WO2000040604A2 - PROCEDES ET COMPOSITIONS PERMETTANT DE MODULER LA LIBERATION DE CYTOKINES PAR LES CELLULES A EXPRESSION DE αEβ¿7? - Google Patents

PROCEDES ET COMPOSITIONS PERMETTANT DE MODULER LA LIBERATION DE CYTOKINES PAR LES CELLULES A EXPRESSION DE αEβ¿7? Download PDF

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WO2000040604A2
WO2000040604A2 PCT/US1999/030992 US9930992W WO0040604A2 WO 2000040604 A2 WO2000040604 A2 WO 2000040604A2 US 9930992 W US9930992 W US 9930992W WO 0040604 A2 WO0040604 A2 WO 0040604A2
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expressing cell
cadherin
cytokine
integrin
release
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PCT/US1999/030992
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WO2000040604A3 (fr
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Michael B. Brenner
Christina M. Parker
Michelle Woldemar Carr
Anu Arya
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The Brigham And Women's Hospital, Inc.
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Priority to AU23904/00A priority Critical patent/AU2390400A/en
Publication of WO2000040604A2 publication Critical patent/WO2000040604A2/fr
Publication of WO2000040604A3 publication Critical patent/WO2000040604A3/fr

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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/06—Methods of screening libraries by measuring effects on living organisms, tissues or cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/53—Colony-stimulating factor [CSF]
    • G01N2333/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/555—Interferons [IFN]
    • G01N2333/57—IFN-gamma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • This invention relates to methods and compositions for modulating cytokine release by ⁇ E ⁇ 7 -expressing cells, such as T lymphocytes.
  • the invention relates to methods and compositions for modulating the Th2 response of ⁇ E ⁇ 7 - expressing cells for treating disorders that are characterized by aberrant cytokine levels.
  • Integrins represent one of the best characterized families of adhesion receptors. Integrins are glycoprotein heterodimers which contain a noncovalently- associated ⁇ and ⁇ subunit. There are more than fourteen known ⁇ subunits and eight known ⁇ subunits which can pair to form at least twenty different integrin molecules. Several distinct integrin chains are capable of pairing with one type of ⁇ chain to form a ⁇ chain subfamily. Parker et al.
  • IEL intra-epithelial T lymphocytes
  • HML-1 human mucosal lymphocyte 1 antigen
  • IEL intestinal IEL
  • lamina propria T lymphocytes which lie between the epithelial basement membrane and the muscularis mucosae
  • the HML-1 antigen contains an chain (designated ⁇ E , for "epithelial associated") associated with a ⁇ 7 chain (Parker, C. et al. (1992) Proc. Natl. Acad. Sci. USA 89:1924).
  • ⁇ E ⁇ 7 integrin functions in immune system modulation by playing a role in the adhesion of peripheral lymphocytes to endothelium and in homing to lymph nodes, little, if any, is known regarding the function of ⁇ E ⁇ 7 integrin as a cell signaling molecule.
  • ⁇ E ⁇ 7 integrin in mediating the molecular processes underlying immune system modulation, and to develop improved drug therapies to replace or supplement the existing methods for treating disorders of the immune system.
  • drug therapies would be designed to reduce or prevent immune system dysfunction at its earliest stages.
  • the invention is based on the discovery of a novel function for ⁇ E ⁇ 7 integrin, namely, as a cell signaling molecule for modulating the release of cytokines by ⁇ E ⁇ 7 - expressing cells, particularly T lymphocytes.
  • ⁇ E ⁇ 7 integrin namely, as a cell signaling molecule for modulating the release of cytokines by ⁇ E ⁇ 7 - expressing cells, particularly T lymphocytes.
  • the invention provides compositions and methods for modulating the release of cytokines by ⁇ L ⁇ 7 -expressing cells (e.g., T lymphocytes, dendritic cells, mast cells) and for identifying agents which bind to ⁇ E ⁇ 7 and, thereby, modulate cytokine release.
  • cytokines e.g., T lymphocytes, dendritic cells, mast cells
  • agents which down regulate the release of Th2 cytokines, up regulate the release of Thl cytokines, and/or up regulate the release of IL-12, by stimulated ⁇ E ⁇ 7 -expressing cells are said to "block the Th2 cytokine response" by the T lymphocyte population in contact with the stimulated ⁇ E ⁇ 7 -expressing cells or their products (e.g., released cytokines).
  • Such blocking agents also are referred to herein as "antagonists" of the Th2 cytokine response or as " ⁇ E antagonists".
  • Agents which up regulate the release of Th2 cytokines, down regulate the release of Thl cytokines and/or down regulate the release of IL-12, by the T lymphocyte population in contact with the stimulated E ⁇ 7 -expressing cells or their products (e.g., released cytokines) are said to "enhance the Th2 cytokine response" by the ⁇ E ⁇ 7 -expressing cells.
  • Such agents also are referred to herein as "agonists" of the Th2 cytokine response or as " E agonists”.
  • the invention provides screening methods for identifying agents which block or enhance a Th2 cytokine response by E ⁇ 7 -expressing cells, as well as methods and compositions for using such agents in vitro and in vivo. Accordingly, the compositions and methods disclosed herein are useful for identifying other naturally occurring and synthetic molecules that selectively bind to E ⁇ 7 and. thereby, modulate cytokine release by ⁇ E ⁇ 7 -expressing cells. In addition, the invention provides screening methods to identify novel binding sites on the ⁇ E ⁇ 7 integrin to which the ⁇ E antagonists and ⁇ E agonists bind.
  • Th2 cytokine production by T lymphocytes can be increased directly or indirectly via altered cytokine production by ⁇ E ⁇ 7 -expressing cells.
  • screening methods for identifying lead compounds that modulate cytokine release by stimulated ⁇ E ⁇ 7 - expressing cells or by cells whose cytokine production is modified by ⁇ ⁇ 7 -expressing cells e.g., T lymphocytes, mast cells
  • ⁇ ⁇ 7 -expressing cells e.g., T lymphocytes, mast cells
  • the functional screening methods of the invention measure cytokine release by stimulated ⁇ E ⁇ 7 -expressing cells or by cells whose cytokine production is modified by stimulated ⁇ E ⁇ 7 -expressing cells or stimulated ⁇ E ⁇ 7 -expressing cell products (e.g., released cytokines).
  • a test compound e.g., a putative ⁇ E antagonist or ⁇ E agonist
  • a stimulated E ⁇ 7 -expressing cell refers to a cell which: (1) expresses ⁇ E ⁇ 7 integrin and (2) expresses a cytokine.
  • ⁇ E ⁇ 7 -expressing cells can be stimulated to express a cytokine by contacting the cells with a stimulating agent under conditions which permit the agent to bind to a targeted surface antigen.
  • An exemplary stimulating agent is an antibody, (e.g., an antibody to CD3, optionally, in the presence of a costimulatory molecule such as an antibody to CD28 to further enhance cytokine production), wherein the antibody is contacted with the cells under conditions which permit the antibody to selectively bind to the targeted surface antigen.
  • Other stimulating agents include mitogens (e.g., phytohaemagglutinin, "PHA”); lectins (e.g., concanavalin A, wheat germ agglutinin), phorbol esters, and calcium ionophores.
  • mitogens e.g., phytohaemagglutinin, "PHA”
  • lectins e.g., concanavalin A, wheat germ agglutinin
  • phorbol esters e.g., calcium ionophores.
  • Exemplary conditions for stimulating the ⁇ E ⁇ 7 -expressing cell are provided in the Examples.
  • the method involves: (1) performing a first cytokine release assay to release cytokines by a stimulated ⁇ L ⁇ 7 -expressing cell to obtain a first cytokine release assay result; (2) performing a second cytokine release assay in the presence of one or more test compounds to obtain a second cytokine release assay result; and (3) comparing the first and the second cytokine release assay results to determine whether the test compound modulates cytokine release by the ⁇ E ⁇ 7 -expressing cell.
  • a cytokine release assay result which shows reduced Th2 cytokine release, increased Thl cytokine release, and/or increased IL-12 cytokine release by the ⁇ E ⁇ 7 -expressing cell when the assay is conducted in the presence of the test compound (compared to the assay result obtained when the assay is performed in the absence of the test compound) indicates that the test compound blocks the Th2 cytokine response by the ⁇ E ⁇ 7 -expressing cell, (the test compound is an ⁇ E antagonist).
  • a cytokine release assay result which shows increased Th2 cytokine release, decreased Thl cytokine release, and/or decreased IL012 release by the ⁇ E ⁇ 7 -expressing cell when the assay is conducted in the presence of the test compound (compared to the assay result obtained when the assay is performed in the absence of the test compound) indicates that the test compound enhances the Th2 cytokine response by the ⁇ E ⁇ 7 -expressing cell (the test compound is an ⁇ E agonist).
  • the amount and/or identity of one or more cytokines is determined by performing a cytokine assay, many of which are available and include, ELISA, bioassay, western blot, RIA, and so forth.
  • a cytokine assay many of which are available and include, ELISA, bioassay, western blot, RIA, and so forth.
  • the same types of assays can be used to measure Th2, Thl, and Thl/Th2 cytokines.
  • the cytokine assay is an ELISA which permits high throughput screening of test compounds.
  • the Th2 cytokines which are measured include the interleukins IL-4, IL-5. IL-9, IL-10. IL- 13, and granulocyte/ macrophage-colony stimulating factor (GM-CSF). with IL-4 and IL-10 being the most preferred cytokines.
  • the Thl cytokines which are measured include interferon gamma and IL-2.
  • the Thl/Th2 cytokine, IL-3, and the dendritic cell cytokine, IL-12, also can be measured using any of the above-described assays.
  • a screening method for selecting a test compound that is an ⁇ E antagonist that blocks a Th2 cytokine response is provided.
  • the method involves: (1 ) contacting a stimulated ⁇ E ⁇ 7 -expressing cell with a test compound under conditions to permit the test compound to bind to the ⁇ E ⁇ 7 integrin expressed on the surface of the ⁇ E ⁇ 7 -expressing cell (e.g., T lymphocytes, dendritic cells, mast cells); (2) determining the amount and/or identity of one or more cytokines that are released by the F ⁇ 7 -expressing cell; and (3) selecting as the ⁇ E antagonist a test compound which decreases the release of a Th2 cytokine and/or which increases the release of a Thl cytokine or of IL-12 by the ⁇ E ⁇ 7 -expressing cell compared to the release of such cytokines by ⁇ E ⁇ 7 -expressing cells which have not been treated with the test compound.
  • a stimulated ⁇ E ⁇ 7 -expressing cell under conditions to permit the test compound to bind to the ⁇ E ⁇ 7 integrin expressed on the surface of the ⁇ E
  • the conditions which allow the test compound to bind to the ⁇ E ⁇ 7 integrin expressed on the surface of the ⁇ E ⁇ 7 -expressing cell are the same as those conditions described in U.S. Patent No. 5,160,281, issued to Brenner and Cepek (Brenner and Cepek '281) for permitting an isolated ⁇ E ⁇ 7 integrin or an ⁇ E ⁇ 7 - expressing cell to bind to an E-cadherin or an E-cadherin-expressing cell.
  • Ngents which block a Th2 cytokine response by ⁇ E ⁇ 7 -expressing cells are useful for treating a condition that is mediated by aberrant cytokine levels which present either as an abnormally elevated Th2 cytokine response or as an abnormally reduced level of Thl cytokines in ⁇ E ⁇ 7 -expressing cells.
  • Exemplary conditions that are mediated by an abnormally elevated Th2 cytokine response in ⁇ E ⁇ 7 -expressing cells include allergic diseases (e.g., allergic asthma, allergic conjunctivitis, allergic rhinitis, contact hyper sensitivity); exemplary conditions that are mediated by an abnormally reduced level of Thl cytokines include infectious diseases (e.g., tuberculosis, Helminth infection).
  • Certain inflammatory conditions such as inflammatory bowel disease (IBD) may have an abnormally elevated Th2 cytokine response or an abnormally reduced Th2 cytokine response which can be ascertained by cytokine analysis of biopsy samples and treated accordingly (discussed below).
  • a screening method for selecting a test compound that is an ⁇ E agonist for activating a Th2 cytokine response by ⁇ E ⁇ 7 - expressing cells involves: (1) contacting a stimulated ⁇ E ⁇ 7 - expressing cell with a test compound under conditions to permit the test compound to bind to the ⁇ E ⁇ 7 integrin expressed on the surface of the ⁇ E ⁇ 7 -expressing cell; (2) determining the amount and/or identity of one or more cytokines that are released by the ⁇ E ⁇ 7 -expressing cell; and (3) selecting as the ⁇ E agonist a test compound which increases the release of a Th2 cytokine and/or which decreases the release of a THl cytokine by the ⁇ E ⁇ 7 -expressing cell compared to the release of such cytokines by ⁇ E ⁇ 7 -expressing cells which have not been treated with the test compound.
  • the conditions which permit the test compound to bind to the ⁇ E ⁇ 7 integrin expressed on the surface of the ⁇ E ⁇ 7 -expressing cell are as described in Brenner and Cepek '281 and in the Examples.
  • Agents which enhance a Th2 cytokine response by ⁇ E ⁇ 7 -expressing cells are useful for treating a condition that is mediated by aberrant cytokine levels which present either as a reduction in release of Th2 cytokines by an ⁇ E ⁇ 7 -expressing cell or as an increase of THl cytokines in such cells.
  • exemplary conditions that are mediated by an abnormal decrease in release of Th2 cytokines by ⁇ E ⁇ 7 -expressing cells include inflammatory autoimmune conditions (e.g., rheumatoid arthritis, multiple sclerosis, type 1 diabetes, psoriasis, and inflammatory bowel disease (IBD)).
  • Inflammatory bowel disease can also present as a condition that is mediated by an abnormal increase in the release of Th2 cytokines by ⁇ E ⁇ 7 -expressing cells. Accordingly, to determine whether an ⁇ E ⁇ agonist or an ⁇ E antagonist should be selected for IBD treatment, it is preferably to perform a biopsy and determine which cytokines are being produced by the particular patient's cells and then treat accordingly.
  • a biopsy See, e.g., Strober, W., et al., "Mucosal immunoregulation and inflammatory bowel disease: new insights from murine models of inflammation", Scand. J. Immunol, vol 48(5):453-8 (1998).
  • a screening method to identify additional binding sites on ⁇ E ⁇ 7 which do not bind to any portion of E- cadherin but which play a role in cytokine release by ⁇ E ⁇ 7 -expressing cells is provided.
  • U.S. Patent No. 5,610,281 (Brenner and Cepek '281 ) reports that E- cadherin is the heterotypic cognate of ⁇ E ⁇ 7 integrin, we believe that ⁇ E ⁇ 7 integrin has one or more additional binding sites (some of which bind E-cadherin) which are not involved in intercellular adhesion.
  • first E-cadherin-binding site refers to the ⁇ E ⁇ 7 integrin binding site which is involved in adhesion to an E-cadherin-expressing cell.
  • second E-cadherin- binding site refers to an additional ⁇ E ⁇ 7 integrin binding site which binds to E- cadherin; however, the binding of which does not affect adhesion between an ⁇ L ⁇ 7 - expressing cell (e.g., T lymphocytes, dendritic cells, mast cells) and an E-cadherin- expressing cell.
  • Fragments of E-cadherin which bind to the second E-cadherin- binding site and which do not bind to the first E-cadherin-binding site can be identified, for example, by screening the fragments in an adhesion assay as described in Brenner and Cepek '281 (to identify fragments which do not modulate intercellular adhesion) and in the screening assays disclosed herein (to identify fragments which modulate cytokine release by an ⁇ E ⁇ 7 + -expressing cell).
  • an antibody assay for screening a molecular library is provided.
  • the antibody assay is useful for identifying pharmaceutical lead compounds that modulate cytokine release by ⁇ E ⁇ 7 -expressing cells.
  • the antibody assay is performed, for example, by contacting a stimulated ⁇ E ⁇ 7 - expressing cell with an antibody (known to selectively bind to the ⁇ 7 integrin and block (down regulate) or enhance (up regulate) cytokine release by the ⁇ E ⁇ 7 -expressing cell) in the presence of at least one test compound and determining whether the test compound modulates ⁇ E ⁇ 7 -mediated cytokine release by the ⁇ E ⁇ 7 -expressing cell (e.g., by competing with the antibody or mimicking the antibody).
  • an antibody known to selectively bind to the ⁇ 7 integrin and block (down regulate) or enhance (up regulate) cytokine release by the ⁇ E ⁇ 7 -expressing cell
  • the assay can be performed with unstimulated ⁇ E ⁇ 7 -expressing cells, ⁇ E ⁇ 7 integrin, the ⁇ E subunit, the ⁇ 7 subunit, fragments of the ⁇ E ⁇ 7 integrin or fragments of its subunits, in the presence and absence of at least one test compound by determining whether the test compound affects the binding of the antibody to the ⁇ E ⁇ 7 -expressing cells (or to an isolated ⁇ E ⁇ 7 integrin, its isolated subunits or fragmentSOf the foregoing).
  • the antibody assay is performed under conditions which permit the antibody and the test compound to bind to the ⁇ E ⁇ 7 integrin (expressed on the surface of the cell or isolated ⁇ E ⁇ 7 integrin, its isolated subunits or fragments of the foregoing).
  • a particularly preferred fragment of the ⁇ 1 ⁇ 7 integrin or its subunits is a fusion protein that contains part of the integrin coupled to another protein. More preferably, the fusion protein contains the extracellular domain of the ⁇ E subunit (e.g., the I domain as discussed in U.S. Patent No. 5,594,120, issued to Brenner and Parker) coupled to an immunoglobulin chain).
  • Binding polypeptides other than antibodies also can be used in place of the antibodies in the above-described assay.
  • fragments of E-cadherin or an E-cadherin fusion protein can be substituted for the antibodies in the antibody screening assay to achieve the same purpose.
  • Various types of molecular libraries can be screened in this manner, including peptide libraries and chemical combinatorial libraries. The person of ordinary skill in the art is well versed in the types of various libraries that can be screened.
  • a method for modulating cytokine release by ⁇ E ⁇ 7 -expressing cells in vitro or in vivo is provided.
  • the method involves contacting the stimulated ⁇ E ⁇ 7 -expressing cells with an agent (e.g., an isolated binding polypeptide) that blocks or enhances cytokine release by the ⁇ E ⁇ 7 - expressing cell.
  • an agent e.g., an isolated binding polypeptide
  • the method is performed under conditions which permit the agent to bind to the ⁇ E ⁇ 7 integrin expressed on the surface of the ⁇ E ⁇ 7 -expressing cell. Such conditions are described in the Examples and in Brenner and Cepek '281.
  • a method for treating a subject having a condition that is mediated by an increase in release of Th2 cytokines by an ⁇ E ⁇ 7 -expressing cell or by a decrease in THl cytokines by an ⁇ E ⁇ 7 -expressing cell is- provided.
  • the method involves: (1) administering to the subject an effective amount of a therapeutic agent to block a Th2 cytokine response by the ⁇ E ⁇ 7 -expressing cell.
  • the therapeutic agent does not also modulate adhesion between ⁇ E ⁇ 7 integrin and an E-cadherin.
  • the condition is one which is not mediated by aberrant adhesion between an ⁇ E ⁇ 7 -expressing cell and an E-cadherin expressing cell.
  • exemplary conditions that can be treated in accordance with this method include allergic diseases (e.g., allergic asthma) and infectious diseases (e.g., tuberculosis).
  • the therapeutic agent is an ⁇ E antagonist, more preferably, an E ⁇ 7 - binding polypeptide, most preferably, an antibody or fragment thereof which selectively binds to ⁇ E ⁇ 7 integrin at a site other than the first E-cadherin-binding site.
  • exemplary antibodies which can be evaluated to determine whether they can be used for this purpose include HML-1 ; Be ⁇ Nct-8; Alpha E7-1 , -2, and -3; Fib 504; and M293.
  • the ⁇ E ⁇ 7 - binding polypeptide is a fragment corresponding to the CDR3 region of the exemplary antibodies.
  • the ⁇ E ⁇ 7 - binding polypeptide is a fragment of an extracellular domain of E-cadherin which does not bind to the first E-cadherin-binding site of ⁇ E ⁇ 7 integrin.
  • a method for treating a condition that is mediated by a decrease in ⁇ E ⁇ 7 T lymphocyte release of Th2 cytokines or an increase in THl cytokines or an increase in IL-12 is provided.
  • the method involves (1) administering to the subject an effective amount of a therapeutic agent to enhance a Th2 cytokine response by the ⁇ E ⁇ 7 -expressing cells.
  • the therapeutic agent does not also modulate binding between ⁇ E ⁇ 7 integrin and an E-cadherin.
  • the conditions that can be treated in accordance with this embodiment are not mediated by aberrant adhesion between an ⁇ E ⁇ 7 -expressing cell and an E-cadherin expressing cell.
  • Exemplary conditions include inflammatory autoimmune conditions (e.g., rheumatoid arthritis, multiple sclerosis, type 1 diabetes, psoriasis, and inflammatory bowel disease (IBD)).
  • the therapeutic agent is an E agonist, more preferably, an ⁇ E ⁇ 7 - binding polypeptide such as an antibody or fragment thereof.
  • the ⁇ E ⁇ 7 -binding polypeptide is a fragment of an antibody corresponding to the CDR3 region of an antibody which is useful for this aspect of the invention.
  • the ⁇ E ⁇ 7 -binding polypeptide is a fragment of an extracellular domain of E-cadherin which does not bind to the first E-cadherin-binding site of ⁇ E ⁇ 7 integrin.
  • a pharmaceutical composition for treating a condition that is mediated by aberrant cytokine release by ⁇ E ⁇ 7 -expressing cells includes a pharmaceutically acceptable carrier and a therapeutic agent that is an ⁇ E antagonist or an ⁇ E agonist.
  • the therapeutic agent is an ⁇ E ⁇ 7 -binding polypeptide that selectively binds to ⁇ E ⁇ 7 integrin (more preferably, at a site other than the first E-cadherin-binding site) and, thereby, modulates cytokine release by the ⁇ ⁇ 7 -expressing cell.
  • the therapeutic agent e.g., an isolated binding polypeptide
  • the ⁇ 1 ⁇ 7 - binding polypeptide is an antibody, an antibody fragment or a soluble E-cadherin fragment.
  • isolated binding polypeptides that are capable of modulating (blocking or activating) a Th2 cytokine response by E ⁇ 7 -expressing cells.
  • the isolated binding polypeptide selectively binds to an ⁇ E ⁇ 7 integrin, preferably at a binding site that does not involve E-cadherin mediated intercellular adhesion.
  • the isolated binding polypeptides of the invention include antibodies and antibody fragments which specifically recognize and selectively bind to ⁇ E ⁇ 7 and, thereby, modulate the cytokine release by ⁇ E ⁇ 7 - expressing cells.
  • the antibodies do not modulate E-cadherin-mediated intercellular adhesion.
  • Exemplary antibodies which can be evaluated to determine whether they can be used for this purpose include HML-1 ; BerAct-8; Alpha E7- 1, -2, and -3; Fib 504; and M293.
  • HML-1 HML-1
  • BerAct-8 Alpha E7- 1, -2, and -3
  • Fib 504 Fib 504
  • M293 See also Brenner and Cepek U.S. 5,610,281 and Brenner and Parker 5,594,120, and their respective file histories and documents contained therein for further examples of antibodies that can be evaluated for this purpose.
  • Humanized monoclonal antibodies also can be prepared by those of ordinary skill in the art using no more than routine experimentation. (See, for example, PCT Application No. US93/02479, Publication No. 93/19197, published 9/30/93 and N.
  • the invention also contemplates the use of the antagonists and agonists of the invention in experimental model systems to determine the role that the Th2 cytokine response by ⁇ E ⁇ 7 -expressing cells plays in mediating an immune system disorder.
  • immune system disorders can be induced in animal models such as those described in the Examples.
  • An antagonist or agonist of the invention as described above then is administered to the animal by, e.g., local or systemic administration, followed by monitoring the animal's response, and comparing the response to control animals that have not received the antagonist or agonist of the invention. In this manner, the role that the Th2 cytokine response by ⁇ E ⁇ 7 -expressing cells plays in mediating the disorder can be determined.
  • ⁇ E -/- mice have decreased total cell numbers in the airways as measured by bronchoalveolar lavage (BAL).
  • BAL bronchoalveolar lavage
  • Figure 2 ⁇ E -/- mice have decreased numbers of lymphocytes and eosinophils in the airways. +/+ and ⁇ E -/- mice were sensitized intraperitoneally with OVA then challenged with aerosolized OVA.
  • ⁇ E -/- mice are less reactive to the bronchoconstrictor, methacholine, than ⁇ E +/+ mice.
  • mice were anesthetized, placed in a plethysmograph, and airway resistance in response to increasing doses of methacholine were measured.
  • Each dose response curve was log-transformed and subjected to regression analysis to interpolate the dose required to cause a two-fold increase in lung resistance (log ED 200 R L )-
  • FIG. 4 ⁇ E +/+mice treated with mAb to Ae have decreased total cell numbers in the airways and are less reactive to bronchoconstrictor.
  • Figure 7 Levels of the eosinophils chemoattractant, eotaxin, are reduced in ⁇ E -/-mice or in ⁇ E +/+ mice treated with anti- ⁇ E mAbs.
  • Exotaxin levels from BAL supernatant from ⁇ E -/- vs. ⁇ E +/+ mice (A) or E +/+ mice treated with anti- ⁇ E mAb vs. Control mAb (B) were quantitated using a commercially available ELISA kit; N value is 12;
  • FIG. 8 Splenic T cells derived from ⁇ E -/- mice produced altered profile of cytokines in response to plate bound anti-CD3 as compared to ⁇ ' +/+ T cells.
  • A Spleen derived mononuclear cells were isolated by mechanical disruption and stimulated with varying concentrations of plate bound anti-CD3 ( 145-2C 1 1 ).
  • cytokine concentration was harvested and tested for cytokine concentration by sandwich ELISA, using cytokine antibody pairs available from Pharmingen (IL-2 (JES6-1 A12/JES6-5H4-Bi), IFN- ⁇ (R4-6A2/XMG1.2-Bi) and anti- IL-3 (MP2-8F8/MP2-43D11 -Bi) in comparison with a standard curve determine using recombinant cytokine as described by the manufacturer with the exception that the biotinylated anti-cytokine antibody was detected using streptavidin-alkaline phospatase and para-nitophenyl phosphate as substrate. The outcome of these experiments was graphed.
  • IL-2 JES6-1 A12/JES6-5H4-Bi
  • IFN- ⁇ R4-6A2/XMG1.2-Bi
  • MP2-8F8/MP2-43D11 -Bi anti-IL-3
  • CD4 + T lymphocytes were isolated from ⁇ E +/+ or ⁇ E -/- mice and T cell depleted splenocytes were isolated as antigen presenting cells (APC). Then, the T cells and APC were mixed in the four different possible combinations for in vitro stimulation with plate- bound anti-CD3.
  • the T cells of a given genotype produced IL- 4 similarly when combined with either ⁇ E +/+ or ⁇ E -/- APC.
  • E +/+ T cells produced more IL-4 than ⁇ E -/- T cells.
  • FIG. 9 T. spiralis induced reactive mast cell hyperplasia is accentuated and worm expulsion is accelerated in integrin ⁇ E -/- mice, despite lower numbers of mucosal T lymphocytes. Integrin ⁇ E -/- and ⁇ E +/+ mice of similar genetic background were inoculated with 500 T. spiralis larvae. At each timepoint, 3 mice of each genotype were sacrificed, worm burden was determined, and worm burden was quantitated. Error bars indicate the standard deviation.
  • Figure 10 shows the survival after infection with Mycobacterium tuberculosis for ⁇ E -/- animals and ⁇ E +/+ animals.
  • Th2 cytokine response When T lymphocytes are stimulated, they can differentiate toward either Thl or Th2 cytokine production.
  • the release of Th2 cytokines is referred to herein as a "Th2 cytokine response".
  • the invention is based on the discovery of the role played by ⁇ E ⁇ 7 integrin in mediating cytokine release by E ⁇ 7 -expressing cells and, in particular, of the role played by ⁇ E ⁇ 7 integrin in mediating a Th2 cytokine response in ⁇ E ⁇ 7 -expressing cells.
  • the discovery is based, in part, on the results of in vivo experiments which illustrate the release of cytokines by ⁇ E ⁇ 7 -expressing cells. (See, the Examples).
  • the first in vivo experiment employed an animal model which is recognized by those skilled in the art as predictive of an allergic disease such allergic asthma.
  • the second and third in vivo experiments employed an animal model which also is recognized by those skilled in the art as predictive of an infectious disease.
  • the - invention provides methods for identifying and using both antagonists (" ⁇ E antagonists”) and agonists (" ⁇ E agonists”) which block and enhance, respectively, the Th2 cytokine response by ⁇ E ⁇ 7 -expressing cells in vivo and in vitro.
  • the present invention relates to the discovery that ⁇ F ⁇ 7 integrin plays a role in mediating cytokine release by ⁇ E ⁇ 7 -expressing cells.
  • compositions and methods disclosed herein are useful for identifying other naturally occurring and synthetic molecules that selectively bind to ⁇ E ⁇ 7 and, thereby, modulate cytokine release and for identifying regions of the integrin and integrin subunits to which these molecules bind.
  • screening methods for identifying lead compounds that modulate cytokine release by stimulated ⁇ E ⁇ 7 - expressing cells are provided.
  • the screening methods of the invention measure cytokine release by stimulated ⁇ E ⁇ 7 -expressing cells. These assays are performed under conditions which allow a test compound (e.g., a putative ⁇ E antagonist or ⁇ E agonist) to bind to the ⁇ E ⁇ 7 integrin expressed on the surface of a stimulated ⁇ E ⁇ 7 -expressing cell and, thereby, modulate cytokine release by the cell.
  • a test compound e.g., a putative ⁇ E antagonist or ⁇ E agonist
  • a stimulated ⁇ E ⁇ 7 -expressing cell refers to a cell which: (1) expresses ⁇ E ⁇ 7 integrin and (2) expresses a cytokine.
  • ⁇ E ⁇ 7 -expressing cells can be stimulated to express a cytokine by contacting the cells with a stimulating agent under conditions which permit the agent to bind to a targeted surface antigen.
  • An exemplary stimulating agent is an antibody, (e.g., an antibody to CD3, optionally, in the presence of a costimulatory molecule such as an antibody to CD28 to further enhance cytokine production), wherein the antibody is contacted with the cells under conditions which permit the antibody to selectively bind to the targeted surface antigen.
  • stimulating agents include mitogens (e.g., phytohaemagglutinin, "PHA”); lectins (e.g., concanavalin A, wheat germ agglutinin), phorbol esters, and calcium ionophores.
  • mitogens e.g., phytohaemagglutinin, "PHA”
  • lectins e.g., concanavalin A, wheat germ agglutinin
  • phorbol esters e.g., calcium ionophores.
  • Exemplary conditions for stimulating the ⁇ E ⁇ 7 -expressing cell are provided in the Examples.
  • cytokine assays for determining the amount and/or identity of one or more cytokines are well known to those of ordinary skill in the art and include, e.g., ELISA assays, bioassay, western blot, RIA, and so forth. .
  • the method involves: (1) performing a first cytokine release assay to measure cytokine release by a stimulated E ⁇ 7 - expressing cell to obtain a first cytokine release assay result; (2) performing a second cytokine release assay in the presence of a test compound to obtain a second cytokine release assay result; and (3) comparing the first and the second cytokine release assay results to determine whether the test compound modulates cytokine release by the ⁇ 1 ⁇ -expressing cell.
  • An exemplary cytokine assay to identify and/or measure the amount of cytokines released by the ⁇ E ⁇ 7 -expressing cells is provided in the Examples.
  • cytokine release assay result which shows reduced Th2 cytokine release, increased Thl cytokine release, and/or increased IL-12 cytokine release by the ⁇ F ⁇ 7 -expressing cell when the assay is conducted in the presence of the test compound (compared to the assay result obtained when the assay is performed in the absence of the test compound) indicates that the test compound blocks the Th2 cytokine response by the ⁇ E ⁇ 7 -expressing cell.
  • a cytokine release assay result which shows increased Th2 cytokine release, decreased Thl cytokine release, and/or decreased IL012 release by the ⁇ E ⁇ 7 -expressing cell when the assay is conducted in the presence of the test compound (compared to the assay result obtained when the assay is performed in the absence of the test compound) indicates that the test compound enhances the Th2 cytokine response by the ⁇ E ⁇ 7 -expressing cell.
  • agents which down regulate the release of Th2 cytokines by stimulated E ⁇ 7 -expressing cells are said to "block" the Th2 cytokine response by the ⁇ E ⁇ 7 -expressing cell.
  • blocking agents also are referred to herein as "antagonists" of the Th2 cytokine response or " ⁇ E antagonists”.
  • Agents which up regulate the release of Th2 cytokines by ⁇ E ⁇ 7 -expressing cells are said to "enhance" the Th2 cytokine response by the ⁇ E ⁇ 7 -expressing cell.
  • agents which up regulate the release of Th2 cytokine response by the ⁇ E ⁇ 7 -expressing cell are said to "enhance" the Th2 cytokine response by the ⁇ E ⁇ 7 -expressing cell.
  • agents also are referred to herein as "agonists" of the Th2 cytokine response or " ⁇ E agonists”.
  • the amount and/or identity of one or more cytokines released by the ⁇ E ⁇ 7 - expressing cell is determined by performing a cytokine assay, many of which are available and include, ELISA, bioassay, western blot, RIA, and so forth.
  • a cytokine assay many of which are available and include, ELISA, bioassay, western blot, RIA, and so forth.
  • the same types of assays can be used to measure Th2, Thl, and Thl/Th2 cytokines.
  • the cytokine assay is an ELISA which permits high throughput screening of test compounds.
  • the Th2 cytokines which are measured include the interleukins IL-4, IL-5, IL-9, IL-10, IL-13, and granulocyte/ macrophage-colony stimulating factor (GM-CSF), with IL-4 and IL-10 being the most preferred cytokines.
  • the Thl cytokines which are measured include interferon gamma and IL-2.
  • the Thl/Th2 cytokine, IL-3, and the dendritic cell cytokine, IL-12 also can be measured using any of the above-described assays.
  • a screening method for selecting a test compound that is an ⁇ E antagonist for blocking a Th2 cytokine response by an ⁇ E ⁇ 7 - expressing cell involves: (1) contacting a stimulated E ⁇ 7 - expressing cell with a test compound under conditions to permit the test compound to bind to the ⁇ E ⁇ 7 integrin expressed on the surface of the ⁇ ⁇ 7 -expressing cell (e.g., T lymphocytes, dendritic cells, mast cells); (2) determining the amount and/or identity of one or more cytokines that are released by the ⁇ E ⁇ 7 -expressing cell; and (3) selecting as the ⁇ E antagonist a test compound which decreases the release of a Th2 cytokine and/or which increases the release of a Thl cytokine or of IL-12 by the ⁇ E ⁇ 7 - expressing cell compared to the release of such cytokines by ⁇ E ⁇ 7 -expressing cells which have not been contacted with the test
  • the conditions which allow the test compound to bind to the ⁇ E ⁇ 7 integrin expressed on the surface of the ⁇ E ⁇ 7 -expressing cell are the same as those conditions described in U.S. Patent No. 5, 160,281, issued to Brenner and Cepek (Brenner and Cepek '281) for permitting an isolated ⁇ E ⁇ 7 integrin or an E ⁇ 7 -expressing cell to bind to an E-cadherin or an E- cadherin expressing cell. (See also the Examples.)
  • Agents which block a Th2 cytokine response by an ⁇ E ⁇ 7 -expressing cell are useful for treating a condition that is mediated by aberrant cytokine levels which present either as an abnormally elevated Th2 cytokine response or as an abnormally reduced level of THl cytokines in ⁇ E ⁇ 7 -expressing cells.
  • aberrant means an amount which is different from that which is present in the E ⁇ 7 -expressing cells of a subject which has not been diagnosed as having an adverse immune system disorder such as those disclosed herein.
  • Exemplary conditions that are mediated by an abnormally elevated Th2 cytokine response by ⁇ E ⁇ 7 -expressing cells include allergic diseases (e.g., allergic asthma, allergic conjunctivitis, allergic rhinitis, contact hypersensitivity); exemplary conditions that are mediated by an abnormally reduced level of Thl cytokines include infectious diseases (e.g., tuberculosis, Helminth infection).
  • allergic diseases e.g., allergic asthma, allergic conjunctivitis, allergic rhinitis, contact hypersensitivity
  • exemplary conditions that are mediated by an abnormally reduced level of Thl cytokines include infectious diseases (e.g., tuberculosis, Helminth infection).
  • the screening method is performed under conditions which allow the test compound to bind to the ⁇ E ⁇ 7 integrin expressed on the surface of a stimulated ⁇ E ⁇ 7 -expressing cell.
  • Exemplary conditions for stimulating the ⁇ E ⁇ 7 - expressing cell and for allowing the test compound to bind to the ⁇ E ⁇ 7 -expressing cell are provided in the Examples.
  • the binding conditions which allow the test compound to bind to the ⁇ E ⁇ 7 integrin expressed on the surface of the ⁇ E ⁇ 7 -expressing cell are the same as those conditions described in U.S. Patent No.
  • a screening method for selecting a test compound that is an ⁇ E agonist for activating a Th2 cytokine response by an ⁇ E ⁇ 7 -expressing cell is provided.
  • the method involves: (1) contacting a stimulated ⁇ E ⁇ 7 -expressing cell with a test compound under conditions to permit the test compound to bind to the ⁇ E ⁇ 7 integrin expressed on the surface of the ⁇ E ⁇ 7 - expressing cell; (2) determining the amount and/or identity of one or more cytokines that are released by the ⁇ E ⁇ 7 -expressing cell; and (3) selecting as the ⁇ E agonist a test compound which increases the release of a Th2 cytokine and/or which decreases the release of a THl cytokine by the ⁇ E ⁇ 7 -expressing cell compared to the release of such cytokines by ⁇ E ⁇ 7 -expressing cells which have not been contacted with the test compound.
  • Ngents which enhance the Th2 cytokine response by the ⁇ E ⁇ 7 -expressing cell are useful for treating a condition that is mediated by aberrant cytokine levels which present either as a reduction in release of Th2 cytokines by an ⁇ E ⁇ 7 -expressing cell or as an increase of THl cytokines in such cells.
  • exemplary conditions that are mediated by a decrease in release of Th2 cytokines by the ⁇ E ⁇ 7 -expressing cell include inflammatory autoimmune conditions (e.g., rheumatoid arthritis, multiple sclerosis, type 1 diabetes, psoriasis, and inflammatory bowel disease).
  • the screening method is a cytokine release assay which is performed under conditions which allow the test compound to bind to the ⁇ E ⁇ 7 integrin expressed on the surface of a stimulated ⁇ E ⁇ 7 -expressing cell.
  • the conditions which permit the test compound to bind to the ⁇ E ⁇ 7 integrin expressed on the surface of the ⁇ E ⁇ 7 -expressing cell are as described in Brenner and Cepek '281 and in the Examples.
  • an antibody assay for screening a molecular library is provided.
  • the antibody assay is useful for identifying pharmaceutical lead compounds that modulate cytokine release by ⁇ E ⁇ 7 -expressing cells.
  • the antibody assay is performed, for example, by contacting a stimulated E ⁇ 7 - expressing cell with an antibody (known to selectively bind to the ⁇ E ⁇ 7 integrin and block (down regulate) or enhance (up regulate) cytokine release by the ⁇ E ⁇ 7 -expressing cell) in the presence of at least one test compound and determining whether the test compound modulates E ⁇ 7 -mediated cytokine release by the ⁇ E ⁇ 7 -expressing cell (e.g., by competing with the antibody or mimicking the antibody).
  • an antibody known to selectively bind to the ⁇ E ⁇ 7 integrin and block (down regulate) or enhance (up regulate) cytokine release by the ⁇ E ⁇ 7 -expressing cell
  • the assay can be performed with unstimulated ⁇ E ⁇ 7 -expressing cells, ⁇ E ⁇ 7 integrin, the E subunit, the ⁇ 7 subunit, fragments of the ⁇ E ⁇ 7 integrin or fragments of its subunits, in the presence and absence of at least one test compound by determining whether the test compound affects the binding of the antibody to the E ⁇ 7 -expressing cells (or to an isolated ⁇ E ⁇ 7 integrin, its isolated subunits or fragments of the foregoing).
  • the antibody assay is performed under conditions which permit the antibody and the test compound to bind to the E ⁇ 7 integrin (expressed on the surface of the cell or isolated ⁇ E ⁇ 7 integrin, its isolated subunits or fragments of the foregoing).
  • a particularly preferred fragment of the ⁇ E ⁇ 7 integrin or its subunits is a fusion protein that contains part of the integrin coupled to another protein. More preferably, the fusion protein contains the extracellular domain of the ⁇ E subunit (e.g., the "I" domain as discussed in the Brenner and Parker U.S. 5,594,120) coupled to an immunoglobulin chain). Binding polypeptides other than antibodies also can be used in place of the antibodies in the above-described assay.
  • fragments of E-cadherin or an E-cadherin fusion protein can be substituted for the antibodies in this screening assay to achieve the same purpose.
  • Various types of molecular libraries can be screened in this manner, including peptide libraries and chemical combinatorial libraries. The person of ordinary skill inthe art is well versed in the types of various libraries that can be screened.
  • a method for modulating cytokine release by ⁇ E ⁇ 7 -expressing cells in vitro or in vivo involves contacting the stimulated E ⁇ 7 -expressing cells with an antagonist or an agonist that blocks or enhances, respectively, a Th2 cytokine response by the ⁇ E ⁇ 7 - expressing cell.
  • the method is performed under conditions that permit the antagonists or agonists and the integrin to bind selectively to one another. Such conditions are described in the Examples and in Brenner and Cepek '281.
  • a method for treating a subject having a condition that is mediated by an increase in release of Th2 cytokines or by a decrease in THl cytokines by an ⁇ E ⁇ 7 -expressing cell involves: (1) administering to the subject an effective amount of a therapeutic agent to block a Th2 cytokine response by the E ⁇ 7 -expressing cell.
  • the therapeutic agent does not also modulate adhesion between ⁇ E ⁇ 7 integrin and an E-cadherin.
  • the condition is one which is not mediated by aberrant adhesion between an ⁇ E ⁇ 7 -expressing cell and an E-cadherin expressing cell.
  • Exemplary conditions that can be treated in accordance with this method include allergic disease (e.g., allergic asthma) and infectious disease (e.g., tuberculosis).
  • the therapeutic agent is an ⁇ E antagonist, more preferably, an ⁇ E ⁇ 7 - binding polypeptide, and most preferably, an antibody or fragment thereof which selectively binds to ⁇ E ⁇ 7 integrin at a site other than the first E-cadherin-binding site.
  • exemplary antibodies which can be evaluated to determine whether they can be used for this purpose include HML-1; BerAct-8; Alpha E7-1, -2, and -3; Fib 504; and M293.
  • the ⁇ E ⁇ 7 -binding polypeptide is the CDR3 region of a selected binding antibody.
  • the ⁇ E ⁇ 7 -binding polypeptide is a fragment of an extracellular domain of E-cadherin which does not bind to the first E- cadherin-binding site of E ⁇ 7 integrin.
  • a method for treating a condition that is mediated by a decrease in a Th2 cytokine response by an E ⁇ 7 -expressing cell or an increase in THl cytokines or an increase in IL-12 in the ⁇ E ⁇ 7 -expressing cell involves administering to the subject an effective amount of a therapeutic agent to enhance a Th2 cytokine response by the ⁇ E ⁇ 7 -expressing cells.
  • the therapeutic agent does not also modulate binding between ⁇ E ⁇ 7 integrin and an E-cadherin.
  • the conditions that can be treated in accordance with this embodiment are not mediated by aberrant adhesion between an ⁇ E ⁇ 7 - expressing cell and an E-cadherin expressing cell.
  • exemplary conditions include inflammatory autoimmune conditions (e.g., rheumatoid arthritis, multiple sclerosis. type 1 diabetes, psoriasis, and inflammatory bowel disease).
  • the therapeutic agent is an ⁇ F agonist, more preferably, an ⁇ E ⁇ -,- binding polypeptide such as an antibody or fragment thereof.
  • the ⁇ E ⁇ 7 - binding polypeptide is the CDR3 region of an antibody which is useful for this aspect of the invention.
  • the ⁇ E ⁇ 7 -binding polypeptide is a fragment of an extracellular domain of E-cadherin which does not bind to the first E-cadherin-binding site of ⁇ E ⁇ 7 integrin.
  • a pharmaceutical composition for treating a condition that is mediated by aberrant cytokine release by ⁇ E ⁇ 7 -expressing cells includes a pharmaceutically acceptable carrier and a therapeutic agent that is an ⁇ E antagonist or ⁇ E agonist.
  • the ⁇ E antagonist or ⁇ E agonist is an ⁇ E ⁇ 7 -binding polypeptide that selectively binds to ⁇ E ⁇ 7 integrin (more preferably, at a site other than the first E- cadherin-binding site) and, thereby, modulates cytokine release by the ⁇ E ⁇ 7 -expressing cell.
  • the therapeutic agent e.g., an isolated binding polypeptide
  • the ⁇ E ⁇ 7 -binding polypeptide is an antibody, an antibody fragment or a soluble E-cadherin fragment.
  • isolated binding polypeptides that are capable of modulating (blocking or activating) a Th2 cytokine response by ⁇ E ⁇ 7 -expressing cells.
  • the isolated binding polypeptide selectively binds to an ⁇ E ⁇ 7 integrin, preferably at a binding site that does not involve E-cadherin mediated intercellular adhesion.
  • the isolated binding polypeptides of the invention include antibodies and antibody fragments which specifically recognize and selectively bind to ⁇ E ⁇ 7 and, thereby, modulate the cytokine release by ⁇ E ⁇ 7 - expressing cells.
  • the antibodies do not modulate E-cadherin-mediated intercellular adhesion.
  • Exemplary antibodies which can be evaluated to determine whether they can be used for this purpose include HML-1 ; BerAct-8; Alpha E7- 1, -2, and -3; Fib 504; and M293.
  • HML-1 HML-1
  • BerAct-8 Alpha E7- 1, -2, and -3
  • Fib 504 Fib 504
  • M293 See also Brenner and Cepek U.S. 5,610,281 and Brenner and Parker U.S. 5,594,120, and their respective file histories and documents contained therein for further examples of antibodies that can be evaluated for this purpose.
  • Humanized monoclonal antibodies also can be prepared by those of ordinary skill in the art using no more that routine experimentation. (See, for example, PCT Application No. US93/02479, Publication No. 93/19197, published 9/30/93 and N. Lowberg, et al., Nature 368:856-859 (1994), the contents of which publications are incorporated herein by reference).
  • binding polypeptides of the invention are useful in the above-described screening assays for identifying pharmaceutical lead compounds in molecular libraries.
  • a "molecular library” refers to a collection of structurally-diverse molecules. Molecular libraries can be chemically-synthesized or recombinantly-produced.
  • a "molecular library member” refers to a molecule that is contained within the molecular library. Accordingly, screening refers to the process by which single molecules or mixtures of molecules such as library molecules are tested for the ability to modulate (up regulate or down regulate) cytokine release by ⁇ E ⁇ 7 -expressing cells.
  • a "pharmaceutical lead compound” refers to a molecule which is capable of modulating such cytokine release.
  • the screening assays disclosed herein are useful for assessing the ability of a library molecule to block or enhance the Th2 cytokine response in ⁇ E ⁇ 7 -expressing cells.
  • Libraries of molecularly diverse molecules can be prepared using chemical and/or recombinant technology. Such libraries for screening include recombinantly- produced libraries of fusion proteins.
  • An exemplary recombinantly-produced library is prepared by ligating fragments of E-cadherin cDNA into, for example, the pGEX- 2T vector (Pharmacia, Piscataway, NJ). This vector contains the carboxy terminus of glutathione S-transferase (GST) from Schistosoma japonicum.
  • GST glutathione S-transferase
  • Use of the GST- containing vector facilitates purification of GST-E-cadherin fusion proteins from bacterial lysates by affinity chromatography on glutathione sepharose.
  • E-cadherin fusion proteins can be tested for ⁇ E antagonist or ⁇ E agonist activity using, for example, the above-described screening methods. Fusion proteins which block or enhance the Th2 cytokine response by ⁇ E ⁇ 7 -expressing cells can be selected as pharmaceutical lead compounds and/or to facilitate further characterization of the portion of E-cadherin which binds to ⁇ ⁇ 7 and, thereby, modulates cytokine release.
  • An exemplary E-cadherin fusion protein which binds to ⁇ E ⁇ 7 and modulates the Th2 cytokine response of ⁇ E ⁇ 7 -expressing cells is described in the Examples.
  • the isolated binding polypeptides of the invention also include peptides that are related to, or derived from, the extracellular domain of E-cadherin, i.e., amino acids 1-552 (inclusive) of Sequence I.D. Nos.l (cDNA and encoded protein) and 2 (encoded protein) as described in U.S. Patent No. 5,610,281.
  • first E-cadherin-binding site refers to the ⁇ E ⁇ 7 -integrin-binding site which is involved in intercellular adhesion to an E- cadherin-expressing cell.
  • second E-cadherin-binding site refers to an additional ⁇ E ⁇ 7 -integrin-binding site which selectively binds to E-cadherin but which does not does not affect intercellular adhesion between the ⁇ E ⁇ 7 -expressing cell (e.g., T lymphocytes, dendritic cells, mast cells) and an E-cadherin-expressing cell.
  • Fragments of the E-cadherin (e.g., of the extracellular domain) which bind to the second E-cadherin-binding site but which do not bind to the first E-cadherin-binding site can be identified, for example, by screening the fragments in an adhesion assay as described in Brenner and Cepek '281 to identify and eliminate E-cadherin fragments which modulate intercellular adhesion and in the screening assays disclosed herein to identify and select E-cadherin fragments which modulate cytokine release by an ⁇ E ⁇ 7 -expressing cell.
  • the methods and compositions of the invention employ "isolated” peptides or "isolated” nucleic acids which encode the isolated binding polypeptides of the invention.
  • isolated refers to a cloned expression product of an ohgonucleotide; a peptide which is isolated following cleavage from a larger polypeptide; or a peptide that is synthesized, e.g., using solution and/or solid phase peptide synthesis methods as disclosed in, for example, U.S. 5,120,830, the entire contents of which are incorporated herein by reference.
  • isolated peptides embraces peptide fragments of the extracellular E-cadherin domain, and other ⁇ E ⁇ 7 antibodies, as well as functionally equivalent peptide analogs (defined below) of the foregoing peptide fragments.
  • peptide analog refers to a peptide which shares a common structural feature with the molecule to which it is deemed to be an analog.
  • a “functionally equivalent” peptide analog is a peptide analog which further shares a common functional activity with the molecule to which it is deemed an analog.
  • a "functionally equivalent non-peptide analog is a compound which shares a common functional activity with the molecule to which it is deemed an analog, but may or may not share a common structural feature.
  • non-peptide analogs can be identified from non-peptide libraries (e.g., combinatorial chemistry libraries) by identifying molecules which have the desired functional activity.
  • Non-peptide analogs also include compounds which contain amino acids that are coupled to one another with a bond that approximates the same geometric distance as a peptide but which is not susceptible to protease cleavage.
  • the term "functionally equivalent peptide analog” refers to a peptide analog that is capable of modulating cytokine release by ⁇ E ⁇ 7 -expressing cells, e.g., by competing with or mimicking a naturally-occurring ligand for binding to E ⁇ 7 integrin.
  • Functionally equivalent peptide analogs of E-cadherin are identified, for example, in in vitro cytokine release assays (see, e.g., the assay provided in the
  • exemplary “functionally equivalent peptide analogs” include binding polypeptides which include a conservative amino acid substitution.
  • conservative amino acid substitution refers to an amino acid substitution which does not alter the relative charge or size characteristics of the peptide in which the amino acid substitution is made.
  • a "functionally equivalent peptide analog" of an antibody that selectively binds to ⁇ E ⁇ 7 includes the antigen-binding portions of an antibody known to selectively bind to ⁇ E ⁇ 7 integrin, provided that the peptide fragments and analogs are capable of modulating cytokine release by the ⁇ E ⁇ 7 - expressing cell in vivo and/or in vitro.
  • a "functionally equivalent peptide analog" of E-cadherin includes the extracellular domain of E-cadherin, fragments of the extracellular domain and peptide analogs of the extracellular domain (e.g., peptides which contain conservative amino acid substitutions), provided that the E-cadherin peptide fragments and analogs are capable of modulating cytokine release by ⁇ E ⁇ 7 - expressing cell in vivo and/or in vitro.
  • Particularly preferred binding polypeptides are unique fragments of these polypeptides and nucleic acids.
  • a "unique fragment" of a protein or nucleic acid sequence is a fragment which is not currently known to occur elsewhere in nature (except in allelic or allelomorphic variants). Unique fragments act as a "signature" of the gene or protein from which they are derived. A unique fragment will generally exceed 15 nucleotides or 5 amino acids in length.
  • Genbank Genbank
  • EMBL EMBL
  • SWISS-PROT programs such as Eugene.
  • the homology also can be calculated using various, publicly available software tools developed by NCBI (Bethesda, Maryland) that can be obtained through the internet (ftp:/ncbi. nlm.nih.gov/pub/).
  • Exemplary tools include the BLAST system available at http://www.ncbi.nlm.nih.gov.
  • Pairwise and ClustalW alignments (BLOSUM30 matrix setting) as well as Kyte-Doolittle hydropathic analysis can be obtained using the MacVetor sequence analysis software (Oxford Molecular Group).
  • a unique fragment is particularly useful, for example, in generating monoclonal antibodies or in screening genomic DNA or cDNA libraries.
  • the screening methods of the invention provide useful information for the rational drug design of novel agents which are, for example, capable of modulating cytokine release by an ⁇ ⁇ 7 -expressing cell.
  • exemplary procedures for rational drug design are provided in Saragovi, H. et al., (1992) Biotechnology 10:773; Haber E., (1983) Biochem. Pharmacol. 32(13): 1967; and Connolly Y., (1991) Methods of
  • knowledge of the binding regions of ⁇ E ⁇ 7 -binding polypeptides can be used to rationally choose or design compounds which are more potent than the naturally occurring ⁇ E ⁇ 7 -binding polypeptides in eliciting their normal response or which are competitive inhibitors of the interaction between these naturally-occurring binding polypeptides and ⁇ E ⁇ 7 in vivo.
  • test compounds or library members may be altered, e.g., in primary sequence, to produce new and different peptides.
  • new fragments may be produced by site-directed mutagenesis or may be synthesized in vitro. These new fragments then may be tested for their - ability to bind to ⁇ E ⁇ 7 integrin and, by varying their primary sequences and observing the effects, binding polypeptides which alter the cytokine release profile of ⁇ E ⁇ 7 - expressing cells can be produced.
  • nucleic acid and amino acid sequences of the present invention may be used in computer-based modeling systems to predict the secondary and tertiary structure of the ⁇ E ⁇ 7 -binding domains.
  • Such computer-based systems are well known to those of ordinary skill in the art of rational drug design. Based upon the tertiary structure of the binding polypeptides of the invention, it is possible to identify a binding region on the integrin which is involved in its biological activity.
  • peptides or other compounds which include or mimic this structure and/or which are capable of binding to it can be rationally designed.
  • new compounds may be designed which mimic the activity of the ⁇ E ⁇ 7 -binding polypeptides or which act as competitive inhibitors of these binding polypeptides.
  • peptides which include conservative substitutions and coupled proteins in which a peptide of the invention is coupled to a solid support (such as a polymeric bead), a carrier molecule (such as keyhole limpet hemocyanin), a toxin (such as ricin) or a reporter group (such as radiolabel or other tag), also are embraced within the teachings of the invention.
  • a solid support such as a polymeric bead
  • a carrier molecule such as keyhole limpet hemocyanin
  • a toxin such as ricin
  • reporter group such as radiolabel or other tag
  • a therapeutically effective amount means that amount necessary to delay the onset of. inhibit the progression of, or halt altogether the particular condition being treated.
  • a therapeutically effective amount will vary with the subject's age, condition, and sex, as well as the nature and extent of the disease in the subject, all of which can be determined by one of ordinary skill in the art. The dosage may be adjusted by the individual physician or veterinarian, particularly in the event of any complication.
  • a therapeutically effective amount typically varies from 0.01 mg/kg to about 1000 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg, and most preferably from about 0.2 mg//kg to about 20 mg/kg, in one or more dose administrations daily, for one or more days.
  • the therapeutically effective amount of the isolated antagonists and agonists of the invention is that amount effective to inhibit aberrant cytokine release by ⁇ ⁇ 7 - expressing cells as determined by, for example, standard tests known in the art. It is believed that the antagonists and agonists of the invention inhibit aberrant cytokine release in the target cells by binding to ⁇ 1 ⁇ 7 and. thereby, modulating the Th2 cytokine response by an ⁇ E ⁇ 7 -expressing cell.
  • Aberrant cytokine release can be determined using standard assays to measure the release of cytokines by ⁇ E ⁇ 7 -expressing cells obtained from medically sound individuals not suffering from an immune system disorder such as those disclosed herein and comparing the release profile to the release of cytokines by ⁇ E ⁇ 7 -expressing cells obtained from individuals suffering from a condition such as allergic disease, infectious disease, and inflammatory autoimmune conditions that involve ⁇ L ⁇ 7 -expressing cells (e.g., T lymphocytes, dedritic cells, mast cells).
  • a condition such as allergic disease, infectious disease, and inflammatory autoimmune conditions that involve ⁇ L ⁇ 7 -expressing cells (e.g., T lymphocytes, dedritic cells, mast cells).
  • the isolated antagonists or agonists of the invention are administered to the subject in combination with a method for treating the particular condition being treated. See, e.g., Harrisons, Principles of Internal Medicine (McGraw Hill, Inc., New York) for a more detailed description of the above-described conditions and the identification of agents which are used for treating these conditions.
  • the isolated antagonists and agonists of the invention can be administered in combination with agents which inhibit adhesion between ⁇ E ⁇ 7 -expressing cells and E-cadherin expressing cells.
  • agents which inhibit adhesion between ⁇ E ⁇ 7 -expressing cells and E-cadherin expressing cells are described in the Brenner and Cepek '281 patent.
  • Agents which are useful for treating allergic disease, such as allergic asthma are shown in Table A. immediately preceding the claims.
  • Agents which are useful for treating infectious disease, such as tuberculosis are shown in Table B, immediately preceding the claims.
  • Agents which are useful for treating inflammatory autoimmune conditions, such as rheumatoid arthritis, are shown in Table C, immediately preceding the claims.
  • the above-described drug therapies are well known to those of ordinary skill in the art and are administered by modes known to those of skill in the art.
  • the drug therapies are administered in amounts which are effective to achieve the physiological goals (to prevent or reduce the physiological consequences of the conditions being treated), in combination with the isolated antagonists or agonists of the invention.
  • the drug therapies may be administered in amounts which are not capable of preventing or reducing the physiological consequences of the condition when the drug therapies are administered alone but which are capable of preventing or reducing the physiological consequences of the condition when administered in combination with the isolated antagonists or agonists of the invention.
  • the isolated antagonists or agonists of the invention may be administered alone or in combination with the above-described drug therapies as part of a pharmaceutical composition.
  • the invention provides pharmaceutical compositions and methods of preparing same, e.g., by placing an agent(s) of the invention, alone or in combination with another drug(s) in a suitable carrier.
  • a pharmaceutical composition may include the isolated antagonists or agonists in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art.
  • the compositions should be sterile and contain a therapeutically effective amount of the isolated antagonists or agonist of the invention in a unit of weight or volume suitable for administration to a patient.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration into a human or other animal.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • Pharmaceutically acceptable further means a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration.
  • Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.
  • compositions suitable for parenteral administration comprise a sterile aqueous preparation of the antagonists or agonists of the invention, which is preferably isotonic with the blood of the recipient.
  • This aqueous preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1 ,3-butane diol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • fatty acids such as oleic acid may be used in the preparation of injectables.
  • Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
  • a variety of administration routes are available. The particular mode selected will depend, of course, upon the particular drug selected, the severity of the condition being treated, and the dosage required for therapeutic efficacy.
  • the methods of the invention may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects.
  • modes of administration include oral, rectal, topical, nasal, interdermal, or parenteral routes.
  • parenteral includes subcutaneous, intravenous, intramuscular, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. They could, however, be preferred in emergency situations. Oral administration will be preferred for prophylactic treatment because of the convenience to the patient as well as the dosing schedule.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the antagonists or agonists of the invention into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the antagonists or agonists of the invention into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the antagonists or agonists of the invention.
  • Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the antagonists or agonists described above, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include the above-described polymeric systems, as well as polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109.
  • Delivery systems also include non- polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides
  • hydrogel release systems such as lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides
  • sylastic systems such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides
  • peptide based systems such as mono- di- and tri-glycerides
  • wax coatings such as those described in U.S. Patent Nos. 4,452,
  • Long-term sustained release means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days.
  • Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
  • the isolated antagonists or agonists of the invention may be administered alone or in combination with the above-described drug therapies by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be oral, intravenous, intraperitoneal. intramuscular, intra-cavity. subcutaneous, or transdermal.
  • direct administration to the site of ⁇ E ⁇ 7 -expressing cells that are suspected of aberrant cytokine release is preferred.
  • Such local delivery can be accomplished by, for example, aerosol inhalation (allergic asthma), intranasal spray (allergic rhinitis), eye drops (allergic conjunctivitis), topical ointment (psoriasis), intra articular injection (arthritis), and intrathecal injection (multiple sclerosis).
  • Preparations for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the isolated antagonists or agonists of the invention that are nucleic acids can be administered to the subject (any mammalian recipient) using the same modes of administration that currently are used for gene therapy in humans (e.g., adenovirus-mediated gene therapy).
  • adenovirus-mediated gene therapy e.g., U.S. Patent Nos. 5,670,488, entitled “Adenovirus Vector for Gene Therapy”, issued to Gregory et al., and 5,672,344, entitled “Viral-Mediated Gene Transfer System", issued to Kelley et al. which describe patented procedures for performing in vivo gene therapy.
  • a patented procedure for performing ex vivo gene therapy is outlined in U.S.
  • Patent 5,399,346 and in exhibits submitted in the file history of that patent, all of which are publicly available documents.
  • ex vivo gene therapy involves introduction in vitro of a functional copy of a gene, a fragment thereof, or an antisense nucleic acid into a cell(s) of a subject and returning the genetically engineered cell(s) to the subject.
  • the functional copy of the gene, fragment thereof or antisense nucleic acid is under operable control of regulatory elements which permit expression of the gene in the genetically engineered cell(s) or transcription of the antisense nucleic acid.
  • the antagonists and agonists of the invention can be delivered to ⁇ ⁇ 7 -expressing cells, ex vivo or in vivo, to treat conditions that are mediated by aberrant cytokine levels released by ⁇ E ⁇ 7 -expressing cells.
  • Numerous transfection and transduction techniques as well as appropriate expression vectors are well known to those of ordinary skill in the art, some of which are described in PCT application WO95/00654 and in the above-noted United States patents.
  • a vector containing an ⁇ r antagonist nucleic acid or an ⁇ E agonist nucleic acid is delivered to a site of ⁇ E ⁇ 7 -expressing cells (e.g., cells of the intestinal tract, skin, eye, nose, brain, and genitourinary epithelium) in a subject who is a candidate for such gene therapy.
  • the vector genetically modifies the ⁇ E ⁇ 7 -expressing cells in vivo with DNA (RNA) encoding the antagonists or agonists of the invention.
  • RNA DNA
  • Such genetically modified ⁇ E ⁇ 7 -expressing cells are expected to have altered cytokine release in vivo.
  • primary E ⁇ 7 - expressing cells can be obtained from a subject who is a candidate for such gene therapy. Then, such cells can be genetically engineered ex vivo with DNA (RNA) encoding an antagonist or agonist of the invention. Such recombinant cells are expected to block or enhance, respectively, the Th2 cytokine response by the ⁇ E ⁇ 7 - expressing cell in vitro and, if reintroduced to the subject, in vivo.
  • RNA DNA
  • the invention provides isolated oligonucleotides that encode the CDR3 region of antibodies which bind to ⁇ E ⁇ 7 and thereby modulate the Th2 cytokine response by the ⁇ E ⁇ 7 -expressing cell.
  • the invention provides isolated oligonucleotides that encode the extracellular domain of E-cadherin and functionally equivalent peptide fragments and analogs thereof.
  • the invention provides isolated oligonucleotides that are antisense nucleic acids of the E ⁇ 7 integrin or its subunits, and functionally equivalent fragments and analogs thereof.
  • isolated in reference to an ohgonucleotide, means an RNA or DNA polymer, portion of genomic nucleic acid, cDN A or synthetic nucleic acid which, by virtue of its origin or manipulation: (a) is not associated with all of a nucleic acid with which it is associated in nature (e.g., is present in a host cell as a portion of an expression vector); or (b) is linked to a nucleic acid or other chemical moiety other than that to which it is linked in nature; or (c) does not occur in nature.
  • isolated it is further meant a nucleic acid sequence: (i) amplified in vitro by, for example, the polymerase chain reaction (PCR); (ii) synthesized by. for example, chemical synthesis; (iii) recombinantly produced by cloning; or (iv) purified from a more complex molecule or from a mixture of molecules, such as by cleavage and size fractionation.
  • PCR polymerase chain reaction
  • the invention also provides an isolated ohgonucleotide that is capable of hybridizing under stringent conditions to the nucleotide sequence encoding the ⁇ E ⁇ 7 integrin or its subunits (described in U.S. Patent No. 5,594,120 ("Brenner and Parker 120") or to the nucleotide sequence encoding the extracellular domain of E-cadherin (described in U.S. Patent No.
  • hybridizing under stringent conditions is a term of art which refers to the conditions of temperature and buffer concentration which will permit hybridization of a particular ohgonucleotide or nucleic acid to its complementary sequence and not to non-complementary sequences.
  • the exact conditions which constitute “stringent” conditions depend upon the length of the nucleic acid sequence and the frequency of occurrence of subsets of that sequence within other non-identical sequences.
  • Stringent hybridization conditions may include hybridization conditions of from 30 to 60%C and from 5x to O. lx SSC. Highly stringent hybridization conditions may include hybridization at 45%C and 0.1 SSC. Less than stringent conditions are employed to isolate nucleic acid sequences which are substantially similar, allelic or homologous to any given sequence. Exemplary high stringency hybridization conditions are provided in U.S. Patent Application Serial No. 08/199,776, the contents of which are incorporated herein by reference.
  • the isolated ohgonucleotide is capable of hybridizing under stringent conditions to a unique fragment of the foregoing nucleotide sequences.
  • unique fragment refers to a nucleic acid sequence having less than 25%o sequence homology with previously identified nucleic acid sequences. More preferably, the unique fragments have less than 10%> sequence homology with known nucleic acid sequences.
  • unique fragments can be identified by searching the Genbank, PIR and/or Swiss-Prot data bases using the Eugene program available through the Harvard Molecular Biology Core Research Resource, Cambridge. MA. The unique fragments are useful, for example, as probes and primers in nucleic acid hybridization assays and in amplification reactions, respectively.
  • the preferred ohgonucleotide is an antisense ohgonucleotide between about 10 and about 100 nucleotides in length.
  • the antisense ohgonucleotide is capable of hybridizing under high stringency conditions to unique fragments of the ⁇ E ⁇ 7 integrin (or ⁇ E subunit or ⁇ 7 subunit).
  • antisense ohgonucleotide refers to an ohgonucleotide (DNA and/or RNA) that is capable of hybridizing to the naturally- occurring DNA or mRNA encoding the ⁇ E subunit of human integrin.
  • Base-pairing of the antisense ohgonucleotide with the DNA (or RNA) encoding the ⁇ E ⁇ 7 integrin (or ⁇ E subunit or ⁇ 7 subunit) in vivo prevents the cytokine release by preventing transcription (or translation) of ⁇ E ⁇ 7 integrin (or ⁇ E subunit or ⁇ 7 subunit) in ⁇ E ⁇ 7 - expressing cells.
  • host cell refers to a cell, e.g., a prokaryotic or eukaryotic cell which, together with a recombinant vector, comprises an expression system.
  • host cell also embraces a host cell in which the vector or isolated ohgonucleotide has integrated into the host cell nucleic acid.
  • the expression vector includes at least one strand of the above-disclosed isolated ohgonucleotide.
  • the ohgonucleotide is operatively joined to at least one regulatory sequence, e.g., a promoter sequence, an enhancer sequence.
  • a coding sequence e.g., the isolated ohgonucleotide
  • a regulatory sequence are said to be operatively joined when they are linked in such a way as to place expression of the coding sequence under the influence or control of the regulatory sequence.
  • Suitable cell lines include mammalian cells (e.g., Chinese hamster ovary cells
  • CFIO monkey COS10C7 or 19 cell
  • bacterial cells e.g., E. coli, B. subtilis and Pseudomonas strains
  • insect cells e.g., SF9
  • yeast strains Exemplary procedures for obtaining expression of a foreign gene in the above-identified cell lines are disclosed in U.S. 5,211,657, the entire contents of which are incorporated herein by reference. All references, patents and patent publications that are recited in this application are incorporated in their entirety herein by reference.
  • the allergic immune response is characterized by elevated production of IgE and a mucosal inflammatory infiltrate composed of mast cells, eosinophils and lymphocytes that are biased toward production of Th2 type cytokines such as interleukin (IL-4), IL-5. IL-9, IL-10, IL-13, and granulocyte/macrophage-colony stimulating factor (GM-CSF) and the Thl/Th2 cytokine IL-3.
  • Th2 type cytokines such as interleukin (IL-4), IL-5.
  • cytokine production by effector cells such as eosinophils and mast cells, may amplify or even help initiate the allergic response by stimulating the proliferation of responding T cells and facilitating leukocyte recruitment.
  • IL-3 induces mast cell progenitor proliferation that can also be enhanced by IL-9
  • IL-4 stimulates epithelial cell production of eotaxin (a potent chemoattractant for eosinophils) and stimulates B cell IgE and IgG, production
  • IL-5 acts to induce eosinophil progenitor propagation, differentiation and attenuation of apoptosis. It has been difficult to unravel how allergic inflammatory responses are regulated because there are many cell types involved and the cells produce cytokines that modulate the functions of the other cell types involved.
  • Mucosal T lymphocytes microenvironments and mechanisms of localization.
  • the gut associated lymphoid tissues include a large number of lymphocytes present in organized lymphoid structures within and draining the intestinal mucosa (i.e.-Peyer's patches and niesenteric lymph nodes).
  • APC primary antigen/antigen presenting cell
  • Peyer's patch B and T cells are induced to proliferate, transit through lymphatics and the circulation and then selectively extravasate within Peyer's patches and the lamina basement (the region of the mucosa between the epithelium and the muscularis mucosa).
  • T lymphocytes are found either partially or fully on the epithelial side of the epithelial basement membrane.
  • intestinal intraepithelial lymphocytes (ilEL) are >80%> CD8 + and have a restricted repertoire of T-cell receptor (TCR) variable region gene segment usage relative to peripheral blood lymphocytes (PBL).
  • TCR T-cell receptor
  • T lymphocytes Like lamina limbal T lymphocytes, these cells are thought to be terminally differentiated, may recognize antigens presented by intestinal epithelial cells [Bland PW, et al., 1989, Immunology, 68(4):497-502; Mayer L, et al., 1991, Gastroenterology, 100(1):3-12; Bleicher PA, et al., 1990, Science, 250:679-82], and are able to mediate cytotoxicity and to produce cytokines including interleukin (IL)-2, IL-3, interferon (IFN)- ⁇ , tumor necrosis factor (TNF), transforming growth factor (TGF)- ⁇ l, and possibly IL-5 upon TCR stimulation, (reviewed in Guy-Grand D, et al., 1993, Curr.
  • IL interleukin
  • IFN interferon
  • TGF tumor necrosis factor
  • TGF transforming growth factor
  • ilEL Both in mouse and man, ilEL include an increased proportion of TCR ⁇ + that are c-kit and KL dependent [Lefrancois L, et al., 1994, Eur. J. Immunol., 24(3):635-40].
  • This TCR ⁇ + cells may recruit additional T lymphocytes upon epithelial injury through the production of chemotactic cytokines (chemokines) such as lymphotactin.
  • chemokines chemotactic cytokines
  • some ilEL appear to undergo an extrathymic developmental pathway, which escapes the selection that ordinarily occurs in the thymus.
  • extrathymic T cells may be derived from "cryptopatches", small accumulations of c- kif IL-7 receptor + Thy-l Nells within the lamina intestinal adjacent to the crypt epithelium, which lack expression of lineage markers for B cells, T cells, granulocytes/neutrophil/dendritic cells, granulocytes or erythroid lineage but acquire TCR expression upon in vitro culture or adoptive transfer into scid/scid mice. Similar to the intestinal immune response, the primary lymphocyte response in the lung is initiated within bronchial associated lymph nodes and the activated bronchial lymph node cells are thought recirculate and then selectively extravasate within pulmonary post-capillary venules.
  • lymphocytes in contrast to the intestine, there are few pulmonary lymphocytes in animals that have not been exposed to a respiratory antigenic challenge. In general, the homing of lymphocytes is thought to occur via a multi-step process (reviewed in Springer T., 1994, Cell, 76:301-14; Butcher EC, et al., 1996, Science, 272(5258):60-6).
  • the initial interaction between a lymphocyte and the endothelium is a weak tethering. This interaction is manifested by lymphocyte rolling along the endothelium and is generally mediated by a selectin/glycosylated selectin counter-receptor interaction or the ⁇ 4 integrins ⁇ 4 ⁇ 7 and ⁇ 4 ⁇ ,.
  • a chemokine receptor on the lymphocyte cell surface binds to a chemokine associated with the endothelial cell surface to trigger a G-protein dependent increase lymphocyte integrin/ligand binding avidity.
  • This inside-out signaling regulates the binding of ⁇ L ⁇ 2 to intercellular adhesion molecule- 1 (ICAM-1), which is important in the extravasation of lymphocytes within peripheral lymph nodes; of ⁇ 4 ⁇ , to vascular cell adhesion molecule- 1 (VCAM-1), which mediates lymphocyte extravasation within inflamed tissues; and of ⁇ 4 ⁇ 7 to mucosal addressin-cell adhesion molecule- 1 (MAd- CAM-1), which mediates extravasation of lymphocytes within Peyer ' s patches and the intestinal lamina intestinal [Butcher EC, et al., 1996, Science, 272(5258):60-6: Campbell .TJ, et al., 1998, Science, 279(5349):381-4; Lau
  • lymphocyte localization to a selected site within a tissue are less clear. However, this process presumably includes integrin mediated migration and may incorporate chemokine gradients to induce targeted migration toward a selected site, similar to the localization of inflammatory leukocytes in the setting of inflammation. Finally, it is likely that cells are actively retained within a selected location, as lymphatic flow would tend to shift unbound cells to the lymphatics, and as intestinal lymphocytes appear to be shed from the epithelium more slowly than enterocytes [Penney L. et al., 1995, Immunology, 86(2):212-8].
  • ⁇ E ⁇ 7 integrin Distribution and function of the integrin p ⁇ 7 .
  • ⁇ E ⁇ 7 integrin a second ⁇ 7 integrin, ⁇ E ⁇ 7 integrin, based upon its selective expression.
  • This ⁇ E ⁇ 7 integrin is found on >90%> of intestinal intraepithelial lymphocytes (ilEL) and on 45-50% of intestinal lamina intestinal T lymphocytes [Cerf-Bensussan N, et al., 1987. Eur. J. Immunol., 17:1279-85; Kilshaw PJ, et al., 1988, Immunol. Lett, 18(2):149-54; Russell G, et al., 1994, Eur. J. of Immun., 24:2832-41] in both mice and humans.
  • T lymphocytes at other mucosal epithelia and on approximately 40%) of bronchioalveolar lavage T cells from normal people [Erie DJ, et al.. 1994, Am. J. Respir. Cell Mol. Biol., 10(3):237-44] but by ⁇ 5% of peripheral blood T lymphocytes in humans and 15% of splenic T lymphocytes in mice [Lefrancois L, et al., 1994, Eur. J. Immun., 24(3):635-40]. Integrin ⁇ E ⁇ 7 has not been found on B cells. However, ⁇ E ⁇ 7 is expressed on cells of dendritic morphology in rats [Brenan M, et al., 1997, Eur. J.
  • TGF- ⁇ l a cytokine that is produced by intestinal epithelial cells [Babyatsky MW, et al., 1996, Gastroenterology, 1 10(4):975-84] and by ilEL.
  • E ⁇ 7 integrin mediates adhesion to epithelial cells [Cepek KL, et al., 1993, J. Immunol., 150(8):3459-70] through binding to E-cadherin [Cepek KL, et al., 1994, Nature, 372(6502): 190-3; Karecla P, et al., 1995, Eur. J. Immunol.. 25:852-6; Higgins JMG, et al., 1998, J. Cell. Biol.l40(l): 197-210].
  • the cadherins each are characterized by a tissue specific distribution and generally mediate binding to a molecule of the same type on an adjacent cell to binding like cells together [Takeichi M, 1991, Science, 251 :1451-5]. This was the first demonstration that an integrin can bind to a member of the cadherin family of adhesion molecules to mediate tissue specific lymphocyte adhesion.
  • the integrin ⁇ E ⁇ 7 also may mediate adhesion to lamina intestinal endothelial cells (see preliminary results).
  • ⁇ H ⁇ 7 appears to be regulated by inside out signaling, as the ⁇ E ⁇ 7 function is enhanced following stimulation through the T cell receptor [Higgins JMG. et al., 1998. J.
  • Integrin ⁇ ⁇ ; ⁇ 7 also appears to transmit an intracellular signal that modifies the T cell response to stimulation, as anti-human ⁇ E ⁇ 7 mAbs enhance proliferation of anti-CD3 stimulated T cells [Russell G, et al., 1994, Eur. J. Immunol. 24:2832-41] and as one anti-murine ⁇ E antibody stimulates the T cell mediated lysis of Fc receptor expressing target cells [Lefrancois L. et al., 1994, Eur. J. Immunol.,
  • ⁇ E ⁇ 7 expression appeared to modulate cytokine production by spleen derived CD4 + T cells. Because of the known role of ⁇ E ⁇ 7 in lymphocyte localization and function and a possible role of ⁇ E ⁇ 7 in modulating the localization and function of dendritic cells and mast cells, we hypothesized that ⁇ E ⁇ 7 expression is important during the course of Th2 biased disease processes. To test this possibility, two animal models were selected for study. Th2 dependent immune response in a murine model of pulmonary inflammation and airway hyperresponsiveness.
  • Asthma in humans is characterized by an initial bronchoconstriction that appears to result from IgE induced mast cells mediator release and a later bronchial response, occurring within 10-12 hours after allergen exposure, that is typified by pulmonary inflammation with a prominent infiltration of T cells and eosinophils within the airways.
  • a late phase asthma-like condition is induced in mice by systemic OVA sensitization followed by repeated aerosolized OVA challenge ([Foster PS, et al., 1996, J. Exp. Med., 183(1):195-201).
  • the pulmonary response in this model is manifested by marked airway hyperresponsiveness to bronchoconstrictor stimuli, and by a significant peribronchial and perivascular leukocytic infiltrate composed primarily of eosinophils and T cells.
  • OVA aerosol -challenged mice develop increased serum levels of allergen- specific IgE and increased BAL concentrations of Th2 cytokines, such as IL-4, IL-5, IL-10 and IL-13 (Corry DB, et al., 1996, J. Exp. Med., 183(1 ): 109-17; Foster PS, et al., 1996, J. Exp. Med. 183(1): 195-201 ; Garssen J.
  • CENT T cells are a critical cellular element in this murine model since monoclonal antibody mediated CD4 + T cells depletion abolishes airway hyperreactivity and pulmonary eosinophilia ([Nakajima H, et al., 1992, Am. Rev. Respir. Dis.. 146(2):374-7; Gavett SH, et al.,
  • Eosinophils also appear to play an important role in the pulmonary response to OVA challenge based upon the correlation of airway hyperresponsiveness with the eosinophil numbers in the airways.
  • the pulmonary eosinophilia in this murine model is dependent on Th2 cytokines, including IL-4 and IL-5, although the relative contributions of each cytokine varies and may depend on the strain of mouse used for study (Hogan SP, et al.. 1998, Am. J. Respir. Crit Care Med., 157(1):210-8; Hogan SP. et al., 1998. J. Immunol.,
  • eosinophil chemoattractant eotaxin
  • the eosinophil chemoattractant, eotaxin is also involved in eosinophil recruitment in this model as antibody neutralization of eotaxin or targeted disruption of the eotaxin gene results in reduced eosinophil (Gonzalo JA, et al., 1996, J. Clin. Invest., 98(10):2332-45; Lilly CM, et al., 1997, J. Clin. Invest., 99(7):1767- 73).
  • mice deficient in c-kit W/W v
  • mice deficient in c-kit which lack mast cells
  • mice deficient in the Ig ⁇ or Ig ⁇ heavy chain genes mice deficient in the Ig ⁇ or Ig ⁇ heavy chain genes
  • mice deficient in the Ig ⁇ or Ig ⁇ heavy chain genes mice deficient in the Ig ⁇ or Ig ⁇ heavy chain genes (Mehlhop PD, et al., 1997, Proc. Natl. Acad. Sci. U.S.A..
  • the murine model provides a powerful tool to dissect the cellular and molecular components critical to the development of airway hyperresponsiveness and pulmonary inflammation because it replicates the Th2 and eosinophil components of an antigen-induced asthmatic response.
  • ⁇ E -integrin-deficient mice ( ⁇ E -/-) were generated by our laboratory as previously described (Schoen MP, Arya A, Murphy EA, Adams CA, Strauch UG, Agace WW, Marsal J,Donohue JP, Her H, Beier DR, Olson S, Lefrancois L, Brenner MB, Grusby MJ,and Parker CM. Mucosal T lymphocyte numbers are selectively reduced inintegrin alphaE (CD 103)-deficient mice. J. Immunol. 1999; 162:6641- 6649.).
  • the ⁇ E -/- mice originally on a mixed BALB/cJ x 129/Sv background, were backcrossed 5-6 generations with BALB/cJ mice for use in these experiments.
  • mice purchased from Jackson Laboratory (Bar Harbor, ME) were used as wild-type controls and in the antibody treatment studies. Four-five week old male mice were used in all experiments and all mice were housed in microisolator cages under specific-pathogen-free (SPF) conditions. Sensitization and Challenge. Mice were immunized on days 0 and 7 via intraperitoneal (i.p.) injection with 10 ⁇ g chicken ovalbumin (OVA; Grade III; Sigma Chemical Co., St. Louis, MO) mixed with 1 ⁇ g Al(OH), (Sigma) in 0.2 ml PBS. Mice were exposed to either aerosolized OVA (6%.
  • OVA chicken ovalbumin
  • mice were placed in a plastic chamber and the OVA or PBS solution was delivered via nebulizer (PARI-LC Plus, PARI Respiratory Equipment, Inc., Richmond, VA) attached directly to the chamber.
  • nebulizer PARI-LC Plus, PARI Respiratory Equipment, Inc., Richmond, VA
  • continuous airflow was supplied to the nebulizer head ( ⁇ 5.2 L/min) to generate aerosol with a mean particle size of 3.1 ⁇ m.
  • Physiological measurements and inflammatory cell analysis were determined 245 hours after the last aerosol challenge. Animals were 7-8 weeks of age at the completion of the protocol.
  • mice were given 1 mg of mAb by i.p. injection followed by twice weekly i.p. injections of 200 ⁇ g/mouse.
  • the antibodies used were M290 (rat anti-mouse ⁇ E ; IgG2a) and Yl 3-238 (rat anti-mouse p21 ras; IgG2a).
  • Airway hyperresponsiveness was measured as previously described (Martin TR, et al., 1988, J. Appl. Physiol., 64(6):2318-23; De Sanctis GT, et al., 1997, J. Appl. Physiol., 83(3):681-7; De Sanctis GT, et al., 1997, Am. J. Respir. Crit. Care Med., 156(4 Pt. 2):S82-8). Briefly, each mouse was anesthetized with an intraperitoneal injection of pentobarbital sodium (70- 80 mg/kg; Anthony Products Co., Arcadia, CA).
  • a 19-gauge tubing adapter was inserted into the trachea and secured in place with sutures.
  • An internal jugular vein was cannulated with a catheter attached to a Hamilton syringe (Hamilton Co., Reno, NV) and was used to administer methacholine (Acetyl- ⁇ -mefhylcholine chloride; Sigma). Animals were placed in a plethysmograph and methacholine dose-response curves were obtained by intravenous administration of sequentially increasing doses of methylcholine (33 ⁇ g/kg to 1000 ⁇ g/kg) in a 20- to 35- ⁇ l volume.
  • Each dose-response curve was log- transformed and then subjected to regression analysis to interpolate the dose required for a two-fold increase in lung resistance (log ED200 R L ).
  • This dose is used as an index of airway responsiveness; numerically low values of log ED200 R, indicate a high level of sensitivity to the administered agonist and are consistent with an "asthma-like" hyperresponsive phenotype.
  • Bronchoalveolar Lavage BAL. Animals were removed from the plethysmograph and killed by exsanguination while under surgical anesthesia. Blood collected was saved and frozen at 70°C for further later analysis.
  • PBS (1 or 2 ml) with 0.6 mM EDTA was instilled into the lung, via the tracheal cannula. and retrieved using gentle suction.
  • the lavage was centrifuged at 2.000 rpm for 10 min., the supernatant was separated from the cell pellet and aliquots were frozen at 70°C for later analysis of soluble factors.
  • the cell pellets were resuspended in PBS with 10% FCS and total cell counts were made in a hemocytometer. For each sample, approximately 40.000 cells in 100 ⁇ l were centrifuged onto slides at 500 rpm for 5 min. Slides were stained with Wright-Geimsa (Sigma) and differentials were determined by counting >200 cells for each sample. The investigator counting the cells was blinded to the treatment group assignment of each sample.
  • Cytokine and chemokine concentrations in the BAL supernatant were determined using commercial ELISA kits (IL-4, IL-5, IL-12, and IFN- ⁇ ; Endogen, Woburn, MA and IL-10. IL-13, and eotaxin; R & D Systems, Minneapolis. MN). All samples were assayed in duplicate using undiluted BAL supernatant. The sensitivities of each ELISA are indicated in the figure legends. Tissue Sample Collection and Histological Evaluation. The lungs were inflated with OCT (Miles Laboratories Inc., Elkhart, IN) via the tracheal cannula and removed from the thoracic cavity.
  • OCT Miles Laboratories Inc., Elkhart, IN
  • the lobes of the right lung were separated and immediately snap-frozen in OCT for use in frozen sections.
  • the left lung was fixed in 10%o neutral buffered formalin solution (Sigma) for paraffin embedding. Five-micron paraffin sections were stained with hematoxylin and eosin and then examined for overall inflammation by light microscopy.
  • Eosinophil Peroxidase Staining Eosinophil levels in the lung were quantitated based on cyanide-resistant peroxidase staining of paraffin sections (Strath M, Warren DJ, Sanderson CJ. Detection of eosinophil assay. Its use as an assay for eosinophil differentiation factors. J. Immunol. Methods.1985: 83 (2): 209-215.). Briefly, the sections were deparaffinized, fixed in 4%> picric acid for 30 min., and then rinsed briefly in water to remove excess fixative.
  • the fixed slides were incubated for 10 s in cyanide buffer (4.9 mg potassium cyanide in 10 ml PBS, pH 6.0), washed in water, and pre-incubated for 10 min. in 10 ml 0.05 M Tris buffer, p H 7.6, containing 5 mg diaminobenzene and 5% sucrose. Then 0.1 ml of fresh 1% H 2 O 2 was added to the buffer and the slides were incubated for 1 hr at room temperature. After incubation, the slides were washed three times in 0.05 M Tris buffer, counterstained with methylene blue, air dried, and mounted.
  • cyanide buffer 4.9 mg potassium cyanide in 10 ml PBS, pH 6.0
  • IgG subclass specific ELISAs were used to quantitate OVA-specific IgGl and IgG2a Ab levels in serum (Saloga J, Renz J, Lack G, Bradley KL, Greenstein JL, Larsen G and Gelfand EW. Evelopment and transfer of immediate cutaneous hypersensitivity in mice exposed to aerosolized antigen. Journal of Clinical Investigation.1993. 91 : 133-140). Briefly, 96-well Corning ELISA plates were coated with 50 ⁇ l of OVA in bicarbonate buffer (0.1 M NaHCO 3 , pH 8.2) overnight at 4°C.
  • bicarbonate buffer 0.1 M NaHCO 3 , pH 8.2
  • mice Have Decreased Pulmonary Inflammation.
  • groups of ⁇ E +/+ or E -/- mice were sensitized systemically with OV A/alum followed by challenge with aerosolized OVA or PBS.
  • mice challenged with PBS aerosol had few leukocytes in the bronchoalveolar lavage (BAL) (0.13 x 10 6 in ⁇ E +/+ vs. 0.18 x 10 6 in ⁇ E -/-).
  • mice challenged with aerosolized OVA had significant pulmonary inflammation as measured by an increase in the total number of leukocytes recovered from the BAL ( Figure 1).
  • the ⁇ E -/- mice in contrast, had increased numbers of BAL leukocytes relative to the PBS-challenged mice, but this inflammation was reduced by >40%> compared to the E +/+ mice.
  • lungs from aE+/+ and AE-/-mice were harvested, fixed in 10% buffered formalin, embedded in paraffin, and then sections stained with hemotoxylin-eosin to visualize leukocytic infiltrate in lung tissue.
  • Airway Responsiveness is Reduced in ⁇ E -/- Mice.
  • Airway hyperresponsiveness is a characteristic feature of asthma.
  • lung resistance (R L ) in relation to the dose of intravenously infused methacholine, a bronchoconstrictor, was measured.
  • R L lung resistance
  • both ⁇ E -/- and ⁇ +/+ mice exhibited similar low levels of airway reactivity, as quantified by the log ED200 values (2.46 in the ⁇ E -/- mice vs. 2.38 in the ⁇ E +/+ mice; Figure 3).
  • OVA When challenged with OVA, however, both mice demonstrated significant increases in airway reactivity relative to the PBS-challenged mice.
  • the ⁇ F -/- mice were significantly- less responsive than the ⁇ E +/+ mice, with airway resistance measures that approached those of the PBS control (2.20 in the ⁇ E -/- mice vs. 1.90 in the E +/+ mice).
  • the ⁇ E -/- mice were 2-fold less responsive than the ⁇ E +/+ wild-type mice and demonstrated a less "asthma-like" phenotype.
  • mice Treated with Antibody to ⁇ E Exhibit Decreased Pulmonary Inflammation and Airway Hyperresponsiveness.
  • ⁇ E -/- mice mice Treated with Antibody to ⁇ E Exhibit Decreased Pulmonary Inflammation and Airway Hyperresponsiveness.
  • BALB/cJ mice were treated systemically with monoclonal antibody (mAb) to ⁇ E .
  • the total level of pulmonary inflammation was significantly decreased in mice treated with ⁇ E mAb compared sham-injected or isotype-matched control mAb-injected mice ( Figure 4a). This 75%o reduction was even more dramatic than that seen with the ⁇ -/- mice.
  • Analysis of the leukocyte subpopulations in the BAL revealed a 70% decrease in the total number of lymphocytes and an 80% decrease in the total number of eosinophils in the BAL (data not shown); trends similar to those seen using the ⁇ E -/- mice.
  • mice treated with the ⁇ 1 mAb were less responsive than mice treated with control mAb or sham injections (Figure 4b). While these differences in pulmonary resistance were not statistically significant, the trend suggests that, like ⁇ 1 -/- mice, ⁇ mAb-treated mice have less hyperresponsive airways than mice in which the integrin ⁇ E is functional.
  • ⁇ F -/- Mice have Decreased Levels of Th2 Cytokines in BAL. Because the pathology of asthma is frequently found to be associated with increased secretion of Th2 cytokines, including IL-4, IL-5, IL-10, and IL-13, the levels of these cytokines in BAL supernatant were measured.
  • Eotaxin Levels are Reduced in the BAL Supernatant of L -/- Mice
  • the chemokine eotaxin is known to be a powerful chemoattractant for eosinophils and has been implicated in eosinophil recruitment into the lung in murine asthma models. Since ⁇ r -deficiency results in a profound reduction in eosinophil recruitment, both in the lung tissue and in the airways, the levels of eotaxin were measured in the BAL supernatant. In both ⁇ r -/- mice and in BALB/cJ mice treated with mAb to ⁇ E, the concentration of eotaxin was reduced by approximately 75% (Figure 7).
  • eotaxin is secreted by lung epithelium in response to a number of stimuli, including IL-4. and given that ⁇ r on mucosal T lymphocytes binds to E-cadherin expressed on lung epithelium, it was questioned whether the decrease in eotaxin was due to direct T lymphocyte-epithelial cell interaction or to secondary effects such as decreased cytokine secretion.
  • T lymphocytes were isolated from ⁇ E +/+ or 1 -/- mice and T cell depleted splenocytes were isolated as antigen presenting cells (APC). Then, the T cells and APC were mixed in the four different possible combinations for in vitro stimulation with plate-bound anti-CD3.
  • the T cells of a given genotype produced IL-4 similarly when combined with either ⁇ l +/+ or ⁇ r -/- APC.
  • ⁇ 1 +/+ T cells produced more IL-4 than ⁇ L -/- T cells ( Figure 8C).
  • ⁇ r -/- splenic T lymphocyte generate reduced amounts of IL-4 relative to ⁇ 1 +/+ splenic T cells and that it is the ⁇ L genotype of the T cell rather than the APC that determines this difference.
  • These effects were not due to altered proliferation because the T lymphocytes derived from ⁇ E +/+ and ⁇ -/- mice proliferated similarly in response to plate-bound anti-CD3 in these assays.
  • the studies were performed using cells derived from BALB/c mice, as ⁇ E deficiency does not alter overall splenocyte number or the percentage of splenic T cells that express either CD4 or CD8 in this genetic background.
  • B-Adrenergic Agonists Albuterol, Terbutaline, Pirbuterol, Bitolterol,
  • Metaproterenol Isoetharine, Isoproterenol, Epinephrine, Rimiterol,
  • Methylxanthines Theophylline
  • Anticholinergics Atropine Sulphate
  • Adrenocortical Steroids Glucocorticoids: Prednisone, Beclomethasone
  • Anti-asthmatic Ablukast; Ablukast Sodium; Azelastine Hydrochloride ; Bunaprolast; Cinalukast; Cromitrile Sodium; Cromolyn Sodium; Enofelast; Isamoxole; Ketotifen Fumarate; Levcromakalim; Lodoxamide Ethyl ; Lodoxamide Tromethamine; Montelukast Sodium; Ontazolast; Oxarbazole; Oxatomide; Piriprost; Piriprost Potassium; Pirolate; Pobilukast Edamine; Quazolast ; Repirinast ; Ritolukast; Sulukast; Tetrazolast Meglumine; Tiaramide Hydrochloride; Tibenelast Sodium ; Tomelukast; Tranilast; Verlukast; Verofylline; Zarirlukast.
  • Sulfonamides Sulfacetamide
  • Tripelennamine hydrochloride Tripelennamine citrate; Alkylamines:
  • Piperidines Terfenadine. Astemizole;
  • Allergic rhinitis Beclamethasone; Cromolyn Sodium; Flunisolide; HI Class
  • Antihistamines Astemizole, Terfenadine
  • HI receptor Antagonists Astemizole; Brompheniramine maleate; Carbinoxamine maleate; Chlorcyclizine; Chlorpheniramine maleate; Cyclizine hydrochloride;
  • Hydroxyzine hydrochloride Hydroxyzine pamoate; Meclizine hydrochloride;
  • Promethazine hydrochloride Pyrilamine maleate; Tripelennamine citrate;
  • Ephedrine Pseudoephedrine, Phenylpropanolamine
  • Triamcinolone Acetonide Flurandrenolide; Fluocinolone Acetonide;
  • Histamine H2 receptor antagonists Ranitidine; Famotidine; Cimetidine;
  • Anti-allergic Amlexanox; Astemizole; Azelastine Hydrochloride; Eclazolast ;
  • Minocromil Nedocromil ; Nedocromil Calcium ; Nedocromil Sodium ; Nivimedone Sodium; Pemirolast Potassium ; Pentigetide; Pirquinozol; Poisonoak Extract; Probicromil Calcium; Proxicromil; Repirinast ; Tetrazolast Meglumine; Thiazinamium Chloride; Tiacrilast; Tiacrilast Sodium; Tiprinast Meglumine; Tixanox.
  • Antihistaminic Acrivastine; Antazoline Phosphate; Astemizole ; Azatadine
  • Tuberculosis Isoniazid, Rifampin, Ethambutol, Streptomycin, Pyrazinamide, Ethionamide, Aminosalicylic Acid, Cycloserine, Kanamycin, Amikacin, Capreomycin, Viomycin, Thioacetazone.
  • Antimicrobial Aztreonam; Chlorhexidine Gluconate; Imidurea; Lycetamine; Nibroxane; Pirazmonam Sodium; Propionic Acid ; Pyrithione Sodium; Sanguinarium Chloride ; Tigemonam Dicholine.
  • Anti -parasitic Abamectin; Clorsulon; Ivermectin.
  • Anti-infective Difloxacin Hydrochloride ; Lauryl Isoquinolinium Bromide; Moxalactam Disodium; Ornidazole; Pentisomicin; Sarafloxacin Hydrochloride; Protease inhibitors of HIV and other retroviruses; Integrase Inhibitors of FIIV and other retroviruses; Cefaclor (Ceclor); Acyclovir (Zovirax); Norfloxacin (Noroxin); Cefoxitin (Mefoxin); Cefuroxime axetil (Ceftin); Ciprofloxacin (Cipro).
  • Anti-infective, topical Alcohol; Aminacrine Hydrochloride; Benzethonium Chloride : Bithionolate Sodium; Bromchlorenone; Carbamide Peroxide; Cetalkonium Chloride; Cetylpyridinium Chloride : Chlorhexidine Hydrochloride; Clioquinol ; Domiphen Bromide; Fenticlor; Fludazonium Chloride; Fuchsin, Basic; Furazolidone ; Gentian Violet; Halquinols; Hexachlorophene : Hydrogen Peroxide; Ichthammol; Imidecyl Iodine; Iodine; Isopropyl Alcohol; Mafenide Acetate; Meralein Sodium; Mercufenol Chloride; Mercury, Ammoniated; Methylbenzethonium Chloride; Nitrofurazone; Nitromersol; Octenidine Hydrochloride; Oxychlorosene; Oxychlorosene Sodium; Parach
  • Rheumatoid arthritis Antimalarials; Auranofin; Nurothioglucose; Azathioprine; Bindarit; Cyclophosphamide; Diclofenac; Etodolac; Fenoprofen; Flurbiprofen; Gold compounds; Glucocorticoids; Ibuprofen; Indomethacin; Ketoprofen; Lobenzarit Sodium; Meclofenamate; Methotrexate; ⁇ abumetone; Naproxen; Oxaprozin; D-penicillamine; Phenylbutazone; Pirazolac; Piroxicam; Prinomide Tromethamine; Salicylic Acid; Seprilose; Sulfasalazine; Sulindac; Tolmetin.
  • Piroxicam Propionic Acid derivatives
  • Salicylates Salicylates
  • Sulindac Sulindac
  • Tolmetin Tolmetin
  • Multiple Sclerosis ACTH; Acetazolamide; Amantidine; Amitriptyline: Baclofen; Carbamazepine; Dantrolene; Fampridine; Interferon-B; Methylprednisone; Phenytoin; Prednisone.
  • IDDM Type I Diabetes
  • Antidiabetic Acetohexamide; Buformin; Butoxamine Hydrochloride ; Camiglibose; Chlorpropamide; Ciglitazone; Englitazone Sodium; Etoformin Hydrochloride; Gliamilide; Glibornuride; Glicetanile Sodium; Gliflumide; Glipizide; Glucagon; Glyburide; Glyhexamide; Glymidine Sodium; Glyoctamide; Glyparamide; Insulin; Insulin.
  • Insulin Human Dalanated; Insulin Human; Insulin Human, Isophane; Insulin Human Zinc; Insulin Human Zinc, Extended; Insulin, Isophane; Insulin Lispro; Insulin, Neutral; Insulin Zinc; Insulin Zinc, Extended; Insulin Zinc, Prompt; Linogliride; Linogliride Fumarate; Metformin; Methyl Palmoxirate; Palmoxirate Sodium; Pioglitazone Hydrochloride; Pirogliride Tartrate; Proinsulin Human; Seglitide Acetate; Tolazamide; Tolbutamide; Tolpyrramide; Troglitazone; Zopolrestat
  • Antidiarrheal Anticholinergics; Bismuth Subsalicylate; Ciprofloxacin; Clonidine; Codeine; Diphenoxylate Hydrochloride; Loperamide; Methylprednisolone; Metronidazole; Norfloxacin; Rolgamidine; Sulfamethoxazole; Sulfasalazine; Tetracycline; Trimethoprim; an immunoglobulin preparation from bovine colostrum; Anti-inflammatory Agents; Adrenal Glucocorticoids; Azathioprine; 6- Mercaptopurine ;
  • Psoriasis Acitretin; Adrenocorticosteroids; Anthralin; Azaribine; Calcipotriene; Coal Tar; Cycloheximide; Enazadrem Phosphate; Etretinate; Glucocorticoids; Liarozole Fumarate; Lonapalene; Methotrexate; Psoralens: Methoxsalen, Trioxsalen; Salicylic Acid; Tepoxalin.
  • Adrenocorticosteroids Ciprocinonide; Desoxycorticosterone Acetate; Desoxycorticosterone Pivalate; Dexamethasone Acetate; Fludrocortisone Acetate; Flumoxonide; Hydrocortisone Hemisuccinate; Methylprednisolone Hemisuccinate; Naflocort; Procinonide; Timobesone Acetate; Tipredane.
  • Glucocorticoids Alclometasone Dipropionate; Amcinonide; Beclomethasone Dipropionate; Betamethasone; Betamethasone Acetate; Betamethasone Benzoate; Betamethasone Dipropionate; Betamethasone Sodium Phosphate; Betamethasone Valerate; Carbenoxolone Sodium; Clobetasol Propionate; Clocortolone Acetate; Clocortolone Pivalate; Cloprednol; Coritosterone; Corticotropin; Corticotropin, Repository; Corticotropin Zinc Hydroxide; Cortisol; Cortisone Acetate; Cortivazol; Descinolone Acetonide; Desonide; Desoximetaso ⁇ e; Desoxycortisol; Dexamethasone; Dexamethasone Acetate; Dexamethasone Sodium Phosphate; Diflucortolone; Diflucortolone Pivalate; Diflora
  • Anti-inflammatory Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac ; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen ; Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;
  • Analgesic Acetaminophen; Alfentanil Hydrochloride; Aminobenzoate Potassium; Aminobenzoate Sodium; Anidoxime; Anileridine; Anileridine Hydrochloride; Anilopam Hydrochloride; Anirolac; Antipyrine; Aspirin; Benoxaprofen; Benzydamine Hydrochloride; Bicifadine Hydrochloride; Brifentanil Hydrochloride; Bromadoline Maleate; Bromfenac Sodium; Buprenorphine Hydrochloride; Butacetin; Butixirate; Butorphanol; Butorphanol Tartrate; Carbamazepine; Carbaspirin Calcium; Carbiphene Hydrochloride; Carfentanil Citrate; Ciprefadol Succinate; Ciramadol; Ciramadol Hydrochloride; Clonixeril; Clonixin; Codeine ; Codeine Phosphate; Codeine

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Abstract

L'invention concerne des procédés et des compositions permettant de moduler la libération de cytokines par les cellules à expression de αEβ7. L'invention concerne également des polypeptides de liaison et autres molécules qui se lient sélectivement à αEβ7, bloquant ou améliorant la réponse en cytokines Th2 par les cellules considérées. Par ailleurs, les antagonistes et les agonistes décrits dans l'invention sont utiles dans les essais de criblage visant à identifier les principaux composés pharmaceutiques capables de moduler la libération de cytokines par les cellules en question. L'invention concerne enfin des procédés et des compositions pharmaceutiques permettant de modifier in vitro et in vivo la réponse en cytokines Th2 par lesdites cellules.
PCT/US1999/030992 1999-01-08 1999-12-28 PROCEDES ET COMPOSITIONS PERMETTANT DE MODULER LA LIBERATION DE CYTOKINES PAR LES CELLULES A EXPRESSION DE αEβ¿7? WO2000040604A2 (fr)

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WO2006026759A3 (fr) * 2004-09-03 2006-10-05 Genentech Inc Antagonistes anti-beta7 humanises et utilisations de ceux-ci
NO20171287A1 (no) * 2004-09-03 2007-06-01 Genentech Inc Humaniserte anti-beta7-antagonister og anvendelser av disse
NO20171288A1 (no) * 2004-09-03 2007-06-01 Genentech Inc Anti-beta7-antistoffer, nukleinsyrer som koder for antistoffene, vektorer, vertsceller og fremgangsmåte for fremstilling av antistoffene.
US7528236B2 (en) 2004-09-03 2009-05-05 Genentech, Inc. Humanized anti-beta7 antagonists and uses therefor
US7687605B2 (en) 2004-09-03 2010-03-30 Genentech, Inc. Humanized anti-beta7 antagonists and uses therefor
EP2322556A1 (fr) * 2004-09-03 2011-05-18 Genentech, Inc. Antagonistes anti-béta 7 humanisés et utilisations associées
AU2005279720B2 (en) * 2004-09-03 2011-07-28 Genentech, Inc. Humanized anti-beta7 antagonists and uses therefor
US8124082B2 (en) 2004-09-03 2012-02-28 Genentech, Inc. Humanized anti-beta7 antagonists and uses therefor
RU2453558C2 (ru) * 2004-09-03 2012-06-20 Дженентек, Инк. Гуманизированные антагонистические антитела против бета7 и их применение
US8779100B2 (en) 2004-09-03 2014-07-15 Genentech, Inc. Humanized anti-beta7 antagonists and uses therefor
US8835133B2 (en) 2004-09-03 2014-09-16 Genentech, Inc. Humanized anti-beta7 antagonists and uses therefor
CN103304667B (zh) * 2004-09-03 2015-04-08 健泰科生物技术公司 人源化的抗-β7拮抗剂及其应用
EP2990422A1 (fr) * 2004-09-03 2016-03-02 Genentech, Inc. Antagonistes anti-béta 7 humanisés et utilisations associées
NO341308B1 (no) * 2004-09-03 2017-10-02 Genentech Inc Humaniserte anti-beta7-antagonister og anvendelser av disse i terapeutisk og/eller profylaktisk behandling av sykdom, samt til fremstilling av et medikament, og en sammensetning.
NO342319B1 (no) * 2004-09-03 2018-05-07 Genentech Inc Anti-beta7-antistoffer, nukleinsyrer som koder for antistoffene, vektorer, vertsceller og fremgangsmåte for fremstilling av antistoffene.
NO342491B1 (no) * 2004-09-03 2018-06-04 Genentech Inc Humaniserte anti-beta7-antagonister og anvendelser av disse
EP3530673A1 (fr) * 2004-09-03 2019-08-28 Genentech, Inc. Antagonistes anti-béta 7 humanisés et utilisations associées

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