of the gastrointestinal tract, with 40% ⁇ 50% of cases affecting the small bowel.
  • CD is characterized by patchy, transmural inflammation, ulcers, and granulomatous lesions that are interspersed with healthy sections of bowel (skip lesions).
  • the disease is progressive; uncontrolled inflammation develops into structuring or penetrating complications such as prestenotic dilatation, obstruction (stricturing), and intra-abdominal or perianal fistulae and abscesses (penetrating).
  • Clinical signs and symptoms include chronic diarrhea, abdominal pain, cachexia, abdominal mass, or tenderness as well as the overt signs of fistulae.
  • CSs Systemic corticosteroids
  • Immunosuppressants e.g., azathioprine [AZA], 6-mercaptopurine [6-MP], or methotrexate [MTX]
  • Is immunosuppressants
  • AZA azathioprine
  • 6-MP 6-mercaptopurine
  • MTX methotrexate
  • Immunosuppressants are given with or without a steroid bridge, depending on a patient’s symptoms during the 2-4-month onset of IS efficacy.
  • budesonide presents a less toxic, more tolerable bridge because of its low systemic bioavail ability resulting from a rapid first-pass metabolism.
  • mAbs tumor necrosis factor
  • TNF tumor necrosis factor
  • Anti-TNFs are also associated with significant side effects, including serious infection, opportunistic infection, lupus-like reactions, and an increased risk of lymphoma (Siegal CA, et al., Therap Adv Gastroenterol 2:245-51, 2009).
  • Tolerability concerns include infusion reactions (occurring in 9%-17% of patients treated with infliximab, see de Vries HS, et al., Br J Clin Pharmacol 71 :7-19, 2011) and injection site reactions (occurring in 10% of patients receiving adalimumab, see van der Heijde D, et al., Arthritis Rheum. 54:2136-46, 2006).
  • infusion reactions occurring in 9%-17% of patients treated with infliximab, see de Vries HS, et al., Br J Clin Pharmacol 71 :7-19, 2011
  • injection site reactions occurring in 10% of patients receiving adalimumab, see van der Heijde D, et al., Arthritis Rheum. 54:2136-46, 2006.
  • 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 0 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 recrui tment 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.
  • beta7 containing integrins i.e., alpha4beta7 and alphaEbeta7
  • the beta7 containing integrins 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 anti-integrins are another class of biologies approved for the treatment of CD.
  • Natalizumab is an anti-integrin approved in the U.S. only for the treatment of moderate to severely active CD
  • the use of natalizumab, which blocks both ⁇ 4 ⁇ 1 and ⁇ 4 ⁇ 7 has been limited due to concerns that inhibition of a4pl/VCAM-l binding increases the risk of progressive multifocal leukoencephalopathy (PML) a rare but serious infection of the CNS.
  • PML progressive multifocal leukoencephalopathy
  • Vedolizumab is the most recently approved gut-selective anti-integrin for CD, but this targets only the «4p7 integrin receptor, inhibiting T-lymphocyte binding to the adhesion molecule MAdCAM-1, and is administered as an intravenous (IV) infusion.
  • the ⁇ 4 ⁇ 7 integrin (the target of vedolizumab) 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.
  • leukocyte rolling and firm adhesion to the mucosal endothelium is initiated by signals from chemokines and is mediated via mucosal addressin cell adhesion molecule (MAd CAM)- 1 -associated sialyl Lewis X.
  • MAd CAM 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., suprd).
  • 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)).
  • oc407 binds preferentially to high endothelial venules expressing MAdCAM-1 and vascular ceil adhesion molecule (VCAM)-l, as well as to the extracellular matrix molecule fibronectin fragment CS-1 (Chan et al., J Biol Chem 267:8366-70 (1992); Ruegg et al., J Cell Biol 17: 179-89 (1992); Berlin et al., Cell 74:185-95 (1993)).
  • VCAM vascular ceil adhesion molecule
  • T lymphocytes and associated with mucosal tissues is the ⁇ E ⁇ 7 integrin, otherwise known as CD 103.
  • the ⁇ E ⁇ 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)).
  • ⁇ E ⁇ 7 The expression of ⁇ E ⁇ 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- ⁇ E ⁇ 7 antibody treatment has been reported to attenuate experimental colitis in mice, implicating a role for ⁇ E ⁇ 7 + lymphocytes in experimental models of IBD (Ludviksson et al., J Immunol 162:4975-82 (1999)).
  • Etrolizumab a subcutaneously administered mAb, is a novel anti-integrin which unlike vedolizumab, targets both the ⁇ 4 ⁇ 7 and ⁇ E ⁇ 7 receptors that regulate trafficking, and retention of T-cell subsets in the intestinal mucosa, respectively.
  • etrolizumab offers the potential of an additive therapeutic effect in CD via a dual mechanism of action (MOA), without generalized immunosuppression.
  • MOA mechanism of action
  • Etrolizumab binds with high affinity to ⁇ 4 ⁇ 7 (Holzmann B, et al., Cell 56:37-46, 1989; Hu M, et al..
  • etrolizumab does not bind to ⁇ 4 ⁇ 1 or inhibit the interaction of cx4pl and VCAM-1 and the distribution and homing of lymphocytes to the CNS and peripheral lymphoid tissue. As such, etrolizumab is not expected to increase the risk of progressive multifocal leukoencephalopathy (PML).
  • Safety assessments for etrolizumab have been completed in adult Phase 1 and Phase 2 studies, in which patients with moderate to severely active UC received either single or multiple doses of IV or subcutaneous (SC) etrolizumab.
  • CDAI Crohn’s Disease Activity Index
  • CDAI ⁇ 150 is defined as clinical remission
  • 150 to 219 is defined as mildly active disease
  • 220 to 450 is defined as moderately active disease
  • above 450 is defined as very severe disease (Best WR, et al., Gastroenterology 77:843-6, 1979).
  • Vedolizumab and natalizumab have been approved on the basis of demonstrated clinical remission, i.e. CDAI ⁇ 150.
  • CDAI has been in use for over 40 years, and has served as the basis for drug approval, it has several limitations as an outcome measure for clinical trials. For example, most of the overall score comes from the patient diary card items (pain, number of liquid bowel movements, and general well-being), which are vaguely defined and not standardized terms (Sandler et al , J. Clin. Epidemiol 41 :451 -8, 1988; Thia et al., Inflamm Bowel Dis 17: 105-11 , 2011). In addition, measurement of pain is based on a four-point scale rather than an updated seven-point scale. The remaining 5 index items contribute very little to identifying an efficacy signal and may be a source of measurement noise.
  • SES- CD Simplied Endoscopic Activity Score for Crohn’s Disease
  • the methods of the invention are based, at least in part, on the surprising and unexpected results of the maintenance phase of a clinical study of the safety and efficacy of etrolizumab treatment in patients with Crohn’s disease. These results showed that in the maintenance phase, the two co-primary endpoints of clinical remission and endoscopic improvement in etrolizumab-treated patients compared to placebo were met. The results also showed that two secondary endpoints of corticosteroid-free remission and endoscopic remission in etrolizumab-treated patients compared to placebo were met.
  • the method comprises administering subcutaneously to the patient an integrin beta7 antagonist for a treatment period of at least 52 weeks or of at least 66 weeks after initiation of an induction therapy, wherein 14 weeks after the initiation of the induction therapy the patient is determined to have acheived a decrease of 70 points or more from a baseline Crohn’s Disease Activity Index (CDAI) score, and wherein the patient maintains clinical remission during the treatment period.
  • CDAI Crohn’s Disease Activity Index
  • the patient maintains endoscopic improvement in addition to maintaining clinical remission.
  • the patient further maintains endoscopic remission, corticosteroid-free clinical remission, or both endoscopic remission and corticosteroid-free clinical remission.
  • the patient receives corticosteroid therapy in addition to the induction therapy during an induction period, wherein the induction period is 14 weeks, and wherein at the end of the induction period the corticosteroid therapy is reduced over time until discontinuation.
  • the corticosteroid therapy is (i) less than or equal to 20 mg of prednisone per day and wherein the corticosteroid therapy is reduced by 2.5 mg prednisone per week until discontinuation or (ii) less than or equal to 6 mg oral budesonide per day and wherein the corticosteroid therapy is reduced by 3 mg oral budesonide every 2 weeks until discontinuation.
  • the integrin beta7 antagonist is a humanized monoclonal anti-integrin beta7 antibody.
  • the anti-integrin beta7 antibody comprises three light chain hypervariable regions (HVRs), HVR-L1, HVR-L2, and HVR-L3, and three heavy chain HVRs, HVR-H1, HVR-H2. and HVR-H3, wherein:
  • HVR-L1 comprises amino acid sequence RASESVDDLLH (SEQ ID NO: 1) ;
  • HVR-L2 comprises amino acid sequence KYASQSIS (SEQ ID NO:2);
  • HVR-L3 comprises amino acid sequence QQGNSLPNT (SEQ ID NO: 3);
  • HVR-H1 comprises amino acid sequence GFFITNNYWG (SEQ ID NO:4);
  • HVR-H2 comprises amino acid sequence GYISYSGSTSYNPSLKS (SEQ ID NO: 5;
  • HVR-H3 comprises amino acid sequence RTGSSGYFDF (SEQ ID NO:6); or amino acid sequence ARTGSSGYFDF (SEQ ID NO:7).
  • the anti-integrin beta7 antibody further comprises a light chain variable region domain comprising the amino acid sequence set forth in SEQ ID NO:8 and a heavy chain variable region domain comprising the amino acid sequence set forth in SEQ ID NO:9.
  • the anti-integrin beta7 antibody comprises a light chain variable region domain comprising the amino acid sequence set forth in SEQ ID NO:8 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:11 or a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 12.
  • the anti-integrin beta7 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10 and a heavy chain variable region domain comprising the amino acid sequence set forth in SEQ ID NO:9.
  • the anti-integrin beta7 antibody further comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11 or a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 12.
  • the anti- integrin beta7 antibody is etrolizumab.
  • the induction therapy comprises one or more therapeutic agents selected from a 5-aminosalicylate, an antibiotic, budesonide, a systemic corticosteroid, a thiopurine, methotrexate, an anti-TNF agent, infliximab, adalimumab, certolizumab pegol, vedolizumab, ustekinumab, natalizumab, efalizumab, etrolizumab, a Janus kinase (JAK) inhibitor, upadacitinib, and filgotinib.
  • a 5-aminosalicylate an antibiotic, budesonide, a systemic corticosteroid, a thiopurine, methotrexate, an anti-TNF agent, infliximab, adalimumab, certolizumab pegol, vedolizumab, ustekinumab, natalizumab, efalizum
  • the patient is determined to have (I) a CDAI score of greater than or equal to 220 and less than or equal to 480 at any time in the seven days prior to initiation of the induction therapy and (2) either an average daily liquid/soft stool frequency (SF) score greater than or equal to 6 or an average daily SF greater than 3 and an average daily abdominal pain (AP) score greater than 1 for the seven consecutive days prior to initiation of the induction therapy.
  • the patient is determined to have active inflammation, wherein the active inflammation is determined as a SES-CD score of greater than or equal to 7 as determined by ileocolonoscopy.
  • the patient has isolated ileitis or post-ileocecal resection and wherein the patient is determined to have active inflammation, wherein the active inflammation is determined as a Simplified Endoscopic Index for Crohn’s Disease (SES-CD) score of greater than or equal to 4 as determined by ileocolonoscopy.
  • SES-CD Simplified Endoscopic Index for Crohn’s Disease
  • the patient is refractory or intolerant to one or more therapies selected from immunosuppressant therapy, corticosteroid therapy, and anti-TNF therapy.
  • the patient has an inadequate response to anti-TNF therapy.
  • the immunosuppressant therapy is selected from 6-mercaptopurine, azathioprine, and methotrexate.
  • the corticosteroid therapy is selected from prednisone and oral budesonide.
  • the anti-TNF therapy is selected from infliximab, adalimumab, and certolizumab pegol.
  • the anti-integrin beta7 antibody is administered at a flat dose of 105 mg every 4 weeks from week 14 after initiation of the induction therapy to at least week 52 or to at least week 66.
  • clinical remission is determined as a liquid/soft stool frequency (SF) mean daily score of less than or equal to three and an abdominal pain (AP) mean daily score of less than or equal to one with no worsening in either SF or AP compared to baseline, averaged over at least four days prior to assessment.
  • SF liquid/soft stool frequency
  • AP abdominal pain
  • the SF mean daily score and AP mean daily score are averaged over seven days prior to assessment.
  • endoscopic improvement is determined by Simplified Endoscopic Index for Crohn’s Disease (SES-CD) score and the SES-CD score is reduced by at least fifty percent compared to the SES-CD score determined at baseline.
  • SES-CD Simplified Endoscopic Index for Crohn’s Disease
  • the patient does not receive treatment with one or more corticosteroids for at least 24 continuous weeks after discontinuing corticosteroid treatment.
  • the patient maintains endoscopic remission and endoscopic remission is determined by SES-CD score and the SES-CD score is less than or equal to four.
  • the patient is an ileal patient and the SES-CD score is less than or equal to two.
  • the SES-CD score contains no segment having a subcategory score that is greater than one.
  • the subcategoiy is selected from size and extent of ulceration, affected surface, and narrowing.
  • methods of treating Crohn’s disease according to any of the embodiments comprise administering the integrin beta7 antagonist using a prefilled syringe or a prefilled syringe and autoinjector combination.
  • FIG. 1 shows the study schema for the Induction Phase of the Phase III clinical studj' as described in Example 1.
  • Anti-TNF anti-tumor necrosis factor
  • CD Crohn’s Disease
  • ETRO etrolizumab
  • IS immunosuppressants
  • SC subcutaneous
  • Wk week.
  • FIG. 2 shows the study schema for the Maintenance Phase of the Phase III clinical study as described in Example 1.
  • CS corticosteroids
  • ETRO etrolizumab
  • OLE open label extension phase
  • PBO placebo
  • q4w every 4 weeks
  • Re-Rx re-randomized
  • Wk week.
  • FIG. 3 shows the medical aid known as the Bristol Stool Scale as described in Example 1.
  • Ranges provided in the specification and appended claims include both end points and all points between the end points. Thus, for example, a range of 2.0 to 3.0 includes 2.0, 3.0, and all points between 2.0 and 3.0.
  • "Treatment,” “treating,” and grammatical variations thereof refer 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 administration, or duration of treatment, with or without addition of a second medication
  • Effective treatment regimen refers to a treatment period en 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.
  • “Clinical remission” as used herein means a liquid/soft stool frequency (SF) mean daily score of less than or equal to three and an abdominal pain (AP) mean daily score of less than or equal to 1, with no worsening in either subscore compared to baseline, averaged over seven days. Assessment of SF and AP are described in Example 1.
  • Corticosteroid-free clinical remission or “corticosteroid-free remission” means maintaining clinical remission or maintaining remission without the use of concomitant corticosteroid treatment
  • “Crohn’s Disease Activity Index” score or “CDAl” score means a composite of eight assessments consisting of the number of liquid or sfot stools, abdominal pain, general well-being, presence of complications, taking diphenoxylate/atropine (LOMOTIL®) or other opiates for diarrhea, presence of an abdominal mass, hematocrit, and percentage deviation from standard weight. Further details are described in Example 1 .
  • “Simple Endoscopic Score for Crohn’s Disease” or “SES-CD” means a composite of four assessments, each rated from zero to three consisting of size of ulcers, proportion of the surface covered by ulcers, proportion of the surface with any other lesions, and presence of narrowings, also referred to as stenosis. Further details are described in Example 1.
  • baseline means a clinical (e.g., signs or symptoms) or laboratory value describing a patient’s condition determined prior to administration of a certain therapeutic agent, e.g., an integrin beta7 antagonist such as etrolizumab.
  • a certain therapeutic agent e.g., an integrin beta7 antagonist such as etrolizumab.
  • clinical or laboratory values for which baseline values can be determined include, but are not limited to, hematology and clinical chemi stry values, such as hemoglobin, hematocrit, platelet count, sodium, potassium, chloride and the like, CD Al score, SES-CD score, liquid/soft stool frequency (SF) or abdominal pain (AP) score, or other patient-reported outcome measures such as 1BDQ or CD-PRO/SS.
  • Endoscopic improvement means a greater than or equal to fifty percent reduction in baseline SES-CD score.
  • Endoscopic remission means SES-CD score that is less than or equal to four (less than or equal to two for ileal patients), with no segment having a subcategory score (e.g , for ulceration size and extent, affected surface, or narrowing) that is greater than one
  • CDAI-70 response means a decrease from CD Al baseline score of at least seventy points.
  • Induction therapy means use of a therapeutic agent, or combination of therapeutic agents, in an initial regimen to treat signs and symptoms of Crohn’s disease. Induction therapy is given for a period of time typically ranging from about six weeks to about fourteen weeks. Induction therapy can refer to the initial regimen for a patient following an initial diagnosis of Crohn’s disease or a regimen following intolerance or inadequate response to a prior regime or the initial regimen in a clinical trial of a therapeutic candidate.
  • Exemplary therapeutic agents that can be used in induction therapy in Crohn’s disease include for example, but not limited to, 5-aminosalicylates, antibiotics, budesonide, systemic corticosteroids, thiopurines, methotrexate, anti-TNF agents such as infliximab, adalimumab, and certolizumab pegol, and other biological agents, including but not limited to vedolizumab, ustekinumab, natalizumab, efalizumab, and etrolizumab, as well as Janus kinase (JAK) inhibitors including but not limited to upadacitinib and filgotinib.
  • 5-aminosalicylates antibiotics, budesonide, systemic corticosteroids, thiopurines, methotrexate
  • anti-TNF agents such as infliximab, adalimumab, and certolizumab pegol
  • Disease worsening means during the Induction Phase of treatment of a patient with an anti-beta7 integrin antagonist such as etrolizumab, the CDAI score ten weeks after initiation of treatment is greater than the patient’s CDAI score at baseline or week zero.
  • CDAI relapse means during the Maintenance Phase of treatment of a patient with an anti-beta7 integrin antagonist such as etrolizumab, at least one of the following criteria is met on two consecutive visits with a health-care provider and at least one of the two consecutive CDAI scores is greater than or equal to 220: (1) the CDAI score is greater than or equal to the baseline or week zero score; (2) the CDAI score is greater than or equal to one hundred points higher than the score at fourteen weeks after treatment was initiated [0063]
  • sample refers to a composition that is obtained or derived from a patient or 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 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.
  • 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 integrin 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.
  • the 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 (or rhuMAb beta7).
  • 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 “ ⁇ E subunit” or “ ⁇ E 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 et al., (1996) J. Immunol. 156:2851-2857).
  • VCAM-1 or "vascular cell adhesion molecule-1”
  • CD106 refers to a ligand of alpha4beta7 and alpha4betal, expressed on activated endothelium and important in endothelial-leukocyte interactions such as binding and transmigration of leukocytes during inflammation.
  • 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.
  • 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.
  • “lifetime” of a subject refers to the remainder of the life of the subject after starting treatment.
  • the terms “inadequate response,” “intolerance,” and “refractory response” refer to signs and/or symptoms of persistently active disease despite a history of treatment with one or more therapeutics, for example, corticosteroids, immunosuppressants and/or anti-TNFs.
  • the term “pharmaceutical composition” or “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, which is nontoxic to a subject,
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • 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 corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)).
  • 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.
  • framew'ork 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.
  • 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
  • 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 nonproteinaceous solutes.
  • 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 (Hl, H2, H3), and three in the VL (L1, 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 Sendee, 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 H VRs 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 (Hl), 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.
  • "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.
  • a "human consensus framework” is a framework 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 etal. Proc Nat. Acad.
  • variable 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 p-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the P-sheet structure.
  • the hypervari able 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 etal, 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 (K) and lambda (A), 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., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , s, ⁇ , 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.
  • the terms particularly refer to an antibody with heavy chains that contain an Fc region.
  • 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 etal., 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 Fc region.
  • effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of ceil surface receptors (e.g., B cell receptor; BCR), etc.
  • 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.
  • 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.
  • immunosuppressive agent or “immunosuppressant” as used herein for adjunct therapy 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 Ml IC antigens.
  • exemplary nonbiological therapeutic agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Patent No.
  • non-steroidal anti-inflammatory 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 niofetil (MMF); alkylating agents such as cyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde (which masks the MHC antigens, as described in U.S. Patent No.
  • anti-idiotypic antibodies for MHC antigens and MHC fragments include cyclosporine; 6 mercaptopurine; 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; streptokinase; streptodomase; FK506; RS- 61443; chlorambucil; deoxyspergualin; rapamycin.
  • steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogs, e.g
  • Exemplary biological agents include cytokine antibodies and cytokine receptor antibodies, e.g., antagonists including anti- interferon-alpha, -beta, or -gamma antibodies, anti-tumor necrosis factor(TNF)-alpha antibodies (infliximab [e g., REMICADE); adalimumab [e.g., HUMIRA®]), 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-CD18 antibodies; anti- L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies, anti-CD3 or anti- CD4/CD4a antibodies; anti -idiotypic antibodies for MHC antigens and MHC fragments;
  • TGF-beta transforming growth factor-beta
  • RNA or DNA from the host T-cell receptor
  • 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; laneway, Nature, 341: 482 (1989); and WO 91/01133
  • BAFF antagonists such as BAFF or BR3 antibodies or immunoadhesins
  • biologic agents that interfere with T cell helper signals such as anti-CD40 receptor or anti-CD40 ligand (CD 154), including blocking antibodies to CD40-CD40 ligand. (e.g., Durie etal,.
  • corticosteroid-free means that a patient, e.g. a patient with Crohn’s disease, did not use corticosteroids to treat the disease or symptoms of the disease during the time which the patient is corticosteroid-free. For example, a patient with Crohn’s disease who is corticosteroid-free for 12 months did not use corticosteroids for 12 months to treat symptoms of Crohn’s disease.
  • 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.
  • the expression “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 sam e 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).
  • a “TNF-alpha inhibitor” herein is 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 (SIMPONI®), and certolizumab pegol (CIMZIA®).
  • 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
  • Examples of synthetic corticosteroids include prednisone, prednisolone (including methylprednisolone), dexamethasone triamcinolone, budesonide, 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, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmacological agents and their metabolites, transcriptional and translation control sequences, and the like.
  • 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.
  • the beta7 integrin antagonists are anti-integrin beta7 antibodies.
  • Monoclonal antibodies may be made using the hybridoma method first described by Kohler etal., 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.
  • 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 el 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 chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.
  • 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.
  • monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using e.g., the techniques described in McCafferty etal., Nature, 348:552-554 (1990). Clackson etal., 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
  • the 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-integrin beta7 antibodies are Fib504, Fib 21, 22, 27, 30 (Tidswell, M. J Immunol. 1997 Aug 1 ; 159(3): 1497-505) or humanized derivatives thereof.
  • Humanized antibodies of Fib504 was disclosed in detail in U.S. Patent Publication No. 20060093601 (issued as U S Patent No 7,528,236), the content of which is incorporated by reference in its entirety (also see discussion below).
  • the anti-integrin beta7 antibodies of the invention may further comprise humanized 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.
  • CDR complementary determining region
  • Fv framework residues of the human immunoglobulin 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 etal., Nature, 321 :522-525 (1986); Riechmann etal., Nature, 332:323-327 (1988); Verhoeyen etal., Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • 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.
  • HAMA response human anti-mouse antibody
  • 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); Chothia 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 etal, Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)).
  • 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. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • the hypervariable region residues are directly and most substantially- involved in influencing antigen binding.
  • Exemplary humanized anti-integrin beta7 antibodies include, but are not limited to rhuMAb Beta7, also referred to as etrolizumab, 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 ⁇ I 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 (also referred to as etrolizumab) is a potent in vitro blocker of the cellular interaction between ⁇ 4 ⁇ 7 and its ligands (mucosal addressin cell adhesion molecule-1 [MAdCAM]-!, vascular cell adhesion molecule [VCAM]-1, and fibronectin) as well as the interaction between ⁇ E ⁇ ? and its ligand (E-cadherin).
  • rhuMAb Beta7 (also referred to as etrolizumab) binds reversibly, with similar high affinity, to p7 on lymphocytes from rabbits, cynomolgus monkeys, and humans. It also binds to mouse P7 with high affinity.
  • the heavy chain may lack a C-terminal lysine (HC.vl, SEQ ID NO:11)) or may include a C-terminal lysine (HC.v2, SEQ ID NO: 12).
  • C-terminal lysine residues can be clipped during manufacturing processes.
  • 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 X1776 (ATCC 31,537), and E. coll 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. fragili s (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.
  • wickeramii ATCC 24,178
  • K. waltii ATCC 56,500
  • K. drosophilarum ATCC 36,906
  • K. thermotolerans K. marxianus
  • yarrowia EP 402,226
  • Pichia pastoris EP 183,070
  • Candida Trichoderma reesia
  • Neurospora crassa Neurospora crassa
  • Schwanniomyces such as Schwanniomyces occidentalis
  • 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 califomica 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 CV1 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 etal.. Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VE RO-76, ATCC CRL- 1587); human cervical carcinoma cells (ITELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • Host cells are transformed with the above-described expression or cloning vectors for anti-beta7 integrin antibody production and cultured in conventional nutrient media modified as 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 Fl 0 (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 GENTAMYCIN.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 wall be apparent to the ordinarily skilled artisan.
  • the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, 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.
  • sodium acetate pH 3.5
  • EDTA EDTA
  • PMSF phenylmethylsulfonylfluoride
  • Cell debris can be removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • 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 antibody comprises a C.sub.H3 domain
  • the Bakerbond ABX.TM. resin J. T. Baker, Phillipsburg, N.J ) is useful for purification.
  • 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).
  • the therapeutic agent is an anti-integrin beta7 antibody, also referred to as etrolizumab. Etrolizumab as an IgGl antibody.
  • the anti-integrin beta7 antibody comprises three heavy chain HVRs, HVR-H1 (SEQ ID NO:4), HVR-H2 (SEQ ID NO:5), and HVR-H3 (selected from SEQ ID NO:6 and SEQ ID NO:7).
  • the anti-integrin beta7 antibody comprises three light chain HVRs, HVR-L1 (SEQ ID NO:1), HVR-L2 (SEQ ID NO:2), and HVR-L3 (SEQ ID NO:3).
  • the anti- integrin beta7 antibody comprises three heavy chain HVRs and three light chain HVRs, HVR-H1 (SEQ ID NO:4), HVR-H2 (SEQ ID NO: 5), and HVR-H3 (selected from SEQ ID NO:6 and SEQ ID NO :7), HVR-L1 (SEQ ID NO:1), HVR-L2 (SEQ ID NO:2), and HVR-L3 (SEQ ID NO:3).
  • the anti-integrin beta7 antibody comprises a variable heavy chain region, VH, having an amino acid sequence of SEQ ID NOV. In one embodiment, the anti-integrin beta7 antibody comprises a variable light chain region, VL, having the amino acid sequence of SEQ ID NO:8. In one embodiment, the anti-integrin beta7 antibody comprises a variable heavy chain region, VH, having an amino acid sequence of SEQ ID NOV and a variable light chain region, VL, having an amino acid sequence of SEQ ID NO:8. In one embodiment, the anti-integrin beta7 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.
  • the anti-integrin beta7 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 10. In one embodiment, the anti-integrin beta7 antibody comprises a heavy chain having an amino acid sequence selected from SEQ ID NO:11 and SEQ ID NO: 12 and a light chain having the amino acid sequence of SEQ ID NO: 10. Anti-integrin beta7 antibodies are further described in IntnT Pub. No. 2006/026759. [0144] In another aspect, an anti-integrin beta7 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11.
  • VH heavy chain variable domain
  • a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-integrin beta7 antibody comprising that sequence retains the ability to bind to human integrin beta7.
  • a total of 1 to 10 amino acids have been substituted, altered inserted and/or deleted in SEQ ID NO: 11.
  • substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).
  • the anti-integrin beta7 antibody comprises the VH sequence in SEQ ID NO: 11 or SEQ) ID NO: 12, including post- translational modifications of that sequence.
  • an anti-integrin beta7 antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10.
  • VL light chain variable domain
  • a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-integrin beta7 antibody comprising that sequence retains the ability to bind to human integrin beta7.
  • the anti-integrin beta7 antibody comprises the VL sequence in SEQ ID NO: 10, including post-translational modifications of that sequence.
  • the anti-integrin beta7 antibody comprises a VL region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10 and a VH region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11.
  • 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.
  • formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • Administration of the anti-integrin beta7 antibody 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 anti-integrin beta7 antibody may be administered first, followed by the second therapeutic compound.
  • simultaneous administration or administration of the second therapeutic compound prior to the anti-integrin beta7 antibody is also contemplated.
  • systemic corticosteroids e.g., prednisone (or prednisone equivalent) or budesonide
  • immunosuppressants such as azathioprine, 6-mercaptopurine, or methotrexate
  • tumor necrosis factor inhibitors anti-TNFs
  • infliximab adalimumab
  • certolizumab pegol a tumor necrosis factor inhibitor that has been approved for treatment of CD and these are natalizumab and vedolizumab.
  • the treatment of the present invention for Crohn’s disease (CD) in a human subject with CD comprises administering to the subject an effective amount of a therapeutic agent, such as an anti -beta7 integrin antibody, and further comprising administering to the subject an effective amount of a second medicament, that is an immunosuppressant, a corticosteroid, an anti-TNF, a pain-control agent, an antidiarrheal agent, an antibiotic, or a combination thereof.
  • a therapeutic agent such as an anti -beta7 integrin antibody
  • a second medicament that is an immunosuppressant, a corticosteroid, an anti-TNF, a pain-control agent, an antidiarrheal agent, an antibiotic, or a combination thereof.
  • said secondary medicine is selected from the group consisting of 6-mercaptopurine, azathioprine, methotrexate, prednisone (or prednisone equivalent), budesonide, infliximab, adalimumab, and certlizumab pegol.
  • 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 co-administration (concurrent 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 (medicaments) simultaneously exert their biological activities.
  • PK Pharmacokinetics
  • PD Pharmacodynamics
  • 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 & Development (2006)),
  • 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. (Lakshmi Kamath, supra).
  • PK/PD modeling approaches are proving useful in determining relationships between biomarker responses, 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 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').
  • the amount of biomarkers in the peripheral blood 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, di sease 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
  • the purpose of this study is to assess the efficacy and safety of etrolizumab, an anti-integrin with a unique mechanism of action (MO A) that has been shown to inhibit the trafficking and retention of inflammatory T-cells in the intestinal mucosa, via a disruption of ⁇ 4 ⁇ 7/MAdCAM-l, and ⁇ E ⁇ 7/E-cadherin binding.
  • MO A an anti-integrin with a unique mechanism of action
  • etrolizumab had an acceptable safety profile with no clinically significant safety' signals observed.
  • the study design will comprise 1) a Screening Phase (up to 35 days) to determine patients’ eligibility for the study, 2) an Induction Phase (14 weeks), followed by 3) a Maintenance Phase (52 weeks) in patients demonstrating a CDAI-70 response (defined as a decrease from CD Al baseline score of at least 70 points) at the end of the Induction Phase, and 4) a Safety Follow-Up Phase (12 weeks) after administration of the last dose of study drug in the Maintenance Phase for those patients who are not participating in Part 1 of open- label extension of the Study to receive etrolizumab treatment (see Fig. 1 and Fig. 2).
  • patients will be asked to enter an extended PML- monitoring phase (open-label extension study) for 92 weeks.
  • the study population will consist of patients who are refractory or intolerant to one or more of the following therapies: 1) corticosteroid (CS), 2) immunosuppressant (IS), or 3) anti- tumor necrosis factor (TNF) (or inadequate response to anti -tumor necrosis factor [TNF-IR]).
  • Patients who enroll on the basis of refractory or intolerance to CS and/or IS may have been previously exposed to ant-TNFs or be naive to anti-TNFs.
  • Approximately 1150 patients will be randomized into the study from approximately 420 global investigational sites via enrollment into one of three cohorts. The enrollment will be sequential, first into Cohort 1, then Cohort 2, and lastly Cohort 3.
  • the ileocolonoscopy should be performed during the Screening Phase and at least 9 days prior to randomization to allow sufficient time for a central reader scoring and determination of eligibility, and to avoid the ileocolonoscopy bowel preparation influencing the patient reported outcomes used in the determination of baseline SF, AP, and CD Al scores (i.e., abdominal pain, general well-being, and stool frequency).
  • Eligible patients will be enrolled sequentially into one of three cohorts for the 14- week Induction Phase (see Fig. 1).
  • Patients randomized to high-dose etrolizumab will receive one placebo and one high-dose etrolizumab injection at each administration, except at Week 2 where they will receive one high-dose injection. Finally, patients randomized to placebo will receive two placebo injections at every administration, except at Week 2 where they will receive one placebo injection.
  • the randomization in all cohorts will be stratified by concomitant oral CS treatment (yes vs. no), concomitant IS treatment (yes vs. no), baseline CD Al ⁇ 330 (yes vs. no), and prior anti-TNF exposure (yes vs. no).
  • the enrollment will be managed to ensure that the proportion of the TNF-exposed patients in Cohort 3 does not exceed approximately 60% and that the proportion of patients with a CDAI score between > 450 and ⁇ 480 does not exceed approximately 10% in each cohort.
  • Concomitant CD Therapy in the Induction Phase includes any medication (e.g., prescription drugs, over-the- counter drugs, herbal or homeopathic remedies, nutritional supplements) used by a patient from 4 weeks prior to screening to the study completion/early termination visit.
  • medication e.g., prescription drugs, over-the- counter drugs, herbal or homeopathic remedies, nutritional supplements
  • Anti- diarrheal medications are permitted if used to manage chronic diarrhea but every attempt should be made to keep the dose/regimen stable. Any changes in the dose/regimen after baseline (Week 0) must be captured by the investigator and/or patient. Occasional use of nonsteroidal anti-inflammatory drugs (NSAIDs) or acetaminophen is permitted for pain relief (e.g., in the case of headache, arthritis, myalgia, etc.). Prophylactic aspirin use up to 325 mg/day is also permitted.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • acetaminophen is permitted for pain relief (e.g., in the case of headache, arthritis, myalgia, etc.).
  • Prophylactic aspirin use up to 325 mg/day is also permitted.
  • anti-integrins e.g., anti-MAdCAM- 1
  • T- or B- cell-depleting agents with the exception of AZA and 6-MP (or equivalent)
  • TNF antagonists antagonists of IL -23 ⁇ IL-12 (e.g., ustekinumab)
  • anti-metabolites cyclosporine, and tacrolimus.
  • CS enemas/suppositories and/or topical (rectal) 5 -aminosalicylate (5-ASA) preparations are also prohibited medications. Patients taking these medications will be considered nonresponders for the purpose of any endoscopic analyses. These treatments must be discontinued, but patients may continue to receive etrolizumab and may still be eligible for Part 1 (OLE) Study.
  • OLE Part 1
  • This is defined as medication prescribed for new or worsening CD symptoms, and includes any new CS or IS therapy for CD or any increase in dose or regimen of baseline Crohn’s medications.
  • Antibiotics, 5-ASAs, anti-diarrheals, probiotics, herbal/ayurvedic, nutritional and homeopathic supplements are not considered as rescue therapies. Patients requiring rescue medication during the Induction Phase will be considered non-responders for the primary analysis and will not be eligible for the Maintenance Phase.
  • the randomization call may take place between Week 14 (last visit in the Induction Phase) and Week 16, provided the patient has been assessed as eligible for the Maintenance Phase.
  • the randomization will be stratified by CDAI remission at both Weeks 10 and 14 (yes vs. no), induction dose regimen (low dose vs. high dose), concomitant oral CS treatment (yes vs. no), and prior anti-TNF exposure (yes vs. no).
  • the first dose in the Maintenance Phase is administered at the Week 16 clinic visit.
  • CS dose should be tapered starting at Week 14. Patients who cannot tolerate the CS taper without recurrence of CD symptoms or symptoms of steroid withdrawal can receive an increase in CS dose, but this should not exceed the dose administered at randomization.
  • the dose-tapering regimen must be re-initiated within 2 weeks.
  • Clinical relapse is defined as meeting at least one of the following criteria on two consecutive visits (may include unscheduled visits), with at least one of the two consecutive CDAI scores >220: CDAI score > the baseline (Week 0) score or CDAI score >100 points higher than the Week 14 score.
  • All patients who complete their final Maintenance Phase visit at Week 66 may be eligible to enroll in Part 1 (OLE) Study.
  • Patients who do not enroll in Part 1 (OLE) will enter a 12-week Safety Follow-Up Phase, after which they will be asked to enroll in a 92-week extended PML -monitoring phase (Part 2 [SM] Study).
  • Patients who require surgical intervention for CD at any time during the Maintenance Phase will stop study treatment, enter the Safety Follow-Up Phase, and will be asked to enter Part 2 (SM) Study for PML monitoring.
  • Patients who self-withdraw from the Maintenance Phase and do not meet the eligibility criteria for OLE treatment will also enter the Safety Follow-Up Phase and be asked to enter Part 2 ( SM) Study for PML monitoring.
  • Patients who withdraw will complete the early withdrawal assessments listed in the Schedule of Assessments; withdrawn patients will not be replaced.
  • corticosteroid dose should be tapered starting at Week 14 according to the following schedule: ⁇ 20 mg/day prednisone (or equivalent), titrated via dose reduction of 2.5 mg/week until discontinuation; ⁇ 6 mg/day oral budesonide, titrated via dose reduction of 3 mg every 2 -weeks until discontinuation,
  • patients may increase their corticosteroid dose up to their baseline dose (i.e., dose at randomization), but the dose-tapering regimen should be re-initiated within 2 weeks.
  • Patients who are taking concomitant IS therapy (AZA, 6-MP, MTX) must remain on stable doses throughout the study unless dose reduction or discontinuation is required because of a toxicity related to the medication.
  • Doses of anti-diarrheal medication should also be kept stable.
  • Generally accepted criteria for discontinuation of IS due to toxicity include, but are not limited to, acute pancreatitis, severe leukopenia, severe thrombocytopenia, or significant elevations of the liver-associated enzymes from baseline, especially in the presence of an elevated total bilirubin.
  • the ultimate decision to discontinue IS remains at the discretion of the Investigator.
  • This is defined as medication prescribed for new or worsening CD symptoms, and includes: any new CS or IS therapy for CD; increase in dose of IS therapy above the baseline (Week 0) dose; increase in the dose of CS therapy above a patient’s baseline (Week 0) dose (applicable to patients requiring CS at baseline).
  • Antibiotics, 5-ASAs, anti-diarrheals, probiotics, herbal/ayurvedic, nutritional, and homeopathic supplements are not considered as rescue therapies. Patients who require rescue therapy during the Maintenance Phase may be offered early access to the Part 1 OLE Study, if eligible, and will be required to complete the Early Withdrawal visit assessment. Prohibited therapies are as described for the Induction Phase. Study Drug or Placebo Administration
  • Patients will receive etrolizumab or placebo by SC injection according to their treatment assignment. Patients and study personnel will be blinded to study drug assignment (or the etrolizumab dose assignment for Cohort 2 patients in the Induction Phase) for the entire study. In the Induction Phase, study medication will be administered at the investigational site and patients will be trained to self-administer. A health care professional (HCP) will administer the first two doses, with the patient or their caregiver administering subsequent doses under HCP supervision.
  • HCP health care professional
  • patients In the Maintenance Phase after completing Week 16, patients have the option to return to the investigational site or to self-administer/have a caregiver administer their dose Q4W at home within ⁇ 3 days of their scheduled dose unless a PK blood draw is planned, in which case the dose must be administered after the blood draw on the day of visit or within 3 days after the visit.
  • the co-primary efficacy endpoints for this study are the following: (1) Clinical remission, defined as a SF mean daily score ⁇ 3 and an AP mean daily score ⁇ 1 with no worsening in either subscore compared to baseline, averaged over the 7 days prior to visit; and (2) Endoscopic improvement, defined as a >50% reduction from the baseline SES-CD. [0202] Primary efficacy objectives for this study will be analyzed separately for the Induction and Maintenance Phases as outlined following. Induction Phase: To independently evaluate the efficacy of etrolizumab dose regimens compared with placebo in inducing clinical remission and endoscopic improvement at the end of the Induction Phase (Week 14).
  • Secondary objectives for the induction phase of this study are as follows: (1) To evaluate the efficacy of etrolizumab compared with placebo in achieving clinical remission at Week 6; (2) To evaluate the efficacy of etrolizumab compared with placebo in achieving anSES-CD ⁇ 4 ( ⁇ 2 for ileal patients), with no segment having a subcategory score (i.e., for ulceration size and extent, affected surface, or narrowing) that is >1, at Week 14; and (3) To evaluate the reduction in CD signs and symptoms achieved by etrolizumab dose regimens compared with placebo at Week 14 as assessed by the Crohn’s Disease Patient-Reported Outcome Signs and Symptoms (CD-PRO/SS) measure.
  • CD-PRO/SS Crohn’s Disease Patient-Reported Outcome Signs and Symptoms
  • Secondary objectives for the maintenance phase of this study are as follows for patients who achieved CDAI-70 at Week 14, unless otherwise stated: (1) To evaluate the efficacy of etrolizumab compared with placebo in maintaining clinical remission at Week 66 for patients who achieved clinical remission at Week 14; (2) To evaluate the efficacy of etrolizumab compared with placebo in achieving corticosteroid-free clinical remission at Week 66 among patients who were receiving corticosteroids at baseline; (3) To evaluate the efficacy of etrolizumab compared with placebo in maintaining endoscopic improvement at Week 66 for patients who achieved endoscopic improvement at Week 14; (4) To evaluate the efficacy of etrolizumab compared with placebo in achieving a SES-CD ⁇ 4 ( ⁇ 2 for ileal patients), with no segment having a subcategory score(i.e., for ulceration size and extent, affected surface, or narrowing) that is >1, at Week 66; (5) To evaluate the efficacy of etrolizumab
  • the exploratory efficacy objectives for this study are as follows: (1) To evaluate the efficacy of etrolizumab compared with placebo in reducing fecal calprotectin levels at the end of the Induction and Maintenance Phases; (2) To evaluate the efficacy of etrolizumab compared with placebo in reducing C reactive protein (CRP) levels at the end of the Induction and Maintenance Phases; (3) To evaluate the time from Week 14 until experiencing major CD-related events (including hospitalizations, bowel surgeries, and non-study procedures); (4) To evaluate the effect of etrolizumab compared with placebo on the closure of draining fistulas; (5) To evaluate the efficacy of etrolizumab compared with placebo in achieving an SES-CD of 0 by Week 66; (6) To determine the efficacy of etrolizumab compared with placebo in achieving histologic improvement at the end of Induction and Maintenance Phases; (7) To evaluate efficacy of etrolizumab compared with placebo in achieving
  • the safety objectives for this study are to evaluate the overall safety and tolerability of etrolizumab compared with placebo during Induction and Maintenance Phases of therapy; to evaluate the incidence and severity of infection-related adverse events; to evaluate the incidence of malignancies; to evaluate the incidence and severity of immunogenic responses (anti-therapeutic antibodies [ATAs]); and to evaluate the incidence and severity of hypersensitivity reaction events.
  • the pharmacokinetic objectives for this study are to evaluate etrolizumab serum concentrations during the Induction Phase (Week 14) and at several predose timepoints when at steady state during the Maintenance Phase in patients who are re-randomized to etrolizumab; to characterize the interindividual variability and potential covariate effects on etrolizumab serum exposure; to investigate the relationship between serum exposure and clinical response and remission as well as endoscopic changes during the induction and maintenance treatment phases; to characterize the PK profile of etrolizumab in patients with CD and the relationship between serum exposure of etrolizumab and 07 receptor occupancy by etrolizumab on peripheral blood T and B lymphocytes subsets (in a PK/PD substudy).
  • the target population are refractory or intolerant to CS and/or IS therapy and who have either not received prior anti-TNF therapy (TNF-naive) or who have had prior exposure to one or more anti-TNF therapies, and who have had an inadequate response, refractory response, or intolerance to CS and/or IS therapy and/or anti-TNFs.
  • TNF-naive prior anti-TNF therapy
  • anti-TNF therapies and who have had an inadequate response, refractory response, or intolerance to CS and/or IS therapy and/or anti-TNFs.
  • Patients refractory to CS therapy have signs/symptoms of persistently active disease despite a history of at least one 4-week induction regimen including a dose equivalent to > 30 mg/day prednisone for 2 weeks if oral or 1 week if IV or > 9 mg/day oral budesonide.
  • Patients intolerant to CS therapy have a history including, but not limited to, Cushing’s syndrome, osteopenia/osteoporosis, hyperglycemia, insomnia, and infection.
  • Patients refractory to IS therapy have signs/symptoms of persistently active disease despite a history' of at least one 12-week regimen of oral AZA (or equivalent) (> 1 5 mg/kg) or 6-MP (or equivalent) (> 0.75 mg/kg) or MTX (> 15 mg/week).
  • Patients intolerant to IS therapy have a history' of intolerance to AZA (or equivalent), 6-MP (or equivalent), and/or MTX (including, but not limited to, infection, nausea/vomiting, abdominal pain, pancreatitis, liver function test abnormalities, lymphopenia, and thiopurine methyltransferase genetic polymorphism).
  • Inadequate primary non-response to anti-TNF therapies means the patient did not respond (as evidenced by persistent signs/symptoms related to CD after receiving > 2 induction doses of either infliximab [> 5 mg/kg] or adalimumab [160 mg/80 mg or 80 mg/40 mg] or certolizumab pegol [> 400 mg]).
  • Inadequate secondary' non-response to anti-TNF therapies means the patient initially responded to induction therapy with infliximab (> 5 mg/kg) or adalimumab (> 40 mg) or certolizumab pegol (> 400 mg), but experienced signs/symptoms related to recurrence of CD during maintenance.
  • Intolerance to anti-TNF therapy means the patient experienced a significant injection-site reaction, congestive heart failure, infection, or other condition that precluded continuing use of anti-TNF therapy at any time.
  • Patients who have not previously demonstrated inadequate response or intolerance to one or more anti-TNF therapies are eligible to participate in the study provided they are intolerant or refractory to corticosteroid or immunosuppressant therapy.
  • resection for CD completed a surveillance colonoscopy ⁇ 12 months prior to screening if colonic disease of > 10 years’ duration or ⁇ 5 years prior to screening if patient has any risk factors for bowel cancer (surveillance may be performed during screening); (8) have experienced intolerance, refractory disease, or no response (as defined above) to at least one of CS therapy, IS therapy, and/or anti-TNF therapy within 5 years from screening.
  • the exclusion criteria related to gastrointestinal health include: (1) underwent subtotal colectomy with ileorectal anastomosis or underwent total colectomy; (2) short-bowel syndrome; (3) has an ileostomy or colostomy; (4) has evidence of fixed stenosis or small- bowel stenosis with prestenotic dilation that precludes adequate endoscopic assessment of the bowel; (5) diagnosis of UC or indeterminate colitis; (6) suspicion of ischaemic colitis, radiation colitis, or microscopic colitis; (7) evidence of abdominal or perianal abscess; (8) expected to require surgery' to manage CD-related complications during the study; (9) past or present adenomatous colonic polyps; (10) past or present disease-related colonic mucosal dysplasia (prior age-related polyps are acceptable); (11) Sinus tract with evidence for infection (e.g., purulent discharge) in the clinical judgment of the investigator
  • the exclusion criteria related to prior or concomitant therapy include: (1) received any of adalimumab, certolizumab pegol, or infliximab for CD within 8 weeks prior to randomization; (2) any prior treatment with ustekinumab within 14 weeks prior to randomization; (3) any prior treatment with etrolizumab or other anti-integrin agents (including vedolizumab, natalizumab, and efalizumab); (4) any prior treatment with anti- adhesion molecules (e.g., anti-MAdCAM-1); (5) prior treatment with T cell- or B cell-depleting agents (e.g., rituximab, alemtuzumab, or visilizumab) within ⁇ 12 months prior to randomization with the exception of AZA and 6-MP (or equivalent); (6) received any investigational treatment that included investigational vaccines within 12 weeks prior to randomization in the study or five half-lives of the investigational product, whichever is greater; (7) history
  • Prophylactic aspirin use up to 325 mg/'day is permitted, as is occasional use of NSAIDs for conditions such as headache, arthritis, mylagia, and menstrual cramps; (15) if receiving oral CSs, patients will be excluded unless the dose is stable at ⁇ 20mg/day prednisone (or equivalent) for > 2 weeks immediately prior to randomization; (16) if receiving ongoing treatment with oral 5 aminosalicylate (5-ASA), patients will be excluded if the dose is not stable for > 4 weeks immediately prior to randomization; (17) if receiving ongoing treatment with probiotics (e.g., Culturelle, Saccharomyces boulardii) or over-the- counter supplements (e.g., N-acetyl glucosamine, curcumin), patients will be excluded if the dose is not stable for > 2 weeks immediately prior to randomization; (18) if receiving ongoing treatment with ISs (e.g., 6-MP, AZA, or MTX), patients will be excluded if the dose is not stable
  • Patients may continue to receive ongoing treatment with anti-diarrheals (e g., loperamide or diphenoxylate with atropine), preferably achieving a stable dose for > 2 weeks prior to randomization. However, if the patient and/or the treating physician decides to change the dose or course of anti-diarrheals at any time during screening, these patients will be allowed to participate in study.
  • anti-diarrheals e g., loperamide or diphenoxylate with atropine
  • the exclusion criteria related to infection risk include: (1) congenital or acquired immune deficiency; (2) positive ELISA test result for HIV confirmed by Western blot; (3) positive hepatitis C vims (HCV) antibody test result, unless the patient has documented that HCV RNA is undetectable for > 6 months after completing a successful course of HCV antiviral treatment; (4) in the screening hepatitis B assessment, patients who test positive for HBsAg are excluded from the study; patients who test positive for hepatitis B core antibody (HBcAb) but negative for hepatitis B surface antigen (HBsAg) must have a confirmed negative hepatitis B virus (HBV) DNA test result to be eligible for the study and will be required to undergo periodic monitoring for HBV DNA during the study; (5) positive stool test result for ova or parasites or positive stool culture for pathogens at time of screening; (6) evidence of infection with and/or treatment for Clostridium difficile or other intestinal pathogen treatment for C.
  • HCV
  • the exclusion criteria related to general safety include: (1) pregnant or lactating; (2) lack of peripheral venous access; (3) hospitalized within 4 weeks prior to randomization; (4) inability to comply with study protocol, in the opinion of the investigator; (5) significant uncontrolled comorbidity, such as neurological, cardiac, pulmonary, renal, hepatic, endocrine, or GI disorders (other than CD), (6) neurological conditions or diseases that may interfere with monitoring for PML (7) clinically significant abnormalities on screening neurologic examination; (8) history of demyelinating disease; (9) history of major neurological disorders, including stroke, MS, brain tumor, neurodegenerative disease, or poorly controlled epilepsy; (10) history of alcohol, drug, or chemical abuse ⁇ 6 months prior to screening ; (11) conditions other than CD that could require treatment with >20 mg/day of prednisone (or equivalent) during the course of the study; (12) history of cancer, including hematologic malignancy, solid tumors, and carcinoma in situ within 5 years before screening; (13) history of cervical sm
  • Etrolizumab and Placebo Handling [0218] Etrolizumab will be supplied by the Sponsor as a single-use PFS containing 150 mg/mL etrolizumab for SC administration. To preserve the blind to study drug assignment in the Induction Phase, all patients will receive injections from two PFSs at Weeks 0, 4, 8, and 12: a 1-mL PFS with a 0.7-mL injection volume (delivering placebo or 105 mg of etrolizumab) and a 2.25-mL PFS with a 1.4-mL injection volume (delivering placebo or 210 mg of etrolizumab). Depending on dose assignment, one PFS (patients assigned to active drug) or both PFSs (patients assigned to placebo) will contain placebo.
  • Drug product composition for the placebo is exactly the same as that of active drug product without the presence of etrolizumab.
  • the study drug must be stored according to the details on the product label.
  • the drug label indicates the storage temperature.
  • PFSs of study medication should be refrigerated at 2°C-8°C (35.6°F-46.4°F) and protected from excessive light and heat. PFSs should not be frozen, shaken, or stored at room temperature.
  • the PFS containing study drug is stable for no longer than 8 hours at room temperature (up to 30°C [86°F]). If a syringe is left at room temperature for longer than this time, it should not be used.
  • the CD Al quantifies the signs and symptoms of patients with CD.
  • the CD Al consists of eight factors, each factor is summed after adjustment with a weighting factor (Best et al. 1979).
  • the components of the CD Al include number of liquid or soft stools, abdominal pain, general well-being, presence of complications, use of LOMOTIL® (diphenoxylate/atropine) or other opiates for diarrhea, presence of an abdominal mass, hematocrit, and percentage deviation from standard weight.
  • CD Al Of the eight factors of the CD Al, three are patient reported (number of liquid or soft stools, abdominal pain, and general well- being), four are based on physician assessment (presence of complications, use of LOMOTIL® or other opiates for diarrhea, presence of abdominal mass, and percentage of deviation from standard weight, which is based on the patient’s weight at the visit), and one factor is based on a blood test (hematocrit). Patients are to report their abdominal pain severity, loose-stool frequency, and general well-being on the e-diary on a daily basis. The weighted sum of the average scores over 7 days is calculated for the PRO component of the CD Al score.
  • the Bristol Stool Scale is provided to patients as a reference for determining loose stools (Types 6 and 7 on the Bristol Stool Scale; see Fig. 3). Because the ileocolonoscopy preparations can interfere with the assessment of other clinical parameters, e-diary entries used to calculate the complete CD Al should not correspond to day(s) of bowel preparation, endoscopy, or the day after endoscopy.
  • SF liquid or soft stools
  • AP abdominal pain
  • the score is calculated using the unweighted mean number of liquid/very soft stools and mean AP (on a 0-3 scale) for the 7 days prior to the assessment visit.
  • Patients are to report their loose-stool frequency (the Bristol Stool Scale will be provided [Fig. 3]) and abdominal pain severity on the e-diary on a daily basis.
  • the SF and AP scores should not use e-diary entries that correspond to day(s) of bowel preparation, endoscopy, or the day after endoscopy, to avoid interference related with the ileocolonoscopy.
  • the 0-10 point Abdominal Pain Questionnaire is an I I- point numeric, rating scale to assess the worst abdominal pain on a daily basis. A higher score indicates a greater severity of abdominal pain (0 ::: no pain; 10 ::: pain as bad as you can imagine).
  • the APQ has a recall specification of 24 hours.
  • the calculated APQ score should not use e-diary entries that correspond to day(s) of bowel preparation, endoscopy, or the day after endoscopy, to avoid interference related with the ileocolonoscopy.
  • Another assessment of disease activity is the patient-reported outcome tool known as CD-PRO/SS.
  • the development of this PRO is described in Higgins et al., Journal of Patient-Reported Outcomes (2016) 2:24 and the PRO modules and user manual can be found at https(colon)(slash)(slash)www(dot)evidera(dot)com(slash)crohns-disease-patient-reported- outcomes-cd-pro.
  • the 14-item questionnaire contains two domains: CD signs and symptoms and systemic symptoms.
  • the CD-PRO/SS assesses the presence of CD signs and symptoms and, in some cases, the severity or frequency of the symptoms.
  • the CD-PRO/SS measure has a recall specification of 24 hours. Patients will complete the CD-PRO/SS measure for at least 9 consecutive days around each visit as programmed in the e-diary. To allow for the visit window to account for early or delayed visits, patients may complete CD-PRO/SS for up to 12 days.
  • Clinical remission is defined as the mean number of liquid/soft stools for the 7 days prior to the assessment visit (SF) being ⁇ 3 and the mean AP score (on a 0-3 scale) with no worsening in either subscore compared to baseline, for the 7 days prior to the assessment visit (AP) being ⁇ 1.
  • the primary clinical endpoint is based on previous FDA recommendations, the evolving global regulatory environment (new EMA new medicinal products guideline for CD released July 2018; CHMP 2016), and the results generated from the exploratory induction of Cohort 1 in this study. Both FDA and EMA in its new guideline discouraged the use of CDAI as primary endpoint for CD registration studies due to the limitations of the index. Instead, co-primary endpoint composed of patient-reported signs/symptoms and endoscopic measure is encouraged.
  • SF and AP addresses the FDA’s recommendation to use 7-day scores of SF and AP that correlate with CDAI remission. Defining clinical remission and response that use unweighted measures of AP and SF are more easily interpretable by clinicians. patients, and caregivers and are more likely to represent clinically meaningful improvements in symptoms and disease activity for moderate to severe CD patients experiencing different degrees of SF and AP during a flare.
  • Endoscopic improvement defined as a change in SES-CD > 50% from the baseline score (Ferrante M, et al., Gastroenterology 145:978-86, 2013), is a co-primary endpoint.
  • the SES-CD consists of four endoscopic variables (ulcers, ulcerated surface, inflamed surface, and presence of narrowings) that are scored in five ileocolonic segments, TheSES-CD was prospectively developed and validated in patients with mild to severe CD (according to CD Al) by Dapemo et al., Gastrointest. Endosc. 60(4):505-12, 2004.
  • This scoring system was recommended by the FDA and is generally preferred by physicians to other measures because of quantification of ulcer size (rather than a qualitative assessment of ulcer characteristics), determination of the percent ulceration in a segment (rather than determination by a visual analogue scale), and for better inter-rater reliability (inter-class correlation coefficients were 0.83 and 0.71 for SES-CD and Crohn's Disease Endoscopic Index of Severity (CDEIS), respectively; Khanna R, et al., Inflamm Bowel Dis 20: 1850 > 61, 2014). Because the score does not adjust for the number of visible segments, only segments visualized at baseline will be included in the endpoint assessment.
  • this endpoint will be measured at Week 14 and, for maintenance, at Week 66 among patients who achieved a clinical response at Week 14.
  • the endpoint is based on recent post hoc analyses of the SONIC trial which determined a > 50% reduction in SES-CD score to be predictive of CS-free CD Al remission following 50 weeks of treatment with biologic therapy (Ferrante M, et al., Gastroenterology 145:978-86, 2013).
  • This definition is also appropriate when considering the large variability in SES-CD change at the end of a 6-week Induction Phase measured in a sample of 24 placebo patients (with moderate to severely active CD) who were participating in one of two trials of novel biologic agents (Ferrante et al. Gastroenterology 138, Issue 5, Supplement 1, S-358, 2010).
  • the dataset showed that 6 patients achieved both 50% reduction in either SES-CD or ( DEIS score, and at least a 5-point reduction in either SES-CD or CDEIS score.
  • Patients belonging to each subgroup will be identified on the basis of refractory and inadequate-response criteria described herein.
  • Patients with moderate to severely active CD between the ages of 18 and 80 years of age will be studied. This age range is typical of patients enrolled in clinical trials of new investigational agents for CD and reflects the observation that adult CD can become or persist as moderate to severely active disease at any age. Given that the primary clearance mechanism for etrolizumab is neither renal elimination nor first-pass metabolism, the risk of accumulation in patients >65 years of age is considered low and is also mitigated by laboratory exclusions related to poor renal and hepatic function.
  • Patient randomization into the Induction Phase will be stratified by disease activity on the basis of CDAI score ⁇ 330 or >330 (predictive of CDAI response and remission rates with biologic therapy; Sandborn WJ, et al., Aliment Pharmacol Ther 37:204- 13, 2013; Sands BE, et al., Gastroenterology 147:618-27, 2014), CS use, IS use, and prior anti-TNF failure (all indicators of disease activity). These factors are considered sufficient to mitigate the risk of imbalance in disease severity across treatment arms.
  • a challenge of the 14-week Induction Phase is the requirement to keep concomitant CD therapies stable for the duration, so as not to confound the endpoint analysis. This is problematic in the context of patients who require rescue therapy to treat a flare and patients who are responding to treatment but unable to taper their CS dose.
  • the study design addresses this by allowing use of rescue therapy in case of disease worsening, in which case patients would be classified as non-responders for the primary analysis, and restricting the maximum baseline CS to ⁇ 20mg/day prednisone-equivalent dose.
  • increasing the dose of anti-diarrheal drugs should be avoided. Patients should keep their dose of anti-diarrheal medication stable as much as possible because the effect of titrating anti-diarrheal medication on the placebo-response rate on clinical remission is unknown.
  • the Induction Phase includes an exploratory cohort (Cohort 1) that is adequately- sized to assess effect size and accuracy of statistical planning assumptions for the new endpoints in pivotal cohort (Cohort 3), the clinical validity of the dichotomous endpoint definitions, and the testing hierarchy of the endpoints.
  • An active-treatment induction cohort (Cohort 2) is also included in this study to generate a sufficient number of remitters for assessment of endpoints in the Maintenance Phase.
  • the final cohort (Cohort 3) is a pivotal induction cohort, which will generate data for the endpoint analysis of the induction study.
  • Patients achieving a CDAI-70 response at Week 14 without rescue therapy wall be re-randomized into the Maintenance Phase until a sample size of approximately 480 patients in the Maintenance Phase is achieved.
  • Randomization into the Maintenance Phase will be stratified by the use of disease activity (described for the Induction Phase, except CDAI-remission [score ⁇ 150] at both Weeks 10 and 14 will be used instead of CD Al ⁇ 330 or > 330).
  • the randomization will be stratified by assignment to low- or high-dose etrolizumab (allowing an assessment of the impact of low- or highdose induction therapy on the maintenance endpoints). Given the large number of strata, any potential imbalance in the proportion of endoscopic improvers at Week 14 will be handled using a covariate-adjusted analysis for the Week 66 endoscopic-improvement endpoint.
  • the duration of the M aintenance Phase (52 weeks) is considered an appropriate period by the FDA and EMA to establish the benefit of long-term therapy.
  • patients receiving CS during the Induction Phase should undergo weekly dose reductions aligned with recommendations for CS tapering in the current American College of Gastroenterology and European Crohn’s and Colitis Organization guidelines and following the EMA recommendation to avoid a rapid taper.
  • the tapering schedule will allow patients to be assessed for the key secondary outcome of achieving corticosteroid-free clinical remission at Week 66 among patients who enter into maintenance phase
  • Low-dose etrolizumab (105 mg) will be administered Q4W SC (Weeks 0, 4, 8, and 12).
  • a low' dose regimen of 105 mg SC at Q4W is specified for dose ranging in the Induction Phase on the basis of the following considerations: (1) In the Phase II UC trial, a nominal dose of 100 mg (0.7 mL of 150 mg/mL solution via vial and syringe), actual dose of 105 mg, administered Q4W SC, showed a clinically meaningful induction of remission in patients with UC and had a favorable safety profile in the Phase II trial (Vermeire et al., Lancet 384:309-18, 2014); (2) The exposure of 105 mg, administered Q4W SC, was shown to be sufficient for maximal P7-receptor occupancy in both blood and colonic tissue from all patients who provided evaluable samples in the Phase II trial, Id., (3) Population PK/PD modeling predicts that a dose lower than the 105-mg SC Q4W regimen (e g.,
  • CD exhibits a more complex anatomical disease presentation throughout the Gl tract (i.e., transmural inflammation, patchy distribution, and strictures) when compared with UC.
  • a positive exposure-response relationship was reported recently for vedolizumab, an in-class, anti-integrin antibody, following the Induction Phase of a Phase III clinical trial in patients with CD.
  • a low-dose regimen of 105 mg SC at Q4W is specified in the Maintenance Phase on the basis the following considerations: (1) The 105-mg Q4W SC dose planned for the Phase III study in CD is anticipated (by population modeling) to maintain full p7-receptor occupancy at all times in > 85% of patients.
  • a placebo-treated control group will be used in this study to assess the differences in efficacy, safety, and tolerability in patients who receive etrolizumab and background CD therapy compared with patients who receive placebo and background CD therapy.
  • the use of a control group is necessary given the inherent variability in disease flares and the use of subjective assessments, such as the PROs Patients in the control group will undergo the same study assessments as the etrolizumab-treated patients.
  • the 1 :1 ratio for etrolizumab to placebo treated patients is necessary to achieve a statistically powered comparison of primary endpoint between these treatment arms.
  • PROs IBDQ, CD-PRO/SS, EQ-5D, APQ and the loose stool frequency, abdominal pain, and general well-being components of the CD Al
  • the instruments will be translated as required into the local language.
  • PRO data are collected electronically with the use of electronic PRO (ePRO) devices (i.e., e-diary and tablet).
  • the investigator staff will provide the patient with an e-diary and instructions for completing the PRO questionnaires electronically for those PROs that need to be completed outside of the clinic. Patients will also be instructed to contact the site promptly if they have any questions about the use of the e-diary during screening or at any time during the study.
  • PROs are to be completed at the clinic, the patient will fill them out on a tablet. Review electronic data captured by the patient since the previous study visit with the patient at each clinic visit. ePRO data is collected and assessed at visits. During screening, patients will be instructed on how to appropriately use and complete questions on the e-diary. The signs and symptoms of CD, specifically, number of liquid or soft stools, abdominal pain, and general well-being, must be recorded daily throughout the study, including the screening period. To ensure instrument validity and that data standards meet Health Authority requirements, the PROs completed at the sites (IBDQ and EQ-5D) must be administered at the investigational site prior to the completion of other non-PRO assessments and before the patient receives any disease-status information or study drug during that visit.
  • IBDQ and EQ-5D To ensure instrument validity and that data standards meet Health Authority requirements, the PROs completed at the sites (IBDQ and EQ-5D) must be administered at the investigational site prior to the completion of other non-PRO assessments and before the patient receives any disease-status information or
  • CDAI, SF, AP, and CD-PRO/SS were discussed above.
  • the IBDQ assesses patients’ health -related quality of life (HRQOL; Guyatt G, et al., J Clin Epidemiol 42(5):403- 408, 1989; Irvine EJ, J Pediatr Gastroenterol Nutr 28: S23-7, 1999).
  • the 32-item questionnaire contains four domains: bowel symptoms (10 items), systemic symptoms (five items), emotional function (12 items), and social function (five items). The items are scored on a 7-point Likert scale with a higher score indicating better HRQOL.
  • the IBDQ has a recall specification of 2 weeks. Patients complete the IBDQ at the investigational site on a tablet at baseline, at Weeks 0, 14, 44, and 74 prior to the completion of other non-PRO assessments and before the patient receives any disease-status information or study drug during that visit.
  • the EuroQol Five Dimension Questionnaire (EQ-5D) is a generic preference-based HRQOL that provides a single index value for health status (Rabin R, et al., Enn Med 33:337-43, 2001), This tool includes questions about mobility, self-care, usual activities, pain/ discomfort, and anxiety/depression that are used to build a composite of the patient’s health status. Patients complete the EQ-5D at the investigational site on a tablet at Weeks 0, 14, 44, and 74 (or Early Withdrawal visit) prior to the completion of other non- PRO assessments and before the patient receives any disease-status information or study drug during that visit.
  • the Induction and Maintenance Phases will be treated as independent studies.
  • the open-label Cohort 2 serves as a feeder cohort to provide sufficient sample size for the maintenance analysis.
  • Induction data from Cohort 2 will also be considered exploratory and may be analyzed to inform decision making and devel opment of the Statistical Analysis Plan.
  • the sample size for each cohort in the Induction Phase is summarized in Table 3 below. Table 3. Induction Phase Sample Size for Each Cohort.
  • the sample size for Cohort 1 provides approximately 90% power to detect a >20% difference in PRO2 or CD Al remission rates between each etrolizumab arm and placebo (under the assumption of placebo remission rate of ⁇ 15%, similar to results reported in the GEMINI 2 trial of vedolizumab in patients with CD; Sandborn WJ, et al., Aliment Pharmacol Ther 37:204-13, 2013) and approximately 80% power to detect a 15% difference versus placebo in endoscopic response (under the assumption of placebo response rate of ⁇ 10%) and a two-sided chi-square test at the 10% significance level.
  • the pivotal analysis for induction phase will be performed using only patients from Cohort 3.
  • patients will be randomized to receive either placebo, etrolizumab 105 mg or etrolizumab 210 mg in a 2:3:3 ratio
  • the co-primary endpoint will be tested using a Cochran-Mantel-Haenszel (CMH) test at the 5% significance level, where both clinical remission and endoscopy improvement are required to be significant.
  • CMH Cochran-Mantel-Haenszel
  • the sample size for Cohort 3 is expected to provide > 85% power to detect a > 15% difference in rates of clinical remission (SF mean daily score ⁇ 3 and AP mean daily score ⁇ 1) between each etrolizum ab arm and placebo under the assumption of a placebo remission rate of ⁇ 15% and a tw'o-sided test performed at a significance level of 5%. Additionally, Cohort 3 will provide > 80% power to detect a 10% difference in proportions for each etrolizumab arm versus placebo for endoscopic improvement, under the assumption of placebo response rate of ⁇ 5% and a two-sided test at the 5% significance level. The chi- square test was used to confirm the power calculations. [0253] Cohort 2 is sized at approximately 350 patients to achieve a sufficient number of patients for the Maintenance Phase analyses.
  • a total of approximately 480 patients achieving CDAI-70 response at Week 14 will be eligible to enroll into the Maintenance Phase.
  • a co-primary endpoint will be used for the analysis of the Maintenance Phase: clinical remission (SF mean daily score ⁇ 3 and AP mean daily score ⁇ 1) and endoscopic improvement at Week 66.
  • the maintenance co-primary analysis will be performed on all patients re-randomized into the Maintenance Phase who were randomized to etrolizumab (105 mg or 210 mg) in the Induction Phase.
  • the co-primary endpoint will be tested using a CMH test at the 5% significance level, where both clinical remission and endoscopy improvement are required to be significant. Note that to maintain the blind, patients randomized to placebo in the Induction Phase will undergo a sham randomization and will receive placebo in the Maintenance Phase. These patients wall not form part of the pivotal maintenance analysis.
  • Demographic and baseline characteristics such as age, sex, race, region, use of corticosteroids and immunosuppressants, anti-TNF therapy, duration of disease, extent of disease, average daily SF, average daily AP, CDAI, SES-CD, fecal calprotectin, and CRP will be summarized for all randomized patients by treatment arm with use of descriptive statistics for both the Induction and Maintenance Phases. Exposure to study drug (number of study treatments and duration of treatment) will be summarized by treatment arm.
  • the Induction and Maintenance Phases will be treated as independent studies. Patients who are non-evaluable for efficacy at a specific timepoint, due to missing data, will be considered non-responders for all categorical endpoints. In addition, patients requiring rescue therapy, and/or surgical intervention for CD, and/or took prohibited medications (e g., anti-integrins, T- or B- cell-depleting agents, TNF antagonists, antagonists of IL-23 ⁇ IL-12 (e.g., ustekinumab), anti-metabolites, cyclosporine, tacrolimus, immunosuppressant medications such as AZA (or equivalent), 6-MP, and MTX) will be considered non-responders for the analysis.
  • prohibited medications e g., anti-integrins, T- or B- cell-depleting agents, TNF antagonists, antagonists of IL-23 ⁇ IL-12 (e.g., ustekinumab), anti-metabolites, cyclosporine, tacrolimus,
  • subgroup analyses to evaluate the consistency of results across prespecified subgroups (including baseline anti -TNF -status [naive vs. JR], baseline CS status [on CS vs. not on CS], baseline IS status [on IS vs. not on IS], age, sex, fecal calprotectin, C-reactive protein, region) and (2) sensitivity analyses to evaluate the robustness of results to the primary analysis methods (e.g., handling of dropouts, adjusting for cohort).
  • Efficacy analyses for the Induction Phase will be performed separately for each cohort, and will include all patients who were randomized and received at least one dose of study (modified intent-to-treat population [mITT]). Patients will be grouped according to the treatment assigned at randomization.
  • mITT modified intent-to-treat population
  • Cohort 2 is considered a “feeder” cohort to help achieve the necessary’ sample size for the maintenance study. All primary and secondary efficacy parameters will be summarized descriptively for each treatment arm. Demographic and baseline characteristics such as age, sex, race, region, use of corticosteroids and immunosuppressants, duration of disease and CD activity scores will be summarized for each treatment group by use of descriptive statistics.
  • Cohort 3 The co-primary endpoint analysis wall compare for each etrolizumab dose arm versus the placebo arm the proportion of patients who achieve clinical remission or endoscopic improvement at Week 14.
  • All categorical secondary endpoints will be analyzed using the same methodology as the primary endpoint. For all efficacy endpoints, descriptive summary statistics will be provided for each treatment arm. [0266] Continuous endpoints will be analyzed using an analysis of covariance (ANCOVA) model with the stratification variables used at randomization and the baseline value of the studied measure as a covariate.
  • ANCOVA analysis of covariance
  • the co-primary endpoints will each be tested at the 5% significance level, with both required to be significant for the co-primary endpoints to be deemed significant.
  • the overall type I error rate will be maintained at 5% using a hierarchical model for testing of the two etrolizumab doses versus placebo and corresponding key secondary endpoints. The remaining secondary endpoints and all exploratory endpoints will be considered to provide supportive information and no adjustments for multiple comparisons will be performed.
  • the co-primary efficacy endpoints for the Induction Phase are: (1) the proportion of patients in clinical remission at Week 14 and (2) the proportion of patients achieving endoscopic improvement at Week 14. Maintenance Phase
  • Efficacy analyses for the Maintenance Phase will include all etrolizumab induction patients who were randomized into the Maintenance Phase and received at least one dose of study drug (mITT population). Patients will be grouped according to the treatment assigned at randomization into the Maintenance Phase.
  • the difference in proportions between the two treatment arms will be evaluated using the CMH test.
  • the CMH test will include important stratification factors such as those used to randomize patients into the Maintenance Phase.
  • the stratification factors used for re-randomization into the maintenance phase include sustained CD Al remission at Weeks 10 and 14.
  • This stratification factor in the analysis will be replaced with clinical remission at Week 14 for the clinical remission at Week 66 analysis, and endoscopic improvement at Week 14 for the endoscopic improvement at Week 66 analysis.
  • a sensitivity analysis will be conducted to include the sustained CDAI stratification factor used at randomization.
  • the test will be performed at the two-sided 5% significance level.
  • the co-primary efficacy endpoints for the Maintenance Phase are: among patients who achieve CDAI-70 response at Week 14, (1) the proportion of patients in clinical remission at Week 66 and (2) the proportion of patients achieving endoscopic improvement at Week 66
  • Table 5 Demographics and Baseline Treatments During Induction Phase. [0276] As shown in Table 6 below, baseline disease characteristics during the induction phase were mostly balanced across treatment arms. Distributional differences were noted for disease location and fecal calprotectin.

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Abstract

Methods of treating Crohn's disease are provided. Also provided are methods of administering and dosing integrin beta7 antagonists, such as anti-integrin beta7 antibodies. In addition, methods of administrating and dosing such integrin beta7 antagonists to maintain improvement of Crohn's disease, including clinical remission and/or endoscopic improvement, are provided.

Description

METHODS OF TREATING CROHN’S DISEASE USING INTEGRIN BETA7 ANTAGONISTS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of provisional application number 63/278,891 filed November 12, 2021, which is incorporated herein by reference in its entirety,
SEQUENCE LISTING
[0002] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on November 7, 2022, is named P37218-W0_SL.xml and is 12,529 bytes in size.
FIELD
[0003] Methods of treating Crohn’s disease are provided. Also provided are methods of administering and dosing integrin beta7 antagonists, such as anti-integrin beta7 antibodies. In addition, methods of administrating and dosing such integrin beta7 antagonists to maintain improvement of Crohn’s disease, including clinical remission and/or endoscopic improvement, are provided.
BACKGROUND
[0004] Inflammatory bowel disease (IBD) is a chronic inflammatory autoimmune condition of the gastrointestinal (GI) tract, which presents clinically as either ulcerative colitis (UC) or Crohn’s disease (CD), CD is a chronic transmural inflammatory disease with the potential to affect any part of the entire GI tract, and UC is a mucosal inflammation of the colon. Both conditions are characterized clinically by frequent bowel motions, malnutrition, and dehydration, with disruption in the activities of daily living. The etiology of IBD is complex, and many aspects of the pathogenesis remain unclear.
[0005] CD is a chronic, relapsing form of IBD that can affect any portion of the gastrointestinal tract, with 40%~50% of cases affecting the small bowel. CD is characterized by patchy, transmural inflammation, ulcers, and granulomatous lesions that are interspersed with healthy sections of bowel (skip lesions). The disease is progressive; uncontrolled inflammation develops into structuring or penetrating complications such as prestenotic dilatation, obstruction (stricturing), and intra-abdominal or perianal fistulae and abscesses (penetrating). Clinical signs and symptoms include chronic diarrhea, abdominal pain, cachexia, abdominal mass, or tenderness as well as the overt signs of fistulae. The disease course is variable; patients can experience a severe initial flare followed by few symptoms over the next 10 years (43%) or symptoms that are chronic and persistent (19%) or relapsing-remitting (32%) (Baumgart. DC, et al., Lancet 380: 1590-605, 2012).
[0006] The annual incidences of CD reported in Europe, Asia and the Middle East, and North America were 12.7, 5.0, and 20.2 per 100,000 person-years, respectively (Molodecky NA, et al., Gastroenterology 142:46-54, 2012). Current prevalence rates in North America are reported to be 319 per 100,000 persons (Id.). Disease-related mortality in CD accounts for approximately 30% of deaths in this population, resulting from clinical and/or surgical complications that occur early in the disease course or intestinal cancer occurring later. The global incidence of CD is expected to continue increasing substantially, affecting individuals in the most formative and productive years of life, with long-term costs to patients, healthcare systems, and society (Duricova D et al., Inflamm Bowel Dis 16:347-53, 2010).
[0007] To date, there is no cure for CD. Accordingly, the current treatment goals for CD are to induce and maintain symptom improvement, induce mucosal healing, avoid surgery, and improve quality of life (Lichtenstein GR, et al., Am J Gastroenterol 104:465-83, 2009;
Van Assche G, et al., J Grohns Colitis. 4:63-101, 2010).
[0008] Systemic corticosteroids (CSs) have been the mainstay treatment for inducing remission and are effective in approximately 80% of patients (Summers RW, et al., Gastroenterology 77:847-69, 1979; MalchowH, et al., Gastroenterology 86:249-66, 1984). However, they are less effective as a maintenance therapy, with only 28% of patients achieving a prolonged response after 1 year of treatment and 32% of patients becoming steroid dependent (Faubion WA, et al., Gastroenterology 121:255-60, 2001; Peyrin-Biroulet L, et al., Am J Gastroenterol 105:289-97, 2010). Even if patients’ symptoms improve, fewer than 30% are expected to achieve endoscopic improvement with steroid treatment (Modigliani R, et al., Gastroenterology 98:811-8, 1990). The adverse effects of steroids are well documented and 50% of patients will stop their treatment because of this; long-term safety outcomes include osteoporosis, cataracts, and diabetes.
[0009] Immunosuppressants (ISs) (e.g., azathioprine [AZA], 6-mercaptopurine [6-MP], or methotrexate [MTX]), are typically administered to induce remission in patients who are intolerant of or refractory to steroids and to maintain remission in patients who achieve quiescent CD. Immunosuppressants are given with or without a steroid bridge, depending on a patient’s symptoms during the 2-4-month onset of IS efficacy. In patients with ileal or ascending colonic disease, budesonide presents a less toxic, more tolerable bridge because of its low systemic bioavail ability resulting from a rapid first-pass metabolism. An earlier initiation of IS to alter the inflammatory disease course has been advocated over the past 20 years. However, a decrease in the rate of intestinal resections and complications has not been observed during this time (Cosnes J, et al., Gut 54:237-41, 2005). This may reflect poor adoption of this top-down treatment over concern for systemic toxicities including leukopenia, thromobocytopenia, and increased risk for lymphoma with AZA and 6-MP (Prefontaine E, et al., Cochrane Database Sy st Rev (4):CD000545, 2009) and hepatotoxicity and hair loss with MTX (Hausmann J, et al., Inflamm Bowel Dis 16: 1195-202, 2010).
[0010] The development of monoclonal antibodies (mAbs) against tumor necrosis factor (TNF)-oc has provided an additional treatment option. Although anti-TNFs are effective in a significant proportion of patients, efficacy is suboptimal; remission rates after 4 weeks of induction therapy are fewer than 35% and among patients who respond to induction therapy fewer than 50% achieve remission when assessed in maintenance at 20-30 weeks (Peyrin- Biroulet L, et al., Aliment Pharmacol Ther 33:870-9, 2011). Furthermore, 30% of patients are reported to be primary non-responders to anti-TNF therapy when assessed after 4 weeks of induction therapy (Targan SR, et al., N Engl J Med 337: 1029-35, 1997; Sandborn WJ, et al., Ann Intern Med 2007:19;146:829-38. Epub 2007 Apr 30), possibly because of an underlying pathobiology that is not TNF-a driven and as such may benefit from a different mechanistic class of drug. It is estimated that 30%-40% of patients will be secondary non- responders (i.e., initially responsive) but lose response or become intolerant in their first year of treatment (Colombel JF, et al., Gastroenterology 132:52-65, 2007). Secondary non-response has been attributed to the development of neutralizing antibodies, resulting in low drug serum levels, to accelerated drug clearance, or a biological escape mechanism that may benefit from a therapy with a different pharmacological target. Anti-TNFs are also associated with significant side effects, including serious infection, opportunistic infection, lupus-like reactions, and an increased risk of lymphoma (Siegal CA, et al., Therap Adv Gastroenterol 2:245-51, 2009). Tolerability concerns include infusion reactions (occurring in 9%-17% of patients treated with infliximab, see de Vries HS, et al., Br J Clin Pharmacol 71 :7-19, 2011) and injection site reactions (occurring in 10% of patients receiving adalimumab, see van der Heijde D, et al., Arthritis Rheum. 54:2136-46, 2006). Overall, the benefits versus risks are considered acceptable for this drug class, but there continues to be a need for treatments with better benefit-risk profiles that attenuate inflammation and the clinical sequelae and improve the long-term prognosis of patients with CD.
[0011] 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 0 transmembrane subunits that bind specifically to distinct cell adhesion molecules (CAMs) on endothelia, epithelia, and extracellular matrix proteins. In this manner, integrins can function as tissue-specific cell adhesion receptors aiding in the recrui tment 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)). In the immune system, 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. et al., Science, 1996, 272:60-66). The beta7 containing integrins (i.e., 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)
[0012] The anti-integrins are another class of biologies approved for the treatment of CD. Natalizumab is an anti-integrin approved in the U.S. only for the treatment of moderate to severely active CD The use of natalizumab, which blocks both α4β1 and α4β7 has been limited due to concerns that inhibition of a4pl/VCAM-l binding increases the risk of progressive multifocal leukoencephalopathy (PML) a rare but serious infection of the CNS. Vedolizumab is the most recently approved gut-selective anti-integrin for CD, but this targets only the «4p7 integrin receptor, inhibiting T-lymphocyte binding to the adhesion molecule MAdCAM-1, and is administered as an intravenous (IV) infusion. In the pivotal trials for vedolizumab 31% of patients had a clinical response with 6 weeks of induction treatment, defined as > 100-point decrease in the Crohn’s Disease Activity Index [CD Al] score from baseline; up to 39% of the vedolizumab responders achieved remission with 46 weeks of maintenance treatment (defined as CD Al score of ≤ 150), compared with 22% of patients given placebo (Sandborn WJ, et al., N Engl J Med 369(8):711-721, 2013; Sandbom WJ, et al., Aliment Pharmacol Ther 37:204-13, 2013). While vedolizumab shows promise as a new' treatment for CD, there remains a need for a more convenient therapy which is gut selective and achieves better response and remission rates.
[0013] The α4β7 integrin (the target of vedolizumab) 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. 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 (MAd CAM)- 1 -associated sialyl Lewis X. Chemokine signaling induces the α4β7 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., suprd). The numbers ofα4β7+ 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)). oc407 binds preferentially to high endothelial venules expressing MAdCAM-1 and vascular ceil adhesion molecule (VCAM)-l, as well as to the extracellular matrix molecule fibronectin fragment CS-1 (Chan et al., J Biol Chem 267:8366-70 (1992); Ruegg et al., J Cell Biol 17: 179-89 (1992); Berlin et al., Cell 74:185-95 (1993)). Together with constitutively expressed MAdCAM-1 in gut mucosal vessels, the α4β7 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 α4β1 interaction with VCAM-1 (Yednock et al., Nature 356:63- 6 (1992); Rice et al., Neurology 64: 1336-42 (2005)).
[0014] Another member of the 07 integrin family, expressed exclusively on
T lymphocytes and associated with mucosal tissues, is the αEβ7 integrin, otherwise known as CD 103. The αEβ7 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)). The α4β7+ 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)). The expression of αEβ7 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-αEβ7 antibody treatment has been reported to attenuate experimental colitis in mice, implicating a role for αEβ7+ lymphocytes in experimental models of IBD (Ludviksson et al., J Immunol 162:4975-82 (1999)).
[0015] Humanized monoclonal antibodies targeted against the p7 integrin subunit have been described previously See, e.g., Intn’l Patent Pub. No. W02006/026759. One such antibody, etrolizumab (rhuMAb Beta7) is derived from the rat anti-mouse/human monoclonal antibody FIB504 (Andrew DP, et al., J Immunol 153:3847-61, 1994). It was engineered to include human IgG l-heavy chain and K1— light chain frameworks. Intn’l Patent Pub. No. W02006/026759.
[0016] Etrolizumab, a subcutaneously administered mAb, is a novel anti-integrin which unlike vedolizumab, targets both the α4β7 and αEβ7 receptors that regulate trafficking, and retention of T-cell subsets in the intestinal mucosa, respectively. Thus, etrolizumab offers the potential of an additive therapeutic effect in CD via a dual mechanism of action (MOA), without generalized immunosuppression. Etrolizumab binds with high affinity to α4β7 (Holzmann B, et al., Cell 56:37-46, 1989; Hu M, et al.. Proc Natl Acad Sci USA 89:8254-8, 1992) and αEβ7 (Cepek KL, et al., J Immunol 150:3459-70, 1993). By this mechanism, it blocks the homing and retention of leukocyte subpopulations in the intestinal mucosa, which occur via binding with the cell adhesion molecules (MAdCAM-1) and E-cadherin, respectively. As such, it represents a novel gut mucosal-selective anti-trafficking agent whose selectivity may eliminate generalized immunosuppression by preferentially targeting trafficking to the gut rather than to other organs and tissues. Data from multiple, non-clinical, general toxicity studies of up to 6 months duration demonstrated that etrolizumab had no adverse effects in any organ system. See, e.g., Stefanich et al., British Journal of Pharmacology 162: 1855-1870, 2011; Intn'l Patent Pub No. WO 2009/140684.
[0017] It is important to note that unlike natalizumab, etrolizumab does not bind to α4β1 or inhibit the interaction of cx4pl and VCAM-1 and the distribution and homing of lymphocytes to the CNS and peripheral lymphoid tissue. As such, etrolizumab is not expected to increase the risk of progressive multifocal leukoencephalopathy (PML). Safety assessments for etrolizumab have been completed in adult Phase 1 and Phase 2 studies, in which patients with moderate to severely active UC received either single or multiple doses of IV or subcutaneous (SC) etrolizumab. A total of 158 patients have been exposed to etrolizumab with no significant adverse safety signals, including any evidence of increased rates of serious or opportunistic infections, being associated with etrolizumab treatment. Acknowledging that the clinical experience with etrolizumab is limited, no events of PML have been reported in patients treated with etrolizumab.
[0018] To date, the primary outcome measure in Crohn’s Disease clinical trials is the Crohn’s Disease Activity Index (CDAI), which has served as the basis for approval of multiple drug treatments, including for example, vedolizumab and natalizumab. The CDAI was developed by regressing clinician global assessment of disease activity on eighteen potential items representing patient reported outcomes (PROs) (i.e. abdominal pain, pain awakening patient from sleep, appetite), physical signs (i.e average daily temperature, abdominal mass), medication use (i.e. loperamide or opiate use for diarrhea) and a laboratory test (i.e. hematocrit). Backward stepwise regression analysis identified eight independent predictors which are the number of liquid or soft stools, severity of abdominal pain, general well-being, occurrence of extra-intestinal symptoms, need for antidiarrheal drugs, presence of an abdominal mass, hematocrit, and body weight. The final score is a composite of these eight items, adjusted using regression coefficients and standardization to construct an overall CDAI score, ranging from 0 to 600 with higher score indicating greater disease activity. Widely used benchmarks are: CDAI <150 is defined as clinical remission, 150 to 219 is defined as mildly active disease, 220 to 450 is defined as moderately active disease, and above 450 is defined as very severe disease (Best WR, et al., Gastroenterology 77:843-6, 1979). Vedolizumab and natalizumab have been approved on the basis of demonstrated clinical remission, i.e. CDAI ≤ 150.
[0019] Although the CDAI has been in use for over 40 years, and has served as the basis for drug approval, it has several limitations as an outcome measure for clinical trials. For example, most of the overall score comes from the patient diary card items (pain, number of liquid bowel movements, and general well-being), which are vaguely defined and not standardized terms (Sandler et al , J. Clin. Epidemiol 41 :451 -8, 1988; Thia et al., Inflamm Bowel Dis 17: 105-11 , 2011). In addition, measurement of pain is based on a four-point scale rather than an updated seven-point scale. The remaining 5 index items contribute very little to identifying an efficacy signal and may be a source of measurement noise. Furthermore, concerns have been raised about poor criterion validity for the CDAI, a reported lack of correlation between the CDAI and endoscopic measures of inflammation (which may render the CDAI as a poor discriminator of active CD and irritable bowel syndrome) and high reported placebo rates (Korzenik et al., N Engl J Med. 352:2193-201, 2005; Sandborn WJ, et al., N Engl J Med 353:1912-25, 2005; Sandbom WJ, et al., Ann Intern 19; 146: 829-38, 2007, Epub 2007 Apr 30; Kim et al., Gastroenterology 146: (5 supplement 1) S-368, 2014). [0020] It is, thus, generally recognized that additional or alternative measures of CD symptoms are needed. Indeed, registration requirements for new medications in Crohn’s disease for US Food and Drug Administration (FDA) and European Medicines .Agency (EMA) have been in flux recently and have focused on various patient reported outcomes (PROs) (see, e.g., Willet et al., Clin Gastroenterol Hepatol 2014, 12(8): 1246-1256. el683; Khanna et al Aliment Pharmacol Ther. 2015;41(l):77-86) in addition to objective assessment of inflammation as discussed further below.
[0021] An additional means of assessing the extent and severity of Crohn’s Disease is endoscopy Endoscopic lesions typical of Crohn’s disease have been described in numerous studies and include, e.g., aphthoid ulcerations, “punched-out ulcers,” cobblestoning and stenosis. Endoscopic evaluation of such lesions was used to develop the first validated endoscopic score, the Crohn’s Disease Endoscopic Index of Severity (CDEIS) (Mary et al.. Gut 39:983-9, 1989). More recently, because the CDEIS is time-consuming, complicated and impractical for routine use, a Simplied Endoscopic Activity Score for Crohn’s Disease (SES- CD) was developed and validated (Daperno et al., Gastrointest. Endosc. 60(4):505-12, 2004). The SES-C'D consists of four endoscopic variables (size of ulcers, proportion of surface covered by ulcers, proportion of surface with any other lesions (e.g., inflammation), and presence of narrowings [stenosis]) that are scored in five ileocolonic segments, with each variable, or assessment, rated from 0 to 3.
[0022] Notwithstanding these recent advances in the development and validation of new PRO measures and new endoscopy measures of disease extent and severity in CD, no published prospective clinical studies to date are available to determine how to employ such new measures in the design and conduct of a Phase III clinical study prospectively, i.e. a pivotal or registrational study, for the purpose of evaluating the efficacy of a particular dosing regimen of a therapeutic candidate, including for example, etrolizumab, in inducing remission and, importantly, maintaining remission and other signals of disease improvement of CD.
[0023] The invention described herein meets certain of the above-described needs and provides other benefits.
[0024] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety for any purpose. SUMMARY
[0025] The methods of the invention are based, at least in part, on the surprising and unexpected results of the maintenance phase of a clinical study of the safety and efficacy of etrolizumab treatment in patients with Crohn’s disease. These results showed that in the maintenance phase, the two co-primary endpoints of clinical remission and endoscopic improvement in etrolizumab-treated patients compared to placebo were met The results also showed that two secondary endpoints of corticosteroid-free remission and endoscopic remission in etrolizumab-treated patients compared to placebo were met.
[0026] Accordingly, in one aspect, methods of treating a patient with Crohn’s disease are provided. In certain embodiments, the method comprises administering subcutaneously to the patient an integrin beta7 antagonist for a treatment period of at least 52 weeks or of at least 66 weeks after initiation of an induction therapy, wherein 14 weeks after the initiation of the induction therapy the patient is determined to have acheived a decrease of 70 points or more from a baseline Crohn’s Disease Activity Index (CDAI) score, and wherein the patient maintains clinical remission during the treatment period.
[0027] In further embodiments, the patient maintains endoscopic improvement in addition to maintaining clinical remission. In still further embodiments, the patient further maintains endoscopic remission, corticosteroid-free clinical remission, or both endoscopic remission and corticosteroid-free clinical remission.
[0028] In yet further embodiments, the patient receives corticosteroid therapy in addition to the induction therapy during an induction period, wherein the induction period is 14 weeks, and wherein at the end of the induction period the corticosteroid therapy is reduced over time until discontinuation. In some embodiments, the corticosteroid therapy is (i) less than or equal to 20 mg of prednisone per day and wherein the corticosteroid therapy is reduced by 2.5 mg prednisone per week until discontinuation or (ii) less than or equal to 6 mg oral budesonide per day and wherein the corticosteroid therapy is reduced by 3 mg oral budesonide every 2 weeks until discontinuation.
[0029] In certain embodiments, the integrin beta7 antagonist is a humanized monoclonal anti-integrin beta7 antibody. In certain such embodiments, the anti-integrin beta7 antibody comprises three light chain hypervariable regions (HVRs), HVR-L1, HVR-L2, and HVR-L3, and three heavy chain HVRs, HVR-H1, HVR-H2. and HVR-H3, wherein:
(i) HVR-L1 comprises amino acid sequence RASESVDDLLH (SEQ ID NO: 1) ;
(ii) HVR-L2 comprises amino acid sequence KYASQSIS (SEQ ID NO:2);
(iii) HVR-L3 comprises amino acid sequence QQGNSLPNT (SEQ ID NO: 3); (iv) HVR-H1 comprises amino acid sequence GFFITNNYWG (SEQ ID NO:4);
(v) HVR-H2 comprises amino acid sequence GYISYSGSTSYNPSLKS (SEQ ID NO: 5; and
(vi) HVR-H3 comprises amino acid sequence RTGSSGYFDF (SEQ ID NO:6); or amino acid sequence ARTGSSGYFDF (SEQ ID NO:7).
[0030] In certain such embodiments, the anti-integrin beta7 antibody further comprises a light chain variable region domain comprising the amino acid sequence set forth in SEQ ID NO:8 and a heavy chain variable region domain comprising the amino acid sequence set forth in SEQ ID NO:9. In certain embodiments, the anti-integrin beta7 antibody comprises a light chain variable region domain comprising the amino acid sequence set forth in SEQ ID NO:8 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:11 or a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the anti-integrin beta7 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10 and a heavy chain variable region domain comprising the amino acid sequence set forth in SEQ ID NO:9.
[0031] In certain such embodiments, the anti-integrin beta7 antibody further comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11 or a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain such embodiments, the anti- integrin beta7 antibody is etrolizumab.
[0032] In another aspect, methods of treating a patient having moderately to severely active Crohn’s disease prior to administration of the induction therapy are provided. In some embodiments, the induction therapy comprises one or more therapeutic agents selected from a 5-aminosalicylate, an antibiotic, budesonide, a systemic corticosteroid, a thiopurine, methotrexate, an anti-TNF agent, infliximab, adalimumab, certolizumab pegol, vedolizumab, ustekinumab, natalizumab, efalizumab, etrolizumab, a Janus kinase (JAK) inhibitor, upadacitinib, and filgotinib.
[0033] In a further aspect, the patient is determined to have (I) a CDAI score of greater than or equal to 220 and less than or equal to 480 at any time in the seven days prior to initiation of the induction therapy and (2) either an average daily liquid/soft stool frequency (SF) score greater than or equal to 6 or an average daily SF greater than 3 and an average daily abdominal pain (AP) score greater than 1 for the seven consecutive days prior to initiation of the induction therapy. In some embodiments, the patient is determined to have active inflammation, wherein the active inflammation is determined as a SES-CD score of greater than or equal to 7 as determined by ileocolonoscopy. In some embodiments, the patient has isolated ileitis or post-ileocecal resection and wherein the patient is determined to have active inflammation, wherein the active inflammation is determined as a Simplified Endoscopic Index for Crohn’s Disease (SES-CD) score of greater than or equal to 4 as determined by ileocolonoscopy.
[0034] In another aspect, the patient is refractory or intolerant to one or more therapies selected from immunosuppressant therapy, corticosteroid therapy, and anti-TNF therapy. In some embodiments, the patient has an inadequate response to anti-TNF therapy. In some embodiments, the immunosuppressant therapy is selected from 6-mercaptopurine, azathioprine, and methotrexate. In some embodiments, the corticosteroid therapy is selected from prednisone and oral budesonide. In some embodiments, the anti-TNF therapy is selected from infliximab, adalimumab, and certolizumab pegol.
[0035] In yet another aspect, the anti-integrin beta7 antibody is administered at a flat dose of 105 mg every 4 weeks from week 14 after initiation of the induction therapy to at least week 52 or to at least week 66.
[0036] In still another aspect, clinical remission is determined as a liquid/soft stool frequency (SF) mean daily score of less than or equal to three and an abdominal pain (AP) mean daily score of less than or equal to one with no worsening in either SF or AP compared to baseline, averaged over at least four days prior to assessment. In some embodiments, the SF mean daily score and AP mean daily score are averaged over seven days prior to assessment.
[0037] In certain of the above embodiments, endoscopic improvement is determined by Simplified Endoscopic Index for Crohn’s Disease (SES-CD) score and the SES-CD score is reduced by at least fifty percent compared to the SES-CD score determined at baseline.
[0038] In certain of the above embodiments where the patient has discontinued corticosteroid treatment, the patient does not receive treatment with one or more corticosteroids for at least 24 continuous weeks after discontinuing corticosteroid treatment. [0039] In another aspect, the patient maintains endoscopic remission and endoscopic remission is determined by SES-CD score and the SES-CD score is less than or equal to four. In some embodiments, the patient is an ileal patient and the SES-CD score is less than or equal to two. In some embodiments, the SES-CD score contains no segment having a subcategory score that is greater than one. In some embodiments, the subcategoiy is selected from size and extent of ulceration, affected surface, and narrowing. [0040] In yet another aspect, methods of treating Crohn’s disease according to any of the embodiments comprise administering the integrin beta7 antagonist using a prefilled syringe or a prefilled syringe and autoinjector combination.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG, 1 shows the study schema for the Induction Phase of the Phase III clinical studj' as described in Example 1. Anti-TNF = anti-tumor necrosis factor; CD = Crohn’s Disease; ETRO = etrolizumab; IS = immunosuppressants; SC = subcutaneous; Wk = week. [0042] FIG. 2 shows the study schema for the Maintenance Phase of the Phase III clinical study as described in Example 1. CS = corticosteroids; ETRO = etrolizumab; OLE = open label extension phase; PBO = placebo; q4w = every 4 weeks; Re-Rx = re-randomized; Wk = week.
[0043] FIG. 3 shows the medical aid known as the Bristol Stool Scale as described in Example 1.
DETAILED DESCRIPTION
[0044] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N Y. 1992), provide one skilled in the art with a general guide to many of the terms used in the present application.
CERTAIN DEFINITIONS
[0045] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.
[0046] As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a protein” includes a plurality of proteins; reference to “a cell” includes mixtures of cells, and the like.
[0047] Ranges provided in the specification and appended claims include both end points and all points between the end points. Thus, for example, a range of 2.0 to 3.0 includes 2.0, 3.0, and all points between 2.0 and 3.0. [0048] "Treatment," “treating,” and grammatical variations thereof refer 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.
[0049] “Treatment regimen” refers to a combination of dosage, frequency of administration, or duration of treatment, with or without addition of a second medication [0050] “Effective treatment regimen” refers to a treatment regim en that will offer beneficial response to a patient receiving the treatment.
[0051] “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.
[0052] “Clinical remission” as used herein means a liquid/soft stool frequency (SF) mean daily score of less than or equal to three and an abdominal pain (AP) mean daily score of less than or equal to 1, with no worsening in either subscore compared to baseline, averaged over seven days. Assessment of SF and AP are described in Example 1.
[0053] “Corticosteroid-free clinical remission” or “corticosteroid-free remission” means maintaining clinical remission or maintaining remission without the use of concomitant corticosteroid treatment,
[0054] “Crohn’s Disease Activity Index” score or “CDAl” score means a composite of eight assessments consisting of the number of liquid or sfot stools, abdominal pain, general well-being, presence of complications, taking diphenoxylate/atropine (LOMOTIL®) or other opiates for diarrhea, presence of an abdominal mass, hematocrit, and percentage deviation from standard weight. Further details are described in Example 1 .
[0055] “Simple Endoscopic Score for Crohn’s Disease” or “SES-CD” means a composite of four assessments, each rated from zero to three consisting of size of ulcers, proportion of the surface covered by ulcers, proportion of the surface with any other lesions, and presence of narrowings, also referred to as stenosis. Further details are described in Example 1.
[0056] As used herein, “baseline,” means a clinical (e.g., signs or symptoms) or laboratory value describing a patient’s condition determined prior to administration of a certain therapeutic agent, e.g., an integrin beta7 antagonist such as etrolizumab. Examples of clinical or laboratory values for which baseline values can be determined include, but are not limited to, hematology and clinical chemi stry values, such as hemoglobin, hematocrit, platelet count, sodium, potassium, chloride and the like, CD Al score, SES-CD score, liquid/soft stool frequency (SF) or abdominal pain (AP) score, or other patient-reported outcome measures such as 1BDQ or CD-PRO/SS.
[0057] “Endoscopic improvement” means a greater than or equal to fifty percent reduction in baseline SES-CD score.
[0058] “Endoscopic remission” means SES-CD score that is less than or equal to four (less than or equal to two for ileal patients), with no segment having a subcategory score (e.g , for ulceration size and extent, affected surface, or narrowing) that is greater than one [0059] “CDAI-70 response” means a decrease from CD Al baseline score of at least seventy points.
[0060] “Induction therapy” as used herein means use of a therapeutic agent, or combination of therapeutic agents, in an initial regimen to treat signs and symptoms of Crohn’s disease. Induction therapy is given for a period of time typically ranging from about six weeks to about fourteen weeks. Induction therapy can refer to the initial regimen for a patient following an initial diagnosis of Crohn’s disease or a regimen following intolerance or inadequate response to a prior regime or the initial regimen in a clinical trial of a therapeutic candidate. Exemplary therapeutic agents that can be used in induction therapy in Crohn’s disease include for example, but not limited to, 5-aminosalicylates, antibiotics, budesonide, systemic corticosteroids, thiopurines, methotrexate, anti-TNF agents such as infliximab, adalimumab, and certolizumab pegol, and other biological agents, including but not limited to vedolizumab, ustekinumab, natalizumab, efalizumab, and etrolizumab, as well as Janus kinase (JAK) inhibitors including but not limited to upadacitinib and filgotinib.
[0061] “Disease worsening” as used herein, means during the Induction Phase of treatment of a patient with an anti-beta7 integrin antagonist such as etrolizumab, the CDAI score ten weeks after initiation of treatment is greater than the patient’s CDAI score at baseline or week zero.
[0062] “Clinical relapse” as used herein means during the Maintenance Phase of treatment of a patient with an anti-beta7 integrin antagonist such as etrolizumab, at least one of the following criteria is met on two consecutive visits with a health-care provider and at least one of the two consecutive CDAI scores is greater than or equal to 220: (1) the CDAI score is greater than or equal to the baseline or week zero score; (2) the CDAI score is greater than or equal to one hundred points higher than the score at fourteen weeks after treatment was initiated [0063] The term “sample,” as used herein, refers to a composition that is obtained or derived from a patient or 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. For example, the phrase “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.
[0064] “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 integrin 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. These effects can be modulated by any biologically relevant mechanism, including disruption of ligand binding to beta7 subunit or to the alpha4beta7 or alphaEbeta7 dimeric integrin, and/or by disrupting association between the alpha and beta integrin subunits such that formation of the dimeric integrin is inhibited. In one embodiment of the invention, the beta7 antagonist is an anti-beta7 integrin antibody (or anti-beta7 antibody). In one embodiment, the anti-beta7 integrin antibody is a humanized anti -beta7 integrin antibody and more particularly a recombinant humanized monoclonal anti -beta7 antibody (or rhuMAb beta7). In some embodiments, 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.
[0065] By "beta7 subunit" or "β7 subunit" is meant the human β7 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). The 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. 21:2591-2597; Gurish et al., (1992) 149: 1964- 1972; and Shaw, S. K. and Brenner, M. B. (1995) Semin. Immunol. 7:335). 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).
[0066] By "alphaE subunit" or "alphaE integrin subunit" or "αE subunit" or "αE 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)
[0067] "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 et al., (1996) J. Immunol. 156:2851-2857).
[0068] "VCAM-1" or "vascular cell adhesion molecule-1" "CD106" refers to a ligand of alpha4beta7 and alpha4betal, expressed on activated endothelium and important in endothelial-leukocyte interactions such as binding and transmigration of leukocytes during inflammation.
[0069] "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.
[0070] "Inflammatory Bowel Disease" or "IBD" is used interchangeably herein to refer to diseases of the bowel that cause inflammation and/or ulceration and includes without limitation Crohn's disease and ulcerative colitis.
[0071] "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. 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.
[0072] 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.
[0073] 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. In more severe cases, 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. Unfortunately, there can be a very' long delay (up to six months) before onset of action in some patients. 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.
[0074] An "effective dosage" refers to an amount effective, at dosages and for periods of time necessary', to achieve the desired therapeutic or prophylactic result.
[0075] As used herein, the term "patient" refers to any single subject for which treatment is desired. In certain embodiments, the patient herein is a human.
[0076] A “subject” herein is typically a human. In certain embodiments, 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. Typically, the subject is eligible for treatment, e.g., treatment of a gastrointestinal inflammatory disorder.
[0077] As used herein, “lifetime” of a subject refers to the remainder of the life of the subject after starting treatment. [0078] The terms “inadequate response,” “intolerance,” and “refractory response” refer to signs and/or symptoms of persistently active disease despite a history of treatment with one or more therapeutics, for example, corticosteroids, immunosuppressants and/or anti-TNFs. [0079] The term “pharmaceutical composition” or “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.
[0080] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, which is nontoxic to a subject, A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
[0081] The terms "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.
[0082] "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. In one embodiment, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen. In another embodiment, 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. In one embodiment, an antibody fragment is a monovalent antibody that has an in vivo half life substantially similar to an intact antibody. For example, such an antibody fragment may comprise on antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment,
[0083] 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.
[0084] 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 corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)).
[0085] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, 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. In some instances, framew'ork region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, 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. In general, 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. For further details, see Jones et al., Nature 321:522-525 (1986);
Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1 : 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994). [0086] 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 nonproteinaceous solutes. In certain embodiments, 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
[0087] The term "hypervariable region," "HVR," or "HV," when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six hypervariable regions; three in the VH (Hl, H2, H3), and three in the VL (L1, L2, L3). A number of hypervariable region delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Sendee, 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 H VRs are noted below.
Loop Kabat AbM Chothia Contact
LI L24-L34 L24-L34 L26-L32 L30-L36
L2 L50-L56 L50-L56 L50-L52 L46-L55
L3 L89-L97 L89-L97 L91-L96 L89-L96
Hl H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering)
Hl H31-H35 H26-H35 H26-H32 H30-H35 (Chothia Numbering)
H2 H50-H65 H50-H58 H53-H55 H47-H58
H3 H95-H102 H95-H102 H96-H101 H93-H101
[0088] 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 (Hl), 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. [00§9] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.
[0090] 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. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al. In one embodiment, for the VL, the subgroup is subgroup kappa I as in Kabat et al. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al.
[0091] An "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). In certain embodiments, 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 etal. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169: 147-155 (1996); Yelton et al. J. Immunol. 155: 1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al. J. Mol. Biol 226:889-896 (1992).
[0092] The phrase "substantially similar," or "substantially the same," as used herein, denotes a sufficiently high degree of similarity between two numeric values such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values).
[0093] The term “variable” 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 p-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the P-sheet structure. The hypervari able 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 etal, 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).
[0094] 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.
[0095] “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.
[0096] 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.
[0097] The “light chains” of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (A), based on the amino acid sequences of their constant domains.
[0098] Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. 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., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, s, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology 4th ed. (W. B. Saunders, Co., 2000). 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.
[0099] The terms “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. The terms particularly refer to an antibody with heavy chains that contain an Fc region.
[00100] The term “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. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, 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.
[00101] Unless indi cated otherwise, herein the numbering of the residues in an immunoglobulin heavy chain is that of the EU index as in Kabat etal., 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.
[00102] A “functional Fc region” possesses an “effector function” of a native sequence Fc region. Exemplary “effector functions” include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of ceil 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. [00103] 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.
[00104] 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. In certain embodiments, 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. In certain embodiments, 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.
[00105] An “amino acid sequence variant” antibody herein is an antibody with an amino acid sequence which differs from a main species antibody. In certain embodiments, 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. Examples of 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.
[00106] The term "immunosuppressive agent" or “immunosuppressant” as used herein for adjunct therapy 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 Ml IC antigens. Exemplary nonbiological therapeutic agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Patent No. 4,665,077); non-steroidal anti-inflammatory 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 niofetil (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; cyclosporine; 6 mercaptopurine; 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; streptokinase; streptodomase; FK506; RS- 61443; chlorambucil; deoxyspergualin; rapamycin. Exemplary biological agents include cytokine antibodies and cytokine receptor antibodies, e.g., antagonists including anti- interferon-alpha, -beta, or -gamma antibodies, anti-tumor necrosis factor(TNF)-alpha antibodies (infliximab [e g., REMICADE); adalimumab [e.g., HUMIRA®]), 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-CD18 antibodies; anti- L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies, anti-CD3 or anti- CD4/CD4a antibodies; anti -idiotypic antibodies for MHC antigens and MHC fragments; soluble peptide containing a LFA-3 binding domain (WO 90/08187 published Jul. 26, 1990); transforming growth factor-beta (TGF-beta); RNA or DNA from the host; 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; laneway, Nature, 341: 482 (1989); and WO 91/01133); BAFF antagonists such as BAFF or BR3 antibodies or immunoadhesins); biologic agents that interfere with T cell helper signals, such as anti-CD40 receptor or anti-CD40 ligand (CD 154), including blocking antibodies to CD40-CD40 ligand. (e.g., Durie etal,. Science, 261 : 1328-30 (1993); Mohan etal., J. Immunol., 154: 1470-80 (1995)) and CTLA4-Ig (Finck et al., Science, 265: 1225-7 (1994)); and T-cell receptor antibodies (EP 340,109) such as T10B9. [00107] As used herein, “corticosteroid-free” means that a patient, e.g. a patient with Crohn’s disease, did not use corticosteroids to treat the disease or symptoms of the disease during the time which the patient is corticosteroid-free. For example, a patient with Crohn’s disease who is corticosteroid-free for 12 months did not use corticosteroids for 12 months to treat symptoms of Crohn’s disease.
[00108] The term “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.
[00109] A “symptom” of a disease or disorder (e.g., inflammatory bowel disease, e.g., ulcerative colitis or Crohn’s disease) is any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by a subject and indicative of disease. [00110] The expression “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). For example, 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. Similarly, a “therapeutically effective amount” of a combination of an antibody and a second compound refers to an amount of the antibody and an amount of the second compound that, in combination, is effective for preventing, ameliorating, or treating the specified disease or disorder.
[00111] It is to be understood that the terminology “a combination of’ two compounds does not mean that the compounds have to be administered in admixture with each other. Thus, 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 sam e day or different days. Thus the terminology “combination” means two or more compounds are used for the treatment, either individually or in admixture with each other. When 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. In certain embodiments, a compound other than the antibody is administered prior to the antibody. In certain embodiments, a compound other than the antibody is administered after the antibody.
[00112] For the purposes herein, “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).
[00113] A “TNF-alpha inhibitor” herein is an agent that inhibits, to some extent, a biological function of TNF-alpha, generally through binding to TNF-alpha and neutralizing its activity. Examples of TNF inhibitors specifically contemplated herein are etanercept (ENBREL®), infliximab (REMICADE®), adalimumab (HUMIRA®), golimumab (SIMPONI®), and certolizumab pegol (CIMZIA®).
[00114] “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 Examples of synthetic corticosteroids include prednisone, prednisolone (including methylprednisolone), dexamethasone triamcinolone, budesonide, and betamethasone.
[00115] An “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, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmacological agents and their metabolites, transcriptional and translation control sequences, and the like. 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.
[00116] 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.
[00117] A variety of additional terms are defined or otherwise characterized herein.
COMPOSITIONS AND METHODS
A. Anti-integrin Beta7 Antibodies
[0118] In one embodiment, the beta7 integrin antagonists are anti-integrin beta7 antibodies.
I . Monoclonal Antibodies
[0119] Monoclonal antibodies may be made using the hybridoma method first described by Kohler etal., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). [0120] In the hybridoma method, 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. .Alternatively, 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)).
[0121] 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). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (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.
[0122] In certain embodiments, 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. In certain embodiments, 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. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
[0123] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. In certain embodiments, 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).
[0124] The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson el 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. In addition, 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 chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.
[0125] 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. Once isolated, 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. 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 Pluckthun, Immunol. Revs. 130:151-188 (1992).
[0126] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using e.g., the techniques described in McCafferty etal., Nature, 348:552-554 (1990). Clackson etal., 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. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks etal., Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.
[0127] 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). The 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.
[0128] Exemplary anti-integrin beta7 antibodies are Fib504, Fib 21, 22, 27, 30 (Tidswell, M. J Immunol. 1997 Aug 1 ; 159(3): 1497-505) or humanized derivatives thereof. Humanized antibodies of Fib504 was disclosed in detail in U.S. Patent Publication No. 20060093601 (issued as U S Patent No 7,528,236), the content of which is incorporated by reference in its entirety (also see discussion below).
2. Humanized Antibodies
[0129] The anti-integrin beta7 antibodies of the invention may further comprise humanized 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. In some instances, Fv framework residues of the human immunoglobulin 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. In general, 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)].
[0130] Methods for humanizing non-human antibodies are well known in the art. Generally, 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 etal., Nature, 321 :522-525 (1986); Riechmann etal., Nature, 332:323-327 (1988); Verhoeyen etal., 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. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, 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. According to the so-called "best- fit" method, 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); Chothia 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 etal, Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)). It is further important that antibodies be humanized with retention of high binding affinity for the antigen and other favorable biological properties. To achieve this goal, according to certain embodiments, 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. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially- involved in influencing antigen binding.
[0131] Exemplary humanized anti-integrin beta7 antibodies include, but are not limited to rhuMAb Beta7, also referred to as etrolizumab, which is a humanized monoclonal antibody against the integrin subunit β7 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 κI light chain frameworks and is produced by Chinese hamster ovary cells. This antibody binds to two integrins, α4β7 (Holzmann et al. 1989 Cell, 1989;56:37-46; Hu et al., 1992, Proc Natl Acad Sei USA 1992;89:8254-8) and αEβ7 (Cepek et al., 1993 J Immunol 1993;150:3459-70), which regulate trafficking and retention of lymphocyte subsets in the gastrointestinal tract and are involved in inflammatory' bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn’s disease (CD). rhuMAb Beta7 (also referred to as etrolizumab) is a potent in vitro blocker of the cellular interaction between α4β7 and its ligands (mucosal addressin cell adhesion molecule-1 [MAdCAM]-!, vascular cell adhesion molecule [VCAM]-1, and fibronectin) as well as the interaction between αEβ? and its ligand (E-cadherin). rhuMAb Beta7 (also referred to as etrolizumab) binds reversibly, with similar high affinity, to p7 on lymphocytes from rabbits, cynomolgus monkeys, and humans. It also binds to mouse P7 with high affinity. The amino acid sequence as well as the making and using of rhuMAb Beta7 (also referred to as etrolizumab) and its variants are disclosed in detail in e.g., U.S. Patent Application Publication No. 20060093601 (issued as U.S. Patent No. 7,528,236), the content of which is incorporated in its entirety.
[0132] The Table of Sequences in this document provides etrolizumab sequence information. As indicated in the Table of Sequences, the heavy chain may lack a C-terminal lysine (HC.vl, SEQ ID NO:11)) or may include a C-terminal lysine (HC.v2, SEQ ID NO: 12). As is well known in the art, C-terminal lysine residues can be clipped during manufacturing processes.
3. Vectors, Host Cells and Recombinant Methods for Antibody Production
[0133] Also provided are isolated nucleic acids encoding the anti-beta7 antibodies or polypeptide agents described herein, vectors and host cells comprising the nucleic acids and recombinant techniques for the production of the antibodies
[0134] For recombinant production of the antibody, the nucleic acid encoding it may be isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. In another embodiment, 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.
[0135] 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. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. One E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coll W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
[0136] In addition to prokaryotes, 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. However, 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. fragili s (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); 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.
[0137] Suitable host cells for the expression of glycosylated anti-Beta7 antibody are derived from multicellular organisms. Examples of 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 califomica 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.
[0138] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 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 etal.. Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243- 251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VE RO-76, ATCC CRL- 1587); human cervical carcinoma cells (ITELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
[0139] 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.
[0140] 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 Fl 0 (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. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 1 02:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762;
4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. 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 GENTAMYCIN.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 wall be apparent to the ordinarily skilled artisan.
[0141] When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, 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 commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
[0142] 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. 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. Where the antibody comprises a C.sub.H3 domain, the Bakerbond ABX.TM. resin (J. T. Baker, Phillipsburg, N.J ) is useful for purification. 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. Following any preliminary purification step(s), 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).
4. Certain Therapeutic Agents
[0143] A therapeutic agent for the treatment of Crohn’ s Disease is provided herein. In one embodiment, the therapeutic agent is an anti-integrin beta7 antibody, also referred to as etrolizumab. Etrolizumab as an IgGl antibody. In one embodiment, the anti-integrin beta7 antibody comprises three heavy chain HVRs, HVR-H1 (SEQ ID NO:4), HVR-H2 (SEQ ID NO:5), and HVR-H3 (selected from SEQ ID NO:6 and SEQ ID NO:7). In one embodiment, the anti-integrin beta7 antibody comprises three light chain HVRs, HVR-L1 (SEQ ID NO:1), HVR-L2 (SEQ ID NO:2), and HVR-L3 (SEQ ID NO:3). In one embodiment, the anti- integrin beta7 antibody comprises three heavy chain HVRs and three light chain HVRs, HVR-H1 (SEQ ID NO:4), HVR-H2 (SEQ ID NO: 5), and HVR-H3 (selected from SEQ ID NO:6 and SEQ ID NO :7), HVR-L1 (SEQ ID NO:1), HVR-L2 (SEQ ID NO:2), and HVR-L3 (SEQ ID NO:3). In one embodiment, the anti-integrin beta7 antibody comprises a variable heavy chain region, VH, having an amino acid sequence of SEQ ID NOV. In one embodiment, the anti-integrin beta7 antibody comprises a variable light chain region, VL, having the amino acid sequence of SEQ ID NO:8. In one embodiment, the anti-integrin beta7 antibody comprises a variable heavy chain region, VH, having an amino acid sequence of SEQ ID NOV and a variable light chain region, VL, having an amino acid sequence of SEQ ID NO:8. In one embodiment, the anti-integrin beta7 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12. In one embodiment, the anti-integrin beta7 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 10. In one embodiment, the anti-integrin beta7 antibody comprises a heavy chain having an amino acid sequence selected from SEQ ID NO:11 and SEQ ID NO: 12 and a light chain having the amino acid sequence of SEQ ID NO: 10. Anti-integrin beta7 antibodies are further described in IntnT Pub. No. 2006/026759. [0144] In another aspect, an anti-integrin beta7 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-integrin beta7 antibody comprising that sequence retains the ability to bind to human integrin beta7. In certain embodiments, a total of 1 to 10 amino acids have been substituted, altered inserted and/or deleted in SEQ ID NO: 11. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-integrin beta7 antibody comprises the VH sequence in SEQ ID NO: 11 or SEQ) ID NO: 12, including post- translational modifications of that sequence.
[0145] In another aspect, an anti-integrin beta7 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-integrin beta7 antibody comprising that sequence retains the ability to bind to human integrin beta7. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 10. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-integrin beta7 antibody comprises the VL sequence in SEQ ID NO: 10, including post-translational modifications of that sequence.
[0146] In yet another embodiment, the anti-integrin beta7 antibody comprises a VL region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10 and a VH region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11.
B . Pharmaceutical Formulations
[0147] 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, histidine, arginine, or lysine; monosaccharides, di saccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0148] 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.
[0149] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
C. Administration
[0150] Administration of the anti-integrin beta7 antibody 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. For example, the anti-integrin beta7 antibody may be administered first, followed by the second therapeutic compound. However, simultaneous administration or administration of the second therapeutic compound prior to the anti-integrin beta7 antibody is also contemplated.
[0151] The standard of care for subjects with moderately to severely active CD involves therapy with standard doses of: systemic corticosteroids, e.g., prednisone (or prednisone equivalent) or budesonide, immunosuppressants such as azathioprine, 6-mercaptopurine, or methotrexate, or tumor necrosis factor inhibitors (anti-TNFs), such as infliximab, adalimumab, or certolizumab pegol. Other anti-integrin therapies have been approved for treatment of CD and these are natalizumab and vedolizumab.
[0152] In one embodiment, the treatment of the present invention for Crohn’s disease (CD) in a human subject with CD comprises administering to the subject an effective amount of a therapeutic agent, such as an anti -beta7 integrin antibody, and further comprising administering to the subject an effective amount of a second medicament, that is an immunosuppressant, a corticosteroid, an anti-TNF, a pain-control agent, an antidiarrheal agent, an antibiotic, or a combination thereof.
[0153] In an exemplary embodiment, said secondary medicine is selected from the group consisting of 6-mercaptopurine, azathioprine, methotrexate, prednisone (or prednisone equivalent), budesonide, infliximab, adalimumab, and certlizumab pegol.
[0154] All these 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. Thus, the second medicament need not be one medicament, but may constitute or comprise more than one such drug.
[0155] 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.
[0156] The combined administration of a second medicament includes co-administration (concurrent 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 (medicaments) simultaneously exert their biological activities.
E. Design Treatment Regimens
[0157] Drug development is a complex and expensive process. The cost of bringing a new drug to market is estimated to be between $800 million and $1 billion. Less than 10% of drugs in phase I clinical trials make it to the approval phase. Two key reasons why drugs fail at late stages are a lack of understanding of the relationship between dose-concentration response and unanticipated safety events. Given this scenario, it is important to have enabling tools that help predict how a drug will perform in vivo and assist in the success of a clinical therapeutic candidate (Lakshmi Kamath, Drug Discovery and Development;
Modeling Success in PK/PD Testing Drug Discovery & Development (2006)).
[0158] Pharmacokinetics (PK) characterizes the absorption, distribution, metabolism, and elimination properties of a drug. Pharmacodynamics (PD) defines the physiological and biological response to the administered drug. 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 & Development (2006)),
[0159] 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. (Lakshmi Kamath, supra).
[0160] PK/PD modeling approaches are proving useful in determining relationships between biomarker responses, 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 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').
[0161] The amount of biomarkers in the peripheral blood 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.
[0162] Traditional 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. When used more broadly, quantitative techniques such as drug modeling, di sease 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. A variety of PK/PD modeling tools are available to drug development researchers, for example, WinNonlin and the Knowledgebase Server (PKS) developed by Pharsight, Inc. Mountain View, California.
[0163] The foregoing written specification and following examples are considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and following examples and fall within the scope of the appended claims.
[0164] It is understood that the application of the teachings of the present invention to a specific problem or situation will be within the capabilities of one having ordinary' skill in the art in light of the teachings contained herein.
[0165] Further details of the invention are illustrated by the following non-limiting
Examples. The disclosures of all citations in the specification are expressly incorporated herein by reference.
EXAMPLES
Example 1
A PHASE III, RANDOMIZED. DOUBLE -BLIND. PLACEBO-CONTROLLED, MULTICENTER STUDY TO EVALUATE THE EFFICACY AND SAFETY OF ETROLIZUMAB AS AN INDUCTION AND MAINTENANCE TREATMENT FOR PATIENTS WITH MODERATELY TO SEVERELY ACTIVE CROHN’S DISEASE
Description of the Study
Study Rationale
[0166] The purpose of this study is to assess the efficacy and safety of etrolizumab, an anti-integrin with a unique mechanism of action (MO A) that has been shown to inhibit the trafficking and retention of inflammatory T-cells in the intestinal mucosa, via a disruption of α4β7/MAdCAM-l, and αEβ7/E-cadherin binding.
[0167] Although etrolizumab has not been studied in humans with CD as a maintenance therapy, preliminary expression studies of the pharmacological target for etrolizumab, the integrin P7 receptor, on gut CD4+ and CD8+ T cells isolated from resections of patients with UC and patients with CD, suggests that expression levels are similar between both diseases. The reported efficacy of vedolizumab, an anti-a4p7 mAb, in CD demonstrates a role for α4β7 in the pathobiology of this disease (Sandborn WJ, et al., Aliment Pharmacol Ther 37:204— 13, 2013) and it follows from such studies that etrolizumab will be efficacious in CD. Indeed, the results of an induction study (cohort 1 described below) demonstrated that etrolizumab treatment demonstrated clinically meaningful endoscopic improvement compared with placbo 14 weeks after treatment was initiated and symptomatic remission associated with etrolizumab was seen as early as week 6 and was observed consistently through week 14 (Sandborn et al., United Eur Gastroenterol J. 2017,5(8): 1138-50). In addition, because ctEp7 + expression is reportedly elevated in patients with CD (Elewaut D, et al., Acta Gastroenterol Belg 61:288-94, 1998; Oshitani N, et al., Int J Mol Med 12:715-9, 2003) with an observed increase in expression from distal to proximal bowel, the dual MOA of etrolizumab may bring enhanced efficacy in CD without generalized immunosuppression, compared with available anti-integrin and anti-TNF therapies.
[0168] In a global Phase II study in UC patients, etrolizumab was efficacious in treating moderate to severe UC and achieved a placebo-corrected clinical remission rate of 20.5% (p = 0.058) and an endoscopic remission rate of 10.3% (p = 0.004) at 10 weeks after treatment initiation (105 mg (100 mg nominal dose) every 4 weeks [Q4W]) in an all comers population (Vermeire S, et al., Gastroenterology 144,Sl:S-36, 2013; Vermeire S, et al.. Lancet 384: 309- 18, 2014; Lin et al., Gastroenterology 146:307-315, 2014). In addition, etrolizumab had an acceptable safety profile with no clinically significant safety' signals observed.
[0169] Although the above-described Phase II results in UC were encouraging, topline results from five Phase III studies in UC were mixed and disappointing (Roche press release dated .August 10, 2020 available at www(dot)roche(dot)com). Of note, etrolizumab met its primary' endpoint of inducing remission versus placebo in only two of three induction studies (Id). Moreover, etrolizumab failed to meet its primary endpoint (remission) versus placebo as maintenance therapy in people with ulcerative colitis (Id). Accordingly, the efficacy of etrolizumab therapy in inducing remission in UC in some studies failed to predict the efficacy of etrolizumab as a maintenance therapy in UC.
[0170] Likewise, the previously-reported encouraging results in a CD study comparing etrolizumab efficacy in inducing remission versus placebo, which were discussed above (Sandborn et al.. United Eur Gastroenterol J. 2017;5(8): 1138-50), are not predictive of the efficacy of etrolizumab treatment in maintaining remission, including maintaining remission for at least one year. Accordingly, the study design and endpoints discussed below along with the analysis plan for the clinical results are designed to inform efficacy of etrolizumab treatment as both induction and maintenance therapy.
Study Design [0171] This will be a multicenter, Phase III, double-blind, placebo-controlled study evaluating the efficacy, safety, and tolerability of etrolizumab compared with placebo during induction and maintenance treatment of moderate to severely active CD. As recommended by the European Medicines Agency (EMA) new medicinal products guideline for CD (CPMP/EWP/2284/99 Rev. 1) and as employed in U.S. Food and Drug Administration (FDA)-approved registration trials of CD therapies, this study combines induction and maintenance therapy in a re-randomization/withdrawal design to assess the efficacy and safety of etrolizumab treatment for induction and maintenance of remission.
[0172] The study design will comprise 1) a Screening Phase (up to 35 days) to determine patients’ eligibility for the study, 2) an Induction Phase (14 weeks), followed by 3) a Maintenance Phase (52 weeks) in patients demonstrating a CDAI-70 response (defined as a decrease from CD Al baseline score of at least 70 points) at the end of the Induction Phase, and 4) a Safety Follow-Up Phase (12 weeks) after administration of the last dose of study drug in the Maintenance Phase for those patients who are not participating in Part 1 of open- label extension of the Study to receive etrolizumab treatment (see Fig. 1 and Fig. 2). At the completion of the Safety Follow-Up Phase, patients will be asked to enter an extended PML- monitoring phase (open-label extension study) for 92 weeks. An independent Data Monitoring Committee (iDMC) will monitor safety and study conduct on an ongoing basis. [0173] Patients will have moderately to severely active CD as defined in the Screening Phase by clinical signs and symptoms that result in a CD Al score between >220 and ≤480, calculated on the day of randomization, requiring a minimum of 4 days of e-diary PRO data from the 7 days prior to randomization, as well as by either (a) an average stool frequency (SF) > 6 or (b) an average SF > 3 and average abdominal pain (AP) >1, calculated on the day of randomization using electronic diary (e-diary) patient reported outcome (PRO) data from the 7 days prior to randomization. Also required is the presence of active inflammation, defined as a SES-CD score of > 7, or >4 in cases of isolated ileitis or post-ileocecal resection, as determined by a screening ileocolonoscopy scored using the central read model.
[0174] The study population will consist of patients who are refractory or intolerant to one or more of the following therapies: 1) corticosteroid (CS), 2) immunosuppressant (IS), or 3) anti- tumor necrosis factor (TNF) (or inadequate response to anti -tumor necrosis factor [TNF-IR]). Patients who enroll on the basis of refractory or intolerance to CS and/or IS may have been previously exposed to ant-TNFs or be naive to anti-TNFs. [0175] Approximately 1150 patients will be randomized into the study from approximately 420 global investigational sites via enrollment into one of three cohorts. The enrollment will be sequential, first into Cohort 1, then Cohort 2, and lastly Cohort 3.
Screening Phase
[0176] Patients will be evaluated for eligibility in the 35-day Screening Phase (see Fig. 1 and Fig. 2). Major eligibility criteria are noted below.
[0177] During the Screening Phase, patients taking CS therapy must have been on a stable dose of ≤20 mg/day prednisone (or equivalent) or ≤6 mg/day oral budesonide for at least 2 weeks immediately prior to their randomization. Similarly, eligible patients requiring background IS therapy (e.g., azathioprine [AZA] or equivalent, 6-mercaptopurine [6-MP] or equivalent, or methotrexate [MTX]) must be receiving a stable IS dose regimen for at least 8 weeks immediately prior to their randomization. Patients who have been treated with anti- TNF therapy must have discontinued this treatment for at least 8 weeks prior to their randomization.
[0178] The ileocolonoscopy should be performed during the Screening Phase and at least 9 days prior to randomization to allow sufficient time for a central reader scoring and determination of eligibility, and to avoid the ileocolonoscopy bowel preparation influencing the patient reported outcomes used in the determination of baseline SF, AP, and CD Al scores (i.e., abdominal pain, general well-being, and stool frequency).
Induction Phase
[0179] Eligible patients will be enrolled sequentially into one of three cohorts for the 14- week Induction Phase (see Fig. 1).
[0180] Patients enrolled in Cohort 1 (double-blind, placebo-controlled, exploratory cohort; n=300) will be randomized in a 1 :2:2 ratio to receive placebo, etrolizumab 105 mg SC Q4W (low dose), or etrolizumab 210 mg SC (high dose) at Weeks 0, 2, 4, 8, and 12 within a 14-week Induction Phase (note that patients randomized to low-dose etrolizumab will receive a placebo injection at Week 2- see below). Patients enrolled in Cohort 2 (etrolizumab dose-blind, active-treatment cohort; n=350) will be randomized in a 1:1 ratio to receive low-dose or high-dose regimens of etrolizumab. Patients enrolled in Cohort 3 (double-blind, placebo-controlled, pivotal cohort; n=500) will be randomized in a 2:3:3 ratio to receive placebo or etrolizumab low-dose or high-dose. Because the low dose and high dose of etrolizumab are in syringes of different volumes, in order to preserve the blind, patients in all three cohorts will receive two injections at Weeks 0, 4, 8, and 12. Patients randomized to low-dose etrolizumab will receive one placebo (matching high-dose pre-filled syringe) and one low-dose etrolizumab injection at each administration except at Week 2, when they will receive one placebo injection. Patients randomized to high-dose etrolizumab will receive one placebo and one high-dose etrolizumab injection at each administration, except at Week 2 where they will receive one high-dose injection. Finally, patients randomized to placebo will receive two placebo injections at every administration, except at Week 2 where they will receive one placebo injection.
[0181] The randomization in all cohorts will be stratified by concomitant oral CS treatment (yes vs. no), concomitant IS treatment (yes vs. no), baseline CD Al ≤330 (yes vs. no), and prior anti-TNF exposure (yes vs. no). The enrollment will be managed to ensure that the proportion of the TNF-exposed patients in Cohort 3 does not exceed approximately 60% and that the proportion of patients with a CDAI score between > 450 and ≤480 does not exceed approximately 10% in each cohort.
[0182] During the Induction Phase, patients in all cohorts must keep their dose(s) of CS and IS therapy stable (if requiring CS/IS at baseline). Increase in dose of these medications will be considered rescue therapy. Also, even' attempt should be made to keep anti -diarrheal medication at a fixed dose, if required. The impact of titrating anti -diarrheal medication on the placebo response rate for SF and AP has not been studied in a moderate to severely active CD population; dose adjustments may confound the data interpretation. Changes to any concomitant medications must be documented in the electronic Case Report Form (eCRF). [0183] Between and including Weeks 10 and 14, there is an optional escape to an Open Label Extension (OLE) study (Part 1) where patients can receive open label etrolizumab.
This can only be exercised if a patient experiences disease worsening, defined as CDAI Week 10 score greater than the patient’s baseline (Week 0) score.
[0184] At week 14, patients achieving CDAI-70 response without the use of rescue therapy will continue to the Maintenance Phase until a sample size of approximately 480 patients enrolled in the Maintenance Phase has been acheived. Patients not eligible for the Maintenance Phase may be eligible for the Part 1 open label extension (OLE) study. Patients who are in the Induction Phase after closure of enrollment into the Maintenance Phase may enroll in Part 1 (OLE) Study, if eligible, upon completion of the Induction Phase at Week 14 or in the event of disease worsening, as specified above, between Weeks 10 and 14.
[0185] Patients who require surgical intervention for CD at any time during the Induction Phase will stop study treatment, enter the Safety Follow-Up Phase, and will be asked to enter Part 2 Safety Monitoring (SM) Study for PML monitoring. Patients who self-withdraw from the Induction Phase and do not meet the eligibility criteria for OLE treatment will also enter the Safety Follow-Up Phase and be asked to enter Part 2 (SM) Study for PML monitoring. Concomitant CD Therapy in the Induction Phase [0186] Concomitant therapy includes any medication (e.g., prescription drugs, over-the- counter drugs, herbal or homeopathic remedies, nutritional supplements) used by a patient from 4 weeks prior to screening to the study completion/early termination visit. Anti- diarrheal medications are permitted if used to manage chronic diarrhea but every attempt should be made to keep the dose/regimen stable. Any changes in the dose/regimen after baseline (Week 0) must be captured by the investigator and/or patient. Occasional use of nonsteroidal anti-inflammatory drugs (NSAIDs) or acetaminophen is permitted for pain relief (e.g., in the case of headache, arthritis, myalgia, etc.). Prophylactic aspirin use up to 325 mg/day is also permitted. Patients should not be treated for continuing signs and symptoms of CD with any medication that is prohibited to be taken concomitantly with etrolizumab; these include but are not limited to: anti-integrins, anti-adhesion molecules (e.g., anti-MAdCAM- 1), T- or B- cell-depleting agents with the exception of AZA and 6-MP (or equivalent), TNF antagonists, antagonists of IL -23 ± IL-12 (e.g., ustekinumab), anti-metabolites, cyclosporine, and tacrolimus.
[0187] CS enemas/suppositories and/or topical (rectal) 5 -aminosalicylate (5-ASA) preparations are also prohibited medications. Patients taking these medications will be considered nonresponders for the purpose of any endoscopic analyses. These treatments must be discontinued, but patients may continue to receive etrolizumab and may still be eligible for Part 1 (OLE) Study.
Rescue Therapy in the Induction Phase
[0188] This is defined as medication prescribed for new or worsening CD symptoms, and includes any new CS or IS therapy for CD or any increase in dose or regimen of baseline Crohn’s medications. Antibiotics, 5-ASAs, anti-diarrheals, probiotics, herbal/ayurvedic, nutritional and homeopathic supplements are not considered as rescue therapies. Patients requiring rescue medication during the Induction Phase will be considered non-responders for the primary analysis and will not be eligible for the Maintenance Phase.
Maintenance Phase
[0189] At the end of the Induction Phase (Week 14), patients will be assessed for a CDAI score and will undergo a full endoscopy (ileocolonscopy) with central reading to determine a SES-CD score. Every attempt must be made to schedule the ileocolonoscopy to take place at the Week 14 visit or, no later than 5 calendar days after this visit; the procedure must not be scheduled before Week 14. The patient reported outcomes e-diary data (i.e., abdominal pain, general well-being, and stool frequency) that are captured in the 7 days prior to the bowel preparation will be used to calculate the Week 14 SF, AP, and CDAI scores, thus removing any influence of the bowel preparation on these outcomes.
[0190] Patients who received placebo during the Induction Phase and achieved a CDAI- 70 response will undergo a sham randomization to blinded placebo treatment during the Maintenance Phase. Patients who received etrolizumab and achieved a CDAI-70 response at Week 14 without the use of rescue therapy, will be randomized into the Maintenance Phase in a 1:1 ratio to treatment with placebo or etrolizumab 105 mg SC Q4W (see Fig. 2). This will continue until a sample size of approximately 480 patients enrolled in the Maintenance Phase has been achieved.
[0191] The randomization call may take place between Week 14 (last visit in the Induction Phase) and Week 16, provided the patient has been assessed as eligible for the Maintenance Phase. The randomization will be stratified by CDAI remission at both Weeks 10 and 14 (yes vs. no), induction dose regimen (low dose vs. high dose), concomitant oral CS treatment (yes vs. no), and prior anti-TNF exposure (yes vs. no). The first dose in the Maintenance Phase is administered at the Week 16 clinic visit.
[0192] Patients should remain on a stable dose of IS therapy throughout the treatment period, unless dose reduction or discontinuation is required because of a toxicity related to the medication. CS dose should be tapered starting at Week 14. Patients who cannot tolerate the CS taper without recurrence of CD symptoms or symptoms of steroid withdrawal can receive an increase in CS dose, but this should not exceed the dose administered at randomization. The dose-tapering regimen must be re-initiated within 2 weeks.
[0193] During the Maintenance Phase, patients who experience a clinical relapse may have the option of escaping to Part 1 (OLE) Study. Clinical relapse is defined as meeting at least one of the following criteria on two consecutive visits (may include unscheduled visits), with at least one of the two consecutive CDAI scores >220: CDAI score > the baseline (Week 0) score or CDAI score >100 points higher than the Week 14 score.
[0194] All patients who complete their final Maintenance Phase visit at Week 66 may be eligible to enroll in Part 1 (OLE) Study. Patients who do not enroll in Part 1 (OLE) will enter a 12-week Safety Follow-Up Phase, after which they will be asked to enroll in a 92-week extended PML -monitoring phase (Part 2 [SM] Study). Patients who require surgical intervention for CD at any time during the Maintenance Phase will stop study treatment, enter the Safety Follow-Up Phase, and will be asked to enter Part 2 (SM) Study for PML monitoring. Patients who self-withdraw from the Maintenance Phase and do not meet the eligibility criteria for OLE treatment will also enter the Safety Follow-Up Phase and be asked to enter Part 2 ( SM) Study for PML monitoring. Patients who withdraw will complete the early withdrawal assessments listed in the Schedule of Assessments; withdrawn patients will not be replaced.
Management of Concomitant CD Therapy in the Maintenance Phase
[0195] During the Maintenance Phase, corticosteroid dose should be tapered starting at Week 14 according to the following schedule: ≤20 mg/day prednisone (or equivalent), titrated via dose reduction of 2.5 mg/week until discontinuation; ≤6 mg/day oral budesonide, titrated via dose reduction of 3 mg every 2 -weeks until discontinuation,
[0196] If needed, patients may increase their corticosteroid dose up to their baseline dose (i.e., dose at randomization), but the dose-tapering regimen should be re-initiated within 2 weeks.Patients who are taking concomitant IS therapy (AZA, 6-MP, MTX) must remain on stable doses throughout the study unless dose reduction or discontinuation is required because of a toxicity related to the medication. Doses of anti-diarrheal medication should also be kept stable.
[0197] Generally accepted criteria for discontinuation of IS due to toxicity include, but are not limited to, acute pancreatitis, severe leukopenia, severe thrombocytopenia, or significant elevations of the liver-associated enzymes from baseline, especially in the presence of an elevated total bilirubin. The ultimate decision to discontinue IS remains at the discretion of the Investigator.
Rescue Therapy in Maintenance Phase
[0198] This is defined as medication prescribed for new or worsening CD symptoms, and includes: any new CS or IS therapy for CD; increase in dose of IS therapy above the baseline (Week 0) dose; increase in the dose of CS therapy above a patient’s baseline (Week 0) dose (applicable to patients requiring CS at baseline). Antibiotics, 5-ASAs, anti-diarrheals, probiotics, herbal/ayurvedic, nutritional, and homeopathic supplements are not considered as rescue therapies. Patients who require rescue therapy during the Maintenance Phase may be offered early access to the Part 1 OLE Study, if eligible, and will be required to complete the Early Withdrawal visit assessment. Prohibited therapies are as described for the Induction Phase. Study Drug or Placebo Administration
[0199] Patients will receive etrolizumab or placebo by SC injection according to their treatment assignment. Patients and study personnel will be blinded to study drug assignment (or the etrolizumab dose assignment for Cohort 2 patients in the Induction Phase) for the entire study. In the Induction Phase, study medication will be administered at the investigational site and patients will be trained to self-administer. A health care professional (HCP) will administer the first two doses, with the patient or their caregiver administering subsequent doses under HCP supervision. In the Maintenance Phase after completing Week 16, patients have the option to return to the investigational site or to self-administer/have a caregiver administer their dose Q4W at home within ± 3 days of their scheduled dose unless a PK blood draw is planned, in which case the dose must be administered after the blood draw on the day of visit or within 3 days after the visit.
Outcome Measures: Efficacy Objectives
[0200] Analyses of clinical remission will be made on the basis of patient-reported values for liquid/soft stool frequency (SF) and abdominal pain (AP) (derived from the 7 -day average scores), and centrally read, clinician-reported values for endoscopic improvement, as assessed by the Simple Endoscopic Score for Crohn’s Disease (SES-CD), the summed score of four endoscopic variables that are rated in 5 ileocolonic segments of the bowel (Daperno et al., Gastrointest Endosc 2004;60:505-12).
Co-Primary Efficacy Objectives
[0201] The co-primary efficacy endpoints for this study are the following: (1) Clinical remission, defined as a SF mean daily score ≤ 3 and an AP mean daily score ≤1 with no worsening in either subscore compared to baseline, averaged over the 7 days prior to visit; and (2) Endoscopic improvement, defined as a >50% reduction from the baseline SES-CD. [0202] Primary efficacy objectives for this study will be analyzed separately for the Induction and Maintenance Phases as outlined following. Induction Phase: To independently evaluate the efficacy of etrolizumab dose regimens compared with placebo in inducing clinical remission and endoscopic improvement at the end of the Induction Phase (Week 14). Maintenance Phase: To independently evaluate the efficacy of etrolizumab compared with placebo in achieving clinical remission and endoscopic improvement at 1 year of maintenance treatment (Week 66), for patients who achieved a Crohn’s Disease Activity Index (CDAI)-70 response (defined as a decrease of at least 70 points from baseline CD Al) at Week 14. Secondary Objectives
[0203] Secondary objectives for the induction phase of this study are as follows: (1) To evaluate the efficacy of etrolizumab compared with placebo in achieving clinical remission at Week 6; (2) To evaluate the efficacy of etrolizumab compared with placebo in achieving anSES-CD ≤4 (≤2 for ileal patients), with no segment having a subcategory score (i.e., for ulceration size and extent, affected surface, or narrowing) that is >1, at Week 14; and (3) To evaluate the reduction in CD signs and symptoms achieved by etrolizumab dose regimens compared with placebo at Week 14 as assessed by the Crohn’s Disease Patient-Reported Outcome Signs and Symptoms (CD-PRO/SS) measure.
[0204] Secondary objectives for the maintenance phase of this study are as follows for patients who achieved CDAI-70 at Week 14, unless otherwise stated: (1) To evaluate the efficacy of etrolizumab compared with placebo in maintaining clinical remission at Week 66 for patients who achieved clinical remission at Week 14; (2) To evaluate the efficacy of etrolizumab compared with placebo in achieving corticosteroid-free clinical remission at Week 66 among patients who were receiving corticosteroids at baseline; (3) To evaluate the efficacy of etrolizumab compared with placebo in maintaining endoscopic improvement at Week 66 for patients who achieved endoscopic improvement at Week 14; (4) To evaluate the efficacy of etrolizumab compared with placebo in achieving a SES-CD ≤4 (≤2 for ileal patients), with no segment having a subcategory score(i.e., for ulceration size and extent, affected surface, or narrowing) that is >1, at Week 66; (5) To evaluate the efficacy of etrolizumab compared with placebo in achieving durable clinical remission during 1 year of maintenance therapy (i.e., at > 4 of the 6 in-clinic assessment visits that are conducted during the Maintenance Phase at Weeks 24, 28, 32, 44, 56, and 66); (6) To evaluate corticosteroid- free clinical remission at Week 66 (off corticosteroid for at least 24 weeks prior to Week 66) in patients who were receiving corticosteroids at baseline; and (7) To evaluate change in CD signs and symptoms from baseline to Week 66 as assessed by the CD-PRO/SS measure. Exploratory' Objectives
[0205] The exploratory efficacy objectives for this study are as follows: (1) To evaluate the efficacy of etrolizumab compared with placebo in reducing fecal calprotectin levels at the end of the Induction and Maintenance Phases; (2) To evaluate the efficacy of etrolizumab compared with placebo in reducing C reactive protein (CRP) levels at the end of the Induction and Maintenance Phases; (3) To evaluate the time from Week 14 until experiencing major CD-related events (including hospitalizations, bowel surgeries, and non-study procedures); (4) To evaluate the effect of etrolizumab compared with placebo on the closure of draining fistulas; (5) To evaluate the efficacy of etrolizumab compared with placebo in achieving an SES-CD of 0 by Week 66; (6) To determine the efficacy of etrolizumab compared with placebo in achieving histologic improvement at the end of Induction and Maintenance Phases; (7) To evaluate efficacy of etrolizumab compared with placebo in achieving CD Al remission at Week 66; (8) To evaluate change in patient-reported health- related quality of life (HRQOL) from baseline to Week 14 as assessed by the Inflammatory Bowel Disease Questionnaire (IBDQ); (9) To evaluate the change in patient-reported HRQOL from baseline to Week 66 as assessed by the IBDQ; (10) To evaluate etrolizumab compared with placebo through assessment of histologic activity in the ileum and/or colon at Week 14 and at Week 66 (if sample available).
[0206] In addition to analyses addressing the above objectives, additional analyses of CDAI will be conducted. Furthermore, resource utilization and other patient reported outcome analyses will be undertaken using tools including, but not limited, to the EuroQOL Five-Dimension Questionnaire (EQ-5D).
Safety Objectives
[0207] The safety objectives for this study are to evaluate the overall safety and tolerability of etrolizumab compared with placebo during Induction and Maintenance Phases of therapy; to evaluate the incidence and severity of infection-related adverse events; to evaluate the incidence of malignancies; to evaluate the incidence and severity of immunogenic responses (anti-therapeutic antibodies [ATAs]); and to evaluate the incidence and severity of hypersensitivity reaction events.
Pharmacokinetic Objectives
[0208] The pharmacokinetic objectives for this study are to evaluate etrolizumab serum concentrations during the Induction Phase (Week 14) and at several predose timepoints when at steady state during the Maintenance Phase in patients who are re-randomized to etrolizumab; to characterize the interindividual variability and potential covariate effects on etrolizumab serum exposure; to investigate the relationship between serum exposure and clinical response and remission as well as endoscopic changes during the induction and maintenance treatment phases; to characterize the PK profile of etrolizumab in patients with CD and the relationship between serum exposure of etrolizumab and 07 receptor occupancy by etrolizumab on peripheral blood T and B lymphocytes subsets (in a PK/PD substudy).
Study Population; Patients
[0209] The target population are refractory or intolerant to CS and/or IS therapy and who have either not received prior anti-TNF therapy (TNF-naive) or who have had prior exposure to one or more anti-TNF therapies, and who have had an inadequate response, refractory response, or intolerance to CS and/or IS therapy and/or anti-TNFs.
[0210] Patients refractory to CS therapy have signs/symptoms of persistently active disease despite a history of at least one 4-week induction regimen including a dose equivalent to > 30 mg/day prednisone for 2 weeks if oral or 1 week if IV or > 9 mg/day oral budesonide. Patients intolerant to CS therapy have a history including, but not limited to, Cushing’s syndrome, osteopenia/osteoporosis, hyperglycemia, insomnia, and infection.
[0211] Patients refractory to IS therapy have signs/symptoms of persistently active disease despite a history' of at least one 12-week regimen of oral AZA (or equivalent) (> 1 5 mg/kg) or 6-MP (or equivalent) (> 0.75 mg/kg) or MTX (> 15 mg/week). Patients intolerant to IS therapy have a history' of intolerance to AZA (or equivalent), 6-MP (or equivalent), and/or MTX (including, but not limited to, infection, nausea/vomiting, abdominal pain, pancreatitis, liver function test abnormalities, lymphopenia, and thiopurine methyltransferase genetic polymorphism).
[0212] Inadequate primary non-response to anti-TNF therapies means the patient did not respond (as evidenced by persistent signs/symptoms related to CD after receiving > 2 induction doses of either infliximab [> 5 mg/kg] or adalimumab [160 mg/80 mg or 80 mg/40 mg] or certolizumab pegol [> 400 mg]). Inadequate secondary' non-response to anti-TNF therapies means the patient initially responded to induction therapy with infliximab (> 5 mg/kg) or adalimumab (> 40 mg) or certolizumab pegol (> 400 mg), but experienced signs/symptoms related to recurrence of CD during maintenance. Intolerance to anti-TNF therapy means the patient experienced a significant injection-site reaction, congestive heart failure, infection, or other condition that precluded continuing use of anti-TNF therapy at any time. Patients who have not previously demonstrated inadequate response or intolerance to one or more anti-TNF therapies are eligible to participate in the study provided they are intolerant or refractory to corticosteroid or immunosuppressant therapy.
Inclusion Criteria
[0213] Patients must meet the following criteria for study entry': (1) Able and willing to provide written informed consent; (2) 18-80 years of age; (3) Males and females who are not post-menopausal: use of effective contraception during the treatment period and for at least 24 weeks after the last dose of study drug; (4) Diagnosis of CD based on clinical and endoscopic evidence established > 3 months prior to screening visit; (5) Moderately to severely active disease, as defined above in the Study Design section, determined in the Screening Phase; (6) involvem ent of ileum and/or colon with at least four colonic segments traversable by a pediatric endoscope or three segments (coion and/or ileum) for patients who have undergone a bowe! resection for CD, (7) completed a surveillance colonoscopy ≤ 12 months prior to screening if colonic disease of > 10 years’ duration or ≤ 5 years prior to screening if patient has any risk factors for bowel cancer (surveillance may be performed during screening); (8) have experienced intolerance, refractory disease, or no response (as defined above) to at least one of CS therapy, IS therapy, and/or anti-TNF therapy within 5 years from screening.
Exclusion Criteria
[0214] Patients meeting any of the following criteria will be excluded from study entry. The exclusion criteria related to gastrointestinal health include: (1) underwent subtotal colectomy with ileorectal anastomosis or underwent total colectomy; (2) short-bowel syndrome; (3) has an ileostomy or colostomy; (4) has evidence of fixed stenosis or small- bowel stenosis with prestenotic dilation that precludes adequate endoscopic assessment of the bowel; (5) diagnosis of UC or indeterminate colitis; (6) suspicion of ischaemic colitis, radiation colitis, or microscopic colitis; (7) evidence of abdominal or perianal abscess; (8) expected to require surgery' to manage CD-related complications during the study; (9) past or present adenomatous colonic polyps; (10) past or present disease-related colonic mucosal dysplasia (prior age-related polyps are acceptable); (11) Sinus tract with evidence for infection (e.g., purulent discharge) in the clinical judgment of the investigator. Fistulas related to Crohn’s disease are not exclusionary.
[0215] The exclusion criteria related to prior or concomitant therapy include: (1) received any of adalimumab, certolizumab pegol, or infliximab for CD within 8 weeks prior to randomization; (2) any prior treatment with ustekinumab within 14 weeks prior to randomization; (3) any prior treatment with etrolizumab or other anti-integrin agents (including vedolizumab, natalizumab, and efalizumab); (4) any prior treatment with anti- adhesion molecules (e.g., anti-MAdCAM-1); (5) prior treatment with T cell- or B cell-depleting agents (e.g., rituximab, alemtuzumab, or visilizumab) within ≤ 12 months prior to randomization with the exception of AZA and 6-MP (or equivalent); (6) received any investigational treatment that included investigational vaccines within 12 weeks prior to randomization in the study or five half-lives of the investigational product, whichever is greater; (7) history' of moderate or severe allergic or anaphylactic/anaphylactoid reactions to chimeric, human, or humanized antibodies, fusion proteins, or murine proteins or hypersensitivity to etrolizumab (active drug substance) or any of the excipients (L-histidine, L-arginine, succinic acid, polysorbate 20); (8) treatment with corticosteroid enemas/ suppositories and/or topical (rectal) 5-aminiosalicylate (5-ASA) preparations ≤2 weeks prior to randomization; (9) patients who have continued tube feeding, defined formula diets, and/or parenteral alimentation/nutrition as treatment for CD ≥3 weeks prior to randomization; (10) patients who are expected to require tube feeding, defined formula diets, and/or parenteral alimentation/nutrition as treatment for CD during the study; (11) received any live or attenuated vaccines ≤4 weeks prior to randomization; (12) use of IV steroids during screening, with the exception of a single IV steroid dose administered in the Emergency Department; (13) cyclosporine, tacrolimus, sirolimus, or mycophenolate mofetil ≤4 weeks prior to randomization; (14) chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs). Prophylactic aspirin use up to 325 mg/'day is permitted, as is occasional use of NSAIDs for conditions such as headache, arthritis, mylagia, and menstrual cramps; (15) if receiving oral CSs, patients will be excluded unless the dose is stable at ≤ 20mg/day prednisone (or equivalent) for > 2 weeks immediately prior to randomization; (16) if receiving ongoing treatment with oral 5 aminosalicylate (5-ASA), patients will be excluded if the dose is not stable for > 4 weeks immediately prior to randomization; (17) if receiving ongoing treatment with probiotics (e.g., Culturelle, Saccharomyces boulardii) or over-the- counter supplements (e.g., N-acetyl glucosamine, curcumin), patients will be excluded if the dose is not stable for > 2 weeks immediately prior to randomization; (18) if receiving ongoing treatment with ISs (e.g., 6-MP, AZA, or MTX), patients will be excluded if the dose is not stable for > 8 weeks immediately prior to randomization, (19) if receiving ongoing treatment with antibiotics for the treatment of CD, patients will be excluded if the dose is not stable for > 2 weeks immediately prior to randomization. Patients may continue to receive ongoing treatment with anti-diarrheals (e g., loperamide or diphenoxylate with atropine), preferably achieving a stable dose for > 2 weeks prior to randomization. However, if the patient and/or the treating physician decides to change the dose or course of anti-diarrheals at any time during screening, these patients will be allowed to participate in study.
[0216] The exclusion criteria related to infection risk include: (1) congenital or acquired immune deficiency; (2) positive ELISA test result for HIV confirmed by Western blot; (3) positive hepatitis C vims (HCV) antibody test result, unless the patient has documented that HCV RNA is undetectable for > 6 months after completing a successful course of HCV antiviral treatment; (4) in the screening hepatitis B assessment, patients who test positive for HBsAg are excluded from the study; patients who test positive for hepatitis B core antibody (HBcAb) but negative for hepatitis B surface antigen (HBsAg) must have a confirmed negative hepatitis B virus (HBV) DNA test result to be eligible for the study and will be required to undergo periodic monitoring for HBV DNA during the study; (5) positive stool test result for ova or parasites or positive stool culture for pathogens at time of screening; (6) evidence of infection with and/or treatment for Clostridium difficile or other intestinal pathogen treatment for C. difficile infection within 8 weeks prior to the baseline visit; (7) a history of active or latent tuberculosis confirmed by screening test or suspicion of active tuberculosis on chest radiograph taken within 3 months of randomization; (8) history of recurrent opportunistic infections and/or history of severe or disseminated viral infections; (9) any serious opportunistic infections that occurred ≤ 6 months prior to screening; (10) any current or recent signs or symptoms (≤ 8 weeks before screening) of infection; (11) any major episode of infection requiring hospitalization or treatment with intravenous antibiotics ≤ 8 weeks prior to screening or oral antibiotics ≤ 4 weeks prior to screening.
[0217] The exclusion criteria related to general safety include: (1) pregnant or lactating; (2) lack of peripheral venous access; (3) hospitalized within 4 weeks prior to randomization; (4) inability to comply with study protocol, in the opinion of the investigator; (5) significant uncontrolled comorbidity, such as neurological, cardiac, pulmonary, renal, hepatic, endocrine, or GI disorders (other than CD), (6) neurological conditions or diseases that may interfere with monitoring for PML (7) clinically significant abnormalities on screening neurologic examination; (8) history of demyelinating disease; (9) history of major neurological disorders, including stroke, MS, brain tumor, neurodegenerative disease, or poorly controlled epilepsy; (10) history of alcohol, drug, or chemical abuse ≤6 months prior to screening ; (11) conditions other than CD that could require treatment with >20 mg/day of prednisone (or equivalent) during the course of the study; (12) history of cancer, including hematologic malignancy, solid tumors, and carcinoma in situ within 5 years before screening; (13) history of cervical smear result at any time that indicated the presence of adenocarcinoma in situ (AIS), high-grade squamous intraepithelial lesions (HSIL), or cervical intraepithelial neoplasia (C1N) of Grade > 1; (14) history of organ transplant, or cell transplantation; (15) patients for whom a magnetic resonance imaging (MRI) scan is considered unsafe, due to the presence of metal in the body that could a pose hazard during any potential scanning.
Materials and Methods
Etrolizumab and Placebo; Handling [0218] Etrolizumab will be supplied by the Sponsor as a single-use PFS containing 150 mg/mL etrolizumab for SC administration. To preserve the blind to study drug assignment in the Induction Phase, all patients will receive injections from two PFSs at Weeks 0, 4, 8, and 12: a 1-mL PFS with a 0.7-mL injection volume (delivering placebo or 105 mg of etrolizumab) and a 2.25-mL PFS with a 1.4-mL injection volume (delivering placebo or 210 mg of etrolizumab). Depending on dose assignment, one PFS (patients assigned to active drug) or both PFSs (patients assigned to placebo) will contain placebo. At Week 2, all patients will receive a study drug injection from a 2.25-mL PFS with a 1.4-mL injection volume (delivering placebo or 210 mg of etrolizumab). The PFS will contain placebo for patients in the low-dose etrolizumab and placebo arms and active drug for patients in the high-dose etrolizumab arm. In the Maintenance Phase, patients will receive injections from a 1-mL PFS with a 0.7-mL injection volume (delivering placebo or 105 mg of etrolizumab). [0219] Etrolizumab is formulated as 150 mg/mL in 20 mM histidine, 0.2 M arginine succinate, and 0.04% (weight/volume) polysorbate 20, pH 5.8. Each PFS is for single-dose parenteral administration and contains no preservatives.
[0220] Drug product composition for the placebo is exactly the same as that of active drug product without the presence of etrolizumab.
[0221] The study drug must be stored according to the details on the product label. The drug label indicates the storage temperature. PFSs of study medication should be refrigerated at 2°C-8°C (35.6°F-46.4°F) and protected from excessive light and heat. PFSs should not be frozen, shaken, or stored at room temperature. The PFS containing study drug is stable for no longer than 8 hours at room temperature (up to 30°C [86°F]). If a syringe is left at room temperature for longer than this time, it should not be used.
Crohn’s Disease Actvity Assessments
[0222] For each patient, a detailed history’ of CD, including date of diagnosis, disease severity, hospitalizations, and extraintestinal manifestations at screening will be collected. Disease severity will be evaluated using the CDAI, SF, AP, and SES-CD as described above. Additional details for the CDAI are provided in Table 1 below and SF and AP are described below. The SES-CD (see Table 2) is assessed through endoscopy and is a composite of four factors (ulcer size, percentage of ulcerated surface, percentage of surface affected by other lesions, and extent of stenosis) in up to five ileocolonic segments. The CDAI, SF, and AP scores will be calculated at Weeks 0, 10, 14, 24, 28, 32, 44, 56, and 66 (or Early Withdrawal Visit). SES-CD assessments will take place at screening and Weeks 14 and 66 (or Early Withdrawal Visit). Abdominal pain will be additionally assessed throughout the study with the Abdominal Pain Questionnaire (APQ) recorded daily on an e-diary (scale of 0-10 with 0 indicating no pain and 10 indicating pain as bad as you can imagine).
[0223] The CD Al quantifies the signs and symptoms of patients with CD. The CD Al consists of eight factors, each factor is summed after adjustment with a weighting factor (Best et al. 1979). The components of the CD Al include number of liquid or soft stools, abdominal pain, general well-being, presence of complications, use of LOMOTIL® (diphenoxylate/atropine) or other opiates for diarrhea, presence of an abdominal mass, hematocrit, and percentage deviation from standard weight. Of the eight factors of the CD Al, three are patient reported (number of liquid or soft stools, abdominal pain, and general well- being), four are based on physician assessment (presence of complications, use of LOMOTIL® or other opiates for diarrhea, presence of abdominal mass, and percentage of deviation from standard weight, which is based on the patient’s weight at the visit), and one factor is based on a blood test (hematocrit). Patients are to report their abdominal pain severity, loose-stool frequency, and general well-being on the e-diary on a daily basis. The weighted sum of the average scores over 7 days is calculated for the PRO component of the CD Al score. The Bristol Stool Scale is provided to patients as a reference for determining loose stools (Types 6 and 7 on the Bristol Stool Scale; see Fig. 3). Because the ileocolonoscopy preparations can interfere with the assessment of other clinical parameters, e-diary entries used to calculate the complete CD Al should not correspond to day(s) of bowel preparation, endoscopy, or the day after endoscopy.
[0224] Two patient reported factors will be evaluated: the frequency of liquid or soft stools (SF) and abdominal pain (AP). The score is calculated using the unweighted mean number of liquid/very soft stools and mean AP (on a 0-3 scale) for the 7 days prior to the assessment visit. Patients are to report their loose-stool frequency (the Bristol Stool Scale will be provided [Fig. 3]) and abdominal pain severity on the e-diary on a daily basis. As with the CDAI, the SF and AP scores should not use e-diary entries that correspond to day(s) of bowel preparation, endoscopy, or the day after endoscopy, to avoid interference related with the ileocolonoscopy. In addition, the 0-10 point Abdominal Pain Questionnaire (APQ) is an I I- point numeric, rating scale to assess the worst abdominal pain on a daily basis. A higher score indicates a greater severity of abdominal pain (0 ::: no pain; 10 ::: pain as bad as you can imagine). The APQ has a recall specification of 24 hours. As with the CDAI, SF, and /AP scores, the calculated APQ score should not use e-diary entries that correspond to day(s) of bowel preparation, endoscopy, or the day after endoscopy, to avoid interference related with the ileocolonoscopy. [0225] Another assessment of disease activity is the patient-reported outcome tool known as CD-PRO/SS. The development of this PRO is described in Higgins et al., Journal of Patient-Reported Outcomes (2018) 2:24 and the PRO modules and user manual can be found at https(colon)(slash)(slash)www(dot)evidera(dot)com(slash)crohns-disease-patient-reported- outcomes-cd-pro. The 14-item questionnaire (some questions contain supplementary questions regarding severity/frequency) contains two domains: CD signs and symptoms and systemic symptoms. The CD-PRO/SS assesses the presence of CD signs and symptoms and, in some cases, the severity or frequency of the symptoms. The CD-PRO/SS measure has a recall specification of 24 hours. Patients will complete the CD-PRO/SS measure for at least 9 consecutive days around each visit as programmed in the e-diary. To allow for the visit window to account for early or delayed visits, patients may complete CD-PRO/SS for up to 12 days.
Figure imgf000061_0001
Adapted from: Best WR, Becktel JM, Singleton JW, Kern F, Jr. Development of a Crohn’s disease activity index. National Cooperative Crohn’s Disease Study. Gastroenterology 1976; 70(3):439-44.
Figure imgf000062_0001
Rationale for the Primary and Key Secondary Study Endpoints
[0226] The pivotal cohort of this study is designed to assess remission of signs and symptoms as well as endoscopic improvement as co-primary outcome measures in CD. Clinical remission is defined as the mean number of liquid/soft stools for the 7 days prior to the assessment visit (SF) being ≤3 and the mean AP score (on a 0-3 scale) with no worsening in either subscore compared to baseline, for the 7 days prior to the assessment visit (AP) being ≤1. The primary clinical endpoint is based on previous FDA recommendations, the evolving global regulatory environment (new EMA new medicinal products guideline for CD released July 2018; CHMP 2016), and the results generated from the exploratory induction of Cohort 1 in this study. Both FDA and EMA in its new guideline discouraged the use of CDAI as primary endpoint for CD registration studies due to the limitations of the index. Instead, co-primary endpoint composed of patient-reported signs/symptoms and endoscopic measure is encouraged.
[0227] The use of SF and AP addresses the FDA’s recommendation to use 7-day scores of SF and AP that correlate with CDAI remission. Defining clinical remission and response that use unweighted measures of AP and SF are more easily interpretable by clinicians. patients, and caregivers and are more likely to represent clinically meaningful improvements in symptoms and disease activity for moderate to severe CD patients experiencing different degrees of SF and AP during a flare.
[0228] Endoscopic improvement, defined as a change in SES-CD > 50% from the baseline score (Ferrante M, et al., Gastroenterology 145:978-86, 2013), is a co-primary endpoint. The SES-CD consists of four endoscopic variables (ulcers, ulcerated surface, inflamed surface, and presence of narrowings) that are scored in five ileocolonic segments, TheSES-CD was prospectively developed and validated in patients with mild to severe CD (according to CD Al) by Dapemo et al., Gastrointest. Endosc. 60(4):505-12, 2004. This scoring system was recommended by the FDA and is generally preferred by physicians to other measures because of quantification of ulcer size (rather than a qualitative assessment of ulcer characteristics), determination of the percent ulceration in a segment (rather than determination by a visual analogue scale), and for better inter-rater reliability (inter-class correlation coefficients were 0.83 and 0.71 for SES-CD and Crohn's Disease Endoscopic Index of Severity (CDEIS), respectively; Khanna R, et al., Inflamm Bowel Dis 20: 1850 > 61, 2014). Because the score does not adjust for the number of visible segments, only segments visualized at baseline will be included in the endpoint assessment. This means any new narrowing due to inflammation that renders a segment not evaluable for scoring after study treatment will qualify the patient as not achieving endoscopic improvement for the secondary endpoint analysis. Similarly, any improvement in inflammation that renders a segment evaluable for scoring after study treatment will not be reflected in the endpoint assessment. The Statistical Analysis Plan (see below) will describe any sensitivity analysis which are planned to assess the impact of these missing data on the endpoint. Given the limited experience with endoscopic scoring systems in general gastroenterology practice, all ileocolonoscopies in the study will be recorded at the investigational site, but central readers will determine the SES-CD.
[0229] Published studies have used multiple endoscopic endpoints to assess efficacy, which appear to be arbitrarily defined. Mucosal healing, defined qualitatively as an absence of mucosal ulcers, has been studied as a primary or secondary endpoint in a number of Phase IV studies (Rutgeerts P, et al., Gastro 126: 1593-610, 2004 [natahzumab], Rutgeerts P, et al., Gastrointest Endosc 63:433-42, 2006 [infliximab]; Colombel JF, Sandborn WJ, Reinisch W, et al. Infliximab, azathioprine, or combination therapy for Crohn’s disease. et al., N Engl J Med 362: 1383-95, 2010 [infliximab and azathioprine]; Hebuteme et al., Gut 62:201-8, 2013 [certolizumab]; Rutgeerts P, et al., Gastroenterology 142(5): 1047-9, 2012 [adalimumab]). Some studies have defined endoscopic thresholds that represent endoscopic remission without clarity on the clinical significance of these score (Hebuteme et al., Gut 62:201-8, 2013 [certolizumab]; Rutgeerts P, et al., Gastroenterology 142(5): 1047-9, 2012). In this study, endoscopic improvement is defined as the proportion of patients demonstrating a > 50% reduction in SES-CD versus their pretreatment baseline score.
[0230] For induction, this endpoint will be measured at Week 14 and, for maintenance, at Week 66 among patients who achieved a clinical response at Week 14. The endpoint is based on recent post hoc analyses of the SONIC trial which determined a > 50% reduction in SES-CD score to be predictive of CS-free CD Al remission following 50 weeks of treatment with biologic therapy (Ferrante M, et al., Gastroenterology 145:978-86, 2013). This definition is also appropriate when considering the large variability in SES-CD change at the end of a 6-week Induction Phase measured in a sample of 24 placebo patients (with moderate to severely active CD) who were participating in one of two trials of novel biologic agents (Ferrante et al. Gastroenterology 138, Issue 5, Supplement 1, S-358, 2010). The dataset showed that 6 patients achieved both 50% reduction in either SES-CD or ( DEIS score, and at least a 5-point reduction in either SES-CD or CDEIS score.
Rationale for Patient Population
[0231] Patients with uncontrolled, moderate to severely active CD are at risk for developing stricturing or penetrating complications of inflammation, as well as symptoms that are debilitating to quality of life. The treatment goals for CD are to induce and maintain symptom improvement, induce mucosal healing, and improve quality of life. However, for a significant proportion of patients, these goals are not met by current therapies. As such, the study population will include patients who are refractory to one or more of the following therapies: 1) CS, 2) IS, or 3) anti-TNFs (or TNF-IR). Patients who enroll on the basis of refractory or intolerance to CS and/or IS may have been previously exposed to ant-TNFs or be naive to anti-TNFs Data from previous studies of etrolizumab in ulcerative colitis (Rutgeerts, PJ et al., Gut 62: 1122-1130, 2013; Vermeire et al., Lancet 384:309-18, 2014) and the vedolizumab GEMINI 2 and 3 studies (Sandborn WJ, et al., Aliment Pharmacol Ther 37:204-13, 2013: Sands BE, et al., Gastroenterology 147:618-27, 2014) have demonstrated the favorable efficacy of the anti-α4β7 mechanism of action in these patient subgroups.
Patients belonging to each subgroup will be identified on the basis of refractory and inadequate-response criteria described herein. [0232] Patients with moderate to severely active CD between the ages of 18 and 80 years of age will be studied. This age range is typical of patients enrolled in clinical trials of new investigational agents for CD and reflects the observation that adult CD can become or persist as moderate to severely active disease at any age. Given that the primary clearance mechanism for etrolizumab is neither renal elimination nor first-pass metabolism, the risk of accumulation in patients >65 years of age is considered low and is also mitigated by laboratory exclusions related to poor renal and hepatic function.
[0233] In line with EMA guidelines, eligible patients must have an established diagnosis of CD for at least 3 months, with moderately to severely active di sease corroborated by endoscopic evidence of inflammation. This is expected to improve the specificity of the CDAI, SF, and AP assessments and to allow the evaluation of endoscopic improvement endpoint. On the basis of the construction of the SES-CD, patients with isolated ileitis or post-ileocecal resection can be expected to have a lower baseline score compared with patients with ileocolonic disease, regardless of whether the extent of inflammation and ulceration is the same in affected segments. As such, different SES-CD entry scores are proposed for these subgroups of patients. It is acknowledged that patients with fistulizing disease are not well served by therapeutic options. The protocol includes this subgroup to assess the impact of etrolizumab on draining fistulas.
Rationale for the Design of Induction and Maintenance Phases
[0234] Patient randomization into the Induction Phase will be stratified by disease activity on the basis of CDAI score ≤330 or >330 (predictive of CDAI response and remission rates with biologic therapy; Sandborn WJ, et al., Aliment Pharmacol Ther 37:204- 13, 2013; Sands BE, et al., Gastroenterology 147:618-27, 2014), CS use, IS use, and prior anti-TNF failure (all indicators of disease activity). These factors are considered sufficient to mitigate the risk of imbalance in disease severity across treatment arms.
[0235] Assessment of the induction of clinical remissi on and endoscopic improvement at Week 14 is justified on the basis of the observation that anti-integrin therapies have a slower onset of action compared with anti-TNF therapies (Sandborn WI, et al., N Engl J Med 353: 1912-25, 2005; Sandborn WJ, et al., Aliment Pharmacol Ther 37:204-13, 2013), as well as the clinical consensus that the onset of endoscopic improvement is typically observed 16-24 weeks after induction therapy, particularly in treatment refractory patients (a consensus supported by observed data showing that Mayo Clinic Score remission with etrolizumab took up to 14 weeks in TNF-IR patients with UC (see Vermeire et al., Lancet 384:309-18, 2014). A challenge of the 14-week Induction Phase is the requirement to keep concomitant CD therapies stable for the duration, so as not to confound the endpoint analysis. This is problematic in the context of patients who require rescue therapy to treat a flare and patients who are responding to treatment but unable to taper their CS dose. The study design addresses this by allowing use of rescue therapy in case of disease worsening, in which case patients would be classified as non-responders for the primary analysis, and restricting the maximum baseline CS to ≤20mg/day prednisone-equivalent dose. Although not prohibited therapeutic adjustment, increasing the dose of anti-diarrheal drugs should be avoided. Patients should keep their dose of anti-diarrheal medication stable as much as possible because the effect of titrating anti-diarrheal medication on the placebo-response rate on clinical remission is unknown.
[0236] The Induction Phase includes an exploratory cohort (Cohort 1) that is adequately- sized to assess effect size and accuracy of statistical planning assumptions for the new endpoints in pivotal cohort (Cohort 3), the clinical validity of the dichotomous endpoint definitions, and the testing hierarchy of the endpoints. An active-treatment induction cohort (Cohort 2) is also included in this study to generate a sufficient number of remitters for assessment of endpoints in the Maintenance Phase. The final cohort (Cohort 3) is a pivotal induction cohort, which will generate data for the endpoint analysis of the induction study. [0237] Patients achieving a CDAI-70 response at Week 14 without rescue therapy wall be re-randomized into the Maintenance Phase until a sample size of approximately 480 patients in the Maintenance Phase is achieved. Based on expert clinical opinion, patients achieving clinical remission and endoscopic improvement at Week 14 are expected to be a subpopulation within this group. Randomization into the Maintenance Phase will be stratified by the use of disease activity (described for the Induction Phase, except CDAI-remission [score < 150] at both Weeks 10 and 14 will be used instead of CD Al ≤ 330 or > 330). In addition, the randomization will be stratified by assignment to low- or high-dose etrolizumab (allowing an assessment of the impact of low- or highdose induction therapy on the maintenance endpoints). Given the large number of strata, any potential imbalance in the proportion of endoscopic improvers at Week 14 will be handled using a covariate-adjusted analysis for the Week 66 endoscopic-improvement endpoint.
[0238] The duration of the M aintenance Phase (52 weeks) is considered an appropriate period by the FDA and EMA to establish the benefit of long-term therapy. During the first 8 weeks of the Maintenance Phase, patients receiving CS during the Induction Phase should undergo weekly dose reductions aligned with recommendations for CS tapering in the current American College of Gastroenterology and European Crohn’s and Colitis Organization guidelines and following the EMA recommendation to avoid a rapid taper. The tapering schedule will allow patients to be assessed for the key secondary outcome of achieving corticosteroid-free clinical remission at Week 66 among patients who enter into maintenance phase
[0239] An analysis of data from a Phase I study in patients with UC, which compared etrolizumab induction therapy with placebo (Vermeire S, et al., Gastroenterology 144, SI :S- 36, 2013), showed that infection rates were similar between etrolizumab and placebo in the subgroups of patients taking concomitant IS or CS therapy and the subgroups not taking these concomitant treatments. Although this is a small dataset with limited duration of exposure, more data for a longer duration of concomitant exposure are available for vedolizumab. A recent analysis of the vedolizumab development program demonstrated a modest increase in infection rates in patients using IS regardless of whether they received vedolizumab or placebo. In addition, the rates of infection and serious infection in the vedolizumab treatment group were similar in patients who were taking concurrent IS and those who were not (Colombel et al. Gut 2016;0: 1-13.).
[0240] The risk of PML is expected to be lower with etrolizumab compared with other non-gut-selective biologies, with no events of PML reported in the UC Phase II or OLE studies. However, the potential risk of PML and other severe infections has been carefully considered. Patient safety will be monitored throughout the study via assessment of vital signs, safety laboratory assessments, neurological exams, and continuous review of adverse events. Should any patients have anaphylaxis, develop PML, specific malignancy, colonic mucosal dysplasia, or certain specific serious infections, they will be permanently discontinued from the study treatment. During the Maintenance Phase of the study, patients who experience a clinical relapse in their CD status will have the option to enroll into an open-label study of etrolizumab.
[0241] After the final visit in the treatment period, patients not participating in the OLE study will be assessed for safety for an additional 12 weeks starting after the last dose of study drug. This period reflects an adequate timeframe for washout of etrolizumab based on the estimated elimination half-life of 11 days.
Rationale for Etrolizumab Dose, Dose Ranging and Schedule
[0242] Because etrolizumab has been evaluated in patients with moderate to severely active UC in a Phase II study (Vermeire et al.. Lancet. 384:309-18, 2014) in which clinically meaningful induction of disease remission was achieved at 105 mg (nominal dose of 0.7 niL 150mg/mL etrolizumab formulation) administered every four weeks (Q4W) (three doses at Weeks 0, 4, and 8) and 315 mg Q4W +loading dose (LD [LD of 420 mg at Week 0, 315 mg at Weeks 2, 4, and 8]) without significant safety concerns, the dose regimen (105 mg SC Q4W) of etrolizumab is also proposed as one of the doses to be tested in this study. In addition, in the Induction Phase, patients will be randomized to receive placebo, low-dose etrolizumab or high-dose etrolizumab.
[0243] Low-dose etrolizumab (105 mg) will be administered Q4W SC (Weeks 0, 4, 8, and 12). A low' dose regimen of 105 mg SC at Q4W is specified for dose ranging in the Induction Phase on the basis of the following considerations: (1) In the Phase II UC trial, a nominal dose of 100 mg (0.7 mL of 150 mg/mL solution via vial and syringe), actual dose of 105 mg, administered Q4W SC, showed a clinically meaningful induction of remission in patients with UC and had a favorable safety profile in the Phase II trial (Vermeire et al., Lancet 384:309-18, 2014); (2) The exposure of 105 mg, administered Q4W SC, was shown to be sufficient for maximal P7-receptor occupancy in both blood and colonic tissue from all patients who provided evaluable samples in the Phase II trial, Id., (3) Population PK/PD modeling predicts that a dose lower than the 105-mg SC Q4W regimen (e g., 50 mg Q4W SC) will result in the loss of maximal p7-receptor occupancy during the Q4W dosing interval in approximately 44% of patients, and exposure is likely to be in the nonlinear PK range. [0244] In addition to the 105-mg Q4W dose, a higher dose regimen of 210 mg SC at Weeks 0, 2, 4, 8, and 12 is specified for dose ranging in the Induction Phase on the basis of the following considerations: (1) Despite the similarities in pathogenesis, CD exhibits a more complex anatomical disease presentation throughout the Gl tract (i.e., transmural inflammation, patchy distribution, and strictures) when compared with UC. A positive exposure-response relationship was reported recently for vedolizumab, an in-class, anti-integrin antibody, following the Induction Phase of a Phase III clinical trial in patients with CD. Results showed the induction dose is sufficient to reach a complete receptor occupancy in ah patients (Rosario M, et al., (abstract) Crohn’s and Colitis Foundation of America, Advances in Inflammatory’ Bowel Diseases, Abstract P-140, 2013), yet an increase in clinical response/remission was observed in patients with higher drug concentrations (Sandborn WJ, et al., Aliment Pharmacol Ther 37:204-13, 2013; Rosario M, et al., (abstract), European Crohn’s and Colitis Organisation Congress, abstract P489, 2014). These observations from vedolizumab studies suggest that higher etrolizumab exposure may have the potential to offer more clinical benefit in this patient population; (2) in addition to Q4W dose of 210 mg, the additional 210 mg dose at Week 2 is intended to load the etrolizumab exposure upfront to allow exposure achieving steady state faster. Earlier loading dose of anti-TNF agents or anti-integrins antibodies (Rutgeerts P, et al., Gastro 126:1593-610, 2004) were found to be effective in inducing clinical remission (CDAI score < 150), and such a loading dose strategy was also implemented in this study; (3) the proposed higher dose of 210 mg x 5 doses SC (at Weeks 0, 2, 4, 8, and 12) in patients with CD will result in a 2.5-fold total dose separation from the 105 mg Q4W x 4 dose regimen and is predicted to achieve an exposure level 30% lower than that from the 315 mg+420 mg LD cohort studied in the UC
Phase II trial which had an acceptable safety/tolerability profile (see supra). In summary, given the favorable safety profile and positive clinical outcomes observed in the nominal 100-mg Q4W cohort (actual dose 105 mg) in etrolizumab UC Phase II study, it is appropriate to evaluate the 105-mg Q4W dose for CD induction therapy. In addition, given the complex pathophysiology of CD, an observed positive exposure-efficacy relationship for vedolizumab treatment despite a full receptor saturation in the blood, the available safety coverage and an acceptable safety profile of etrolizumab, it is scientifically sound to evaluate a higher dose regimen of 210 mg (Weeks 0, 2, 4, 8, and 12) to further understand the dose-response relationship in patients with CD during the Induction Phase.
Figure imgf000069_0001
[0245] Patients who respond to etrolizumab treatment in the Induction Phase (achieve a decrease from CD Al baseline score of at least 70 points, “'CDAI-70 response”) will be re- randomized to receive 105 mg etrolizumab or placebo Q4W in the Maintenance Phase; patients who respond to placebo in the Induction Phase will continue to receive placebo in the Maintenance Phase. It has been reported that PK profiles of mAbs (anti-TNF or anti- integrins) are similar in UC and CD patient populations (Temant et al., Ther Drug Monit 2008 30:523-9, Fasanamde et al., Eur J Clin Pharmacol 2009;65:1211-28; Awni et al. abstract presented at ECCO, 2013; Feagan et al., N Engl J Med 2013;369:699-710; Sandborn et al., Aliment Pharmacol Ther 2013;37:204-13). A low-dose regimen of 105 mg SC at Q4W is specified in the Maintenance Phase on the basis the following considerations: (1) The 105-mg Q4W SC dose planned for the Phase III study in CD is anticipated (by population modeling) to maintain full p7-receptor occupancy at all times in > 85% of patients. The nominal dose of 100 mg Q4W SC (105 mg actual dose) administered in the UC Phase II study demonstrated an acceptable safety profile (see supra),' (2) the in-class anti-integrin vedolizumab was successful in maintaining remission with an every-8-week regimen that provided an average steady-state trough serum concentration sufficient to maintain maximal receptor occupancy (Rosario M, et al., (abstract) Crohn’s and Colitis Foundation of America, Advances in Inflammatory Bowel Diseases, Abstract P-140, 2013; Sandborn WJ, et al., Aliment Pharmacol Ther 37:204-13, 2013).
Rationale for Control Group
[0246] A placebo-treated control group will be used in this study to assess the differences in efficacy, safety, and tolerability in patients who receive etrolizumab and background CD therapy compared with patients who receive placebo and background CD therapy. The use of a control group is necessary given the inherent variability in disease flares and the use of subjective assessments, such as the PROs Patients in the control group will undergo the same study assessments as the etrolizumab-treated patients. The 1 :1 ratio for etrolizumab to placebo treated patients is necessary to achieve a statistically powered comparison of primary endpoint between these treatment arms.
Patient-Reported Outcomes Assessments
[0247] PROs (IBDQ, CD-PRO/SS, EQ-5D, APQ and the loose stool frequency, abdominal pain, and general well-being components of the CD Al) will be collected to help characterize the patient-reported clinical profile of etrolizumab. The instruments will be translated as required into the local language. PRO data are collected electronically with the use of electronic PRO (ePRO) devices (i.e., e-diary and tablet). The investigator staff will provide the patient with an e-diary and instructions for completing the PRO questionnaires electronically for those PROs that need to be completed outside of the clinic. Patients will also be instructed to contact the site promptly if they have any questions about the use of the e-diary during screening or at any time during the study. For instances when PROs are to be completed at the clinic, the patient will fill them out on a tablet. Review electronic data captured by the patient since the previous study visit with the patient at each clinic visit. ePRO data is collected and assessed at visits. During screening, patients will be instructed on how to appropriately use and complete questions on the e-diary. The signs and symptoms of CD, specifically, number of liquid or soft stools, abdominal pain, and general well-being, must be recorded daily throughout the study, including the screening period. To ensure instrument validity and that data standards meet Health Authority requirements, the PROs completed at the sites (IBDQ and EQ-5D) must be administered at the investigational site prior to the completion of other non-PRO assessments and before the patient receives any disease-status information or study drug during that visit.
[0248] CDAI, SF, AP, and CD-PRO/SS were discussed above. The IBDQ assesses patients’ health -related quality of life (HRQOL; Guyatt G, et al., J Clin Epidemiol 42(5):403- 408, 1989; Irvine EJ, J Pediatr Gastroenterol Nutr 28: S23-7, 1999). The 32-item questionnaire contains four domains: bowel symptoms (10 items), systemic symptoms (five items), emotional function (12 items), and social function (five items). The items are scored on a 7-point Likert scale with a higher score indicating better HRQOL. The IBDQ has a recall specification of 2 weeks. Patients complete the IBDQ at the investigational site on a tablet at baseline, at Weeks 0, 14, 44, and 74 prior to the completion of other non-PRO assessments and before the patient receives any disease-status information or study drug during that visit.
[0249] The EuroQol Five Dimension Questionnaire (EQ-5D) is a generic preference-based HRQOL that provides a single index value for health status (Rabin R, et al., Enn Med 33:337-43, 2001), This tool includes questions about mobility, self-care, usual activities, pain/ discomfort, and anxiety/depression that are used to build a composite of the patient’s health status. Patients complete the EQ-5D at the investigational site on a tablet at Weeks 0, 14, 44, and 74 (or Early Withdrawal visit) prior to the completion of other non- PRO assessments and before the patient receives any disease-status information or study drug during that visit.
Statistical Considerations and Analysis Plan
[0250] For the purpose of statistical analyses, the Induction and Maintenance Phases will be treated as independent studies. The open-label Cohort 2 serves as a feeder cohort to provide sufficient sample size for the maintenance analysis. Induction data from Cohort 2 will also be considered exploratory and may be analyzed to inform decision making and devel opment of the Statistical Analysis Plan. The sample size for each cohort in the Induction Phase is summarized in Table 3 below. Table 3. Induction Phase Sample Size for Each Cohort.
Figure imgf000072_0001
NA = not applicable; No. = number
[0251] The sample size for Cohort 1 (see Table 3) provides approximately 90% power to detect a >20% difference in PRO2 or CD Al remission rates between each etrolizumab arm and placebo (under the assumption of placebo remission rate of ≤ 15%, similar to results reported in the GEMINI 2 trial of vedolizumab in patients with CD; Sandborn WJ, et al., Aliment Pharmacol Ther 37:204-13, 2013) and approximately 80% power to detect a 15% difference versus placebo in endoscopic response (under the assumption of placebo response rate of ≤ 10%) and a two-sided chi-square test at the 10% significance level.
[0252] The pivotal analysis for induction phase will be performed using only patients from Cohort 3. In this pivotal induction cohort, patients will be randomized to receive either placebo, etrolizumab 105 mg or etrolizumab 210 mg in a 2:3:3 ratio The co-primary endpoint will be tested using a Cochran-Mantel-Haenszel (CMH) test at the 5% significance level, where both clinical remission and endoscopy improvement are required to be significant. The sample size for Cohort 3 is expected to provide > 85% power to detect a > 15% difference in rates of clinical remission (SF mean daily score ≤ 3 and AP mean daily score ≤ 1) between each etrolizum ab arm and placebo under the assumption of a placebo remission rate of ≤ 15% and a tw'o-sided test performed at a significance level of 5%. Additionally, Cohort 3 will provide > 80% power to detect a 10% difference in proportions for each etrolizumab arm versus placebo for endoscopic improvement, under the assumption of placebo response rate of ≤ 5% and a two-sided test at the 5% significance level. The chi- square test was used to confirm the power calculations. [0253] Cohort 2 is sized at approximately 350 patients to achieve a sufficient number of patients for the Maintenance Phase analyses.
Table 4. Power Estimates for Primary and Key Secondary Efficacy Analysis in the Pivotal Induction Phase and Maintenace Phase.
Figure imgf000073_0001
All analyses powered using type I error, α=5.0%. a Analysis conducted on all patients in Maintenance Phase who were randomized to etrolizumab in the Induction Phase.
[0254] A total of approximately 480 patients achieving CDAI-70 response at Week 14 will be eligible to enroll into the Maintenance Phase. A co-primary endpoint will be used for the analysis of the Maintenance Phase: clinical remission (SF mean daily score ≤ 3 and AP mean daily score ≤ 1) and endoscopic improvement at Week 66. The maintenance co-primary analysis will be performed on all patients re-randomized into the Maintenance Phase who were randomized to etrolizumab (105 mg or 210 mg) in the Induction Phase. The co-primary endpoint will be tested using a CMH test at the 5% significance level, where both clinical remission and endoscopy improvement are required to be significant. Note that to maintain the blind, patients randomized to placebo in the Induction Phase will undergo a sham randomization and will receive placebo in the Maintenance Phase. These patients wall not form part of the pivotal maintenance analysis.
[0255] Of the approximately 480 patients projected to be enrolled in the Maintenance Phase, approximately 420 patients wall undergo re-randomization to receive either placebo or etrolizumab, and approximately 60 patients will undergo a sham randomization to placebo. These projections are based on the assumption that 50% of patients receiving etrolizumab and 40% of patients receiving placebo during the Induction Phase will be eligible for the Maintenance Phase.
[0256] Assuming a maintenance sample size of 210 patients per arm using a 1 :1 allocation to receive either placebo or etrolizumab 105 mg, the maintenance analyses conducted at the 5% significance level will provide the following power: (1) Clinical remission at Week 66 (SF mean daily score ≤ 3 and AP mean daily score ≤1): at least 90% power to detect a 15% treatment difference assuming a placebo Week 66 remission rate of up to 20%; (2) Endoscopic improvement: approximately 90% power to detect a 15% treatment difference assuming a placebo Week 66 improvement rate of up to 30%.
[0257] Furthermore, the study is designed to provide sufficient power for the following secondary' analyses: (1) Clinical remission at Week 66 among patients achieving clinical remission at Week 14: a sample size of N = 186 (i.e., 93 per arm) will provide 80% power to detect a 20% treatment difference assuming a placebo rate of up to 30%. This assumes a Week 14 clinical remission rate of > 22% among etrolizumab patients.
[0258] Demographic and baseline characteristics such as age, sex, race, region, use of corticosteroids and immunosuppressants, anti-TNF therapy, duration of disease, extent of disease, average daily SF, average daily AP, CDAI, SES-CD, fecal calprotectin, and CRP will be summarized for all randomized patients by treatment arm with use of descriptive statistics for both the Induction and Maintenance Phases. Exposure to study drug (number of study treatments and duration of treatment) will be summarized by treatment arm.
Efficacy Analyses
[0259] For the purpose of statistical analyses, the Induction and Maintenance Phases will be treated as independent studies. Patients who are non-evaluable for efficacy at a specific timepoint, due to missing data, will be considered non-responders for all categorical endpoints. In addition, patients requiring rescue therapy, and/or surgical intervention for CD, and/or took prohibited medications (e g., anti-integrins, T- or B- cell-depleting agents, TNF antagonists, antagonists of IL-23 ± IL-12 (e.g., ustekinumab), anti-metabolites, cyclosporine, tacrolimus, immunosuppressant medications such as AZA (or equivalent), 6-MP, and MTX) will be considered non-responders for the analysis. The following analyses will be performed for the primary efficacy endpoint and key secondary efficacy endpoints: (1) subgroup analyses to evaluate the consistency of results across prespecified subgroups (including baseline anti -TNF -status [naive vs. JR], baseline CS status [on CS vs. not on CS], baseline IS status [on IS vs. not on IS], age, sex, fecal calprotectin, C-reactive protein, region) and (2) sensitivity analyses to evaluate the robustness of results to the primary analysis methods (e.g., handling of dropouts, adjusting for cohort).
Induction Phase
[0260] Efficacy analyses for the Induction Phase will be performed separately for each cohort, and will include all patients who were randomized and received at least one dose of study (modified intent-to-treat population [mITT]). Patients will be grouped according to the treatment assigned at randomization.
[0261] Cohort 1: An exploratory analysis was conducted when all patients in Cohort 1 (n = 300) completed the Week 14 visit or, discontinued early from the study. At this point, the Sponsor was unblinded to patient level data and treatment assignments for patients in Cohort 1 Induction Phase only. Patients, site monitors, and investigators remain blinded to patient- specific treatment assignments. The induction data from Cohort 1 was exploratory in nature and was evaluated prior to the commencement of enrollment to the pivotal Induction Phase of Cohort 3. Maintenance phase data from Cohort 1 patients will form part of the pivotal maintenance phase analysis and remains blinded to all Sponsor personnel and sites until the database lock.
[0262] Cohort 2: Cohort 2 is considered a “feeder” cohort to help achieve the necessary’ sample size for the maintenance study. All primary and secondary efficacy parameters will be summarized descriptively for each treatment arm. Demographic and baseline characteristics such as age, sex, race, region, use of corticosteroids and immunosuppressants, duration of disease and CD activity scores will be summarized for each treatment group by use of descriptive statistics.
[0263] Cohort 3: The co-primary endpoint analysis wall compare for each etrolizumab dose arm versus the placebo arm the proportion of patients who achieve clinical remission or endoscopic improvement at Week 14.
[0264] The difference between each etrolizumab arm and placebo arm will be evaluated using the CMH test statistic stratified by the factors used at randomization. The absolute treatment difference will be provided along with the 95% two-sided confidence interval (CI) estimate.
[0265] All categorical secondary endpoints will be analyzed using the same methodology as the primary endpoint. For all efficacy endpoints, descriptive summary statistics will be provided for each treatment arm. [0266] Continuous endpoints will be analyzed using an analysis of covariance (ANCOVA) model with the stratification variables used at randomization and the baseline value of the studied measure as a covariate.
[0267] The co-primary endpoints will each be tested at the 5% significance level, with both required to be significant for the co-primary endpoints to be deemed significant. The overall type I error rate will be maintained at 5% using a hierarchical model for testing of the two etrolizumab doses versus placebo and corresponding key secondary endpoints. The remaining secondary endpoints and all exploratory endpoints will be considered to provide supportive information and no adjustments for multiple comparisons will be performed. [0268] The co-primary efficacy endpoints for the Induction Phase are: (1) the proportion of patients in clinical remission at Week 14 and (2) the proportion of patients achieving endoscopic improvement at Week 14. Maintenance Phase
[0269] Efficacy analyses for the Maintenance Phase will include all etrolizumab induction patients who were randomized into the Maintenance Phase and received at least one dose of study drug (mITT population). Patients will be grouped according to the treatment assigned at randomization into the Maintenance Phase.
[0270] For all categorical endpoints, the difference in proportions between the two treatment arms will be evaluated using the CMH test. The CMH test will include important stratification factors such as those used to randomize patients into the Maintenance Phase. The stratification factors used for re-randomization into the maintenance phase include sustained CD Al remission at Weeks 10 and 14. As a result of the changes to endpoints introduced in Protocol Version 6, this stratification factor in the analysis will be replaced with clinical remission at Week 14 for the clinical remission at Week 66 analysis, and endoscopic improvement at Week 14 for the endoscopic improvement at Week 66 analysis. A sensitivity analysis will be conducted to include the sustained CDAI stratification factor used at randomization. The test will be performed at the two-sided 5% significance level. The absolute treatment difference will be provided along with the 95% two-sided CI estimate. All categorical secondary endpoints will be analyzed using the same methodology as for the primary endpoint.
[0271] Continuous endpoints will be analyzed using an ANCOVA model with the stratification variables used at randomization and the baseline value of the studied measure as a covariate. [0272] If the primary endpoint is statistically significant, key secondary endpoints will be tested sequentially.
[0273] Patients who are non-evaluable for efficacy at a specific timepoint (e.g., because of missing data or early enrollment into the OLE study) will be considered non-responders for all categorical endpoints.
[0274] The co-primary efficacy endpoints for the Maintenance Phase are: among patients who achieve CDAI-70 response at Week 14, (1) the proportion of patients in clinical remission at Week 66 and (2) the proportion of patients achieving endoscopic improvement at Week 66
Results
Patient Demographics: Induction and Maintenance Phases
[0275] As shown in Table 5 below, baseline characteristics during the induction phase were mostly balanced across treatment arms. Distributional differences were noted for sex (gender) and region.
Table 5. Demographics and Baseline Treatments During Induction Phase.
Figure imgf000078_0001
[0276] As shown in Table 6 below, baseline disease characteristics during the induction phase were mostly balanced across treatment arms. Distributional differences were noted for disease location and fecal calprotectin.
Table 6. Baseline Disease Characteristics During Induction Phase.
Figure imgf000079_0001
[0277] As shown in Table 7 below, baseline characteristics during the maintenance phase were mostly balanced across treatment arms. Distributional differences were noted for sex (gender) and region.
Table 7. Demographics and Baseline Treatments During Maintenance Phase.
Figure imgf000080_0001
Figure imgf000081_0001
[0278] As shown in Table 8 below, baseline disease characteristics during the maintenance phase were mostly balanced across treatment arms
Table 8. Baseline Disease Characteristics During Maintenance Phase.
Figure imgf000081_0002
Figure imgf000082_0002
Induction Phase Resuits
[0279] As shown in Table 9 below, the co-primary endpoint of clinical remission and endoscopic improvement at Week 14 for 210mg of etrolizumab administered at Weeks 0, 2, 4, 8, and 12 compared to placebo in patients with Crohn’s disease in the modified intent-to- treat (mITT) population was not met.
Table 9. Induction Phase Co-Primary Endpoint Results
Figure imgf000082_0001
Figure imgf000082_0003
[0280] Of the 12 secondary endpoints, none showed statistically significant and clinically meaningful results and none showed nominally significant and clinically meaningful results.
[0281] Overall etrolizumab was safe and well tolerated during the induction phase, with no new safety signals.
Maintenance Phase Results
[0282] As shown in Table 10 below, the co-primary endpoint of clinical remission and endoscopic improvement at Week 66 for 105mg of etrolizumab administered every four weeks during the maintenance phase (i.e. after the induction phase ended at Week 14) vs. placebo in patients with Crohn’s disease in the modified intent-to-treat (mITT) population was met. Table 10. Maintenance Phase Co-Primary Endpoint Results.
Figure imgf000083_0001
[0283] Of the seven secondary endpoints, two showed statistically significant and clinically meaningful results, see Table 11 below (indicated with heavy-line boxes). In addition, four of the secondary endpoints showed nominally significant and clinically meaningful results (indicated with light-line boxes).
Table 1 1. Maintenance Phase Secondary Endpoint Results.
Figure imgf000084_0001
Figure imgf000085_0001
[0284] Overall etrolizumab was safe and well tolerated during the maintenance phase, with no new safety signals.
TABLE OF SEQUENCES
Figure imgf000086_0001
Figure imgf000087_0001

Claims

WHAT IS CLAIMED IS:
1. A method of treating a patient with Crohn’s disease, the method comprising administering subcutaneously to the patient an integrin beta7 antagonist for a treatment period of at least 52 weeks or at least 66 weeks after initiation of an induction therapy, whereinl4 weeks after the initiation of the induction therapy the patient is determined to have acheived a decrease of 70 points or more from a baseline Crohn’s Disease Activity Index (CD Al) score, and wherein the patient maintains clinical remission during the treatment period.
2. The method of claim 1, wherein the patient further maintains endoscopic improvement.
3. The method of claim 1 or claim 2, wherein the patient further maintains endoscopic remission, corticosteroid-free clinical remission, or both endoscopic remission and corticosteroid-free clinical remission.
4. The method of any one of claims 1-3, wherein the patient receives corticosteroid therapy in addition to the induction therapy during an induction period, wherein the induction period is 14 weeks, and wherein at the end of the induction period the corticosteroid therapy is reduced over time until discontinuation.
5. The method of claim 4, wherein the corticosteroid therapy is (i) less than or equal to 20 mg of prednisone per day and wherein the corticosteroid therapy is reduced by 2.5 mg prednisone per week until discontinuation or (ii) less than or equal to 6 mg oral budesonide per day and wherein the corticosteroid therapy is reduced by 3 mg oral budesonide every 2 weeks until discontinuation.
6. The method of any one of claims 1-5, wherein the integrin beta7 antagonist is a monoclonal anti-integrin beta7 antibody.
7. The method of claim 6, wherein the anti-integrin beta7 antibody is a chimeric antibody or a humanized antibody.
8. The method of claim 6 or claim 7, wherein the anti-integrin beta7 antibody is an antibody fragment.
9. The method of any one of claims 6-8, wherein the anti-integrin beta7 antibody comprises three light chain hypervariable regions (HVRs), HVR-L1, HVR-L2, and HVR-L3, and three heavy chain HVRs, HVR-H1, HVR-H2, and HVR-H3, wherein:
(i) HVR-LI comprises amino acid sequence RASESVDDLLH (SEQ ID NO:1);
(ii) HVR-L2 comprises amino acid sequence KYASQSIS (SEQ ID NO:2); (iii) HVR-L3 comprises amino acid sequence QQGNSLPNT (SEQ ID NO:3);
(iv) HVR-H1 comprises amino acid sequence GFFITNNYWG (SEQ ID NO:4);
(v) HVR-H2 comprises amino acid sequence GYISYSGSTSYNPSLKS (SEQ ID NO:5); and
(vi) HVR-H3 comprises amino acid sequence RTGSSGYFDF (SEQ ID NO:6); or amino acid sequence ARTGSSGYFDF (SEQ ID NO:7).
10. The method of claim 9, wherein the anti-integrin beta7 antibody comprises a light chain variable region domain comprising the amino acid sequence set forth in SEQ ID NO: 8 and a heavy chain variable region domain comprising the amino acid sequence set forth in SEQ ID NON.
11. The method of claim 9, wherein the anti-integrin beta7 antibody comprises a light chain variable region domain comprising the amino acid sequence set forth in SEQ ID NO:8 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11 or a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 12.
12. The method of claim 9, wherein the anti-integrin beta7 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO:10 and a heavy chain variable region domain comprising the amino acid sequence set forth in SEQ ID NON.
13. The method of claim 9, wherein the anti-integrin beta7 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO:10 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11 or a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 12.
14. The method of any one of claims 9-13, wherein the anti-integrin beta7 antibody is etrolizumab.
15. The method of any one of claims 1-14, wherein the patient has moderately to severely active Crohn’s disease prior to administration of the induction therapy.
16. The method of any one of claims 1-15, wherein the induction therapy comprises one or more therapeutic agents selected from a 5-aminosalicylate, an antibiotic, budesonide, a systemic corticosteroid, a thiopurine, methotrexate, an anti-TNF agent, infliximab, adalimumab, certolizumab pegol, vedolizumab, ustekinumab, natalizumab, efalizumab, etrolizumab, a Janus kinase (JAK) inhibitor, upadacitinib, and filgotinib.
17. The method of claim 15 or claim 16, wherein the patient is determined to have (1) a CD Al score of greater than or equal to 220 and less than or equal to 480 at any time in the seven days prior to initiation of the induction therapy and (2) either an average daily liquid/soft stool frequency (SF) score greater than or equal to 6 or an average daily SF greater than 3 and an average daily abdominal pain (AP) score greater than 1 for the seven consecutive days prior to initiation of the induction therapy,
18. The method of any one of claims 15-17, wherein the patient is determined to have active inflammation, wherein the active inflammation is determined as a SES-CD score of greater than or equal to 7 as determined by ileocolonoscopy.
19. The method of any one of claims 15-18, wherein the patient has isolated ileitis or post-ileocecal resection and wherein the patient is determined to have active inflammation, wherein the active inflammation is determined as a Simplified Endoscopic Index for Crohn’s Disease (SES-CD) score of greater than or equal to 4 as determined by ileocolonoscopy.
20. The method of any one of claims 15-19, wherein the patient is refractor}' or intolerant to one or more therapies selected from immunosuppressant therapy, corticosteroid therapy, and anti-TNF therapy.
21. The method of any one of claims 15-19, wherein the patient has an inadequate response to anti-TNF therapy.
22. The method of claim 20, wherein the immunosuppressant therapy is selected from 6-mercaptopurine, azathioprine, and methotrexate.
23. The method of claim 20, wherein the corticosteroid therapy is selected from prednisone and oral budesonide.
24. The method of claim 20 or claim 21, wherein the anti-TNF therapy is selected from infliximab, adalimumab, and certolizumab pegol.
25. The method of any one of claims 1-24, wherein the anti-integrin beta7 antibody is administered at a flat dose of 105 mg every 4 weeks from week 14 after initiation of the induction therapy to at least week 52 or to at least week 66.
26. The method of any one of claims 1-25, wherein clinical remission is determined as a liquid/soft stool frequency (SF) mean daily score of less than or equal to three and an abdominal pain (AP) mean daily score of less than or equal to one with no worsening in either SF or AP compared to baseline, averaged over four days prior to assessment or averaged over seven days prior to assessment.
27. The method of any one of claims 2-26, wherein endoscopic improvement is determined by Simplified Endoscopic Index for Crohn’s Disease (SES-CD) score and wherein the SES-CD score is reduced by at least fifty percent compared to the SES-CD score determined at baseline.
28. The method of any one of ciaims 3-27, wherein the patient has discontinued corticosteroid treatment and wherein the patient does not receive treatment with one or more corticosteroids for at ieast 24 continuous weeks after discontinuing corticosteroid treatment.
29. The method of any one of claims 3-28, wherein the endoscopic remission is determined by SES-CD score and wherein the SES-CD score is less than or equal to four.
30. The method of claim 29, wherein the patient is an ileal patient and the SES-CD score is less than or equal to two.
31 . The method of claim 29 or claim 30, wherein the SES-CD score contains no segment having a subcategory score that is greater than one
32. The method of claim 31, wherein the subcategory is selected from size and extent of ulceration, affected surface, and narrowing.
33. The method according to any one of the preceding claims, wherein the integrin beta7 antagonist is administered using a prefilled syringe or a prefilled syringe and autoinjector combination.
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