US20020004482A1 - Treatment of acute lung injury and fibrosis with antagonists of avbeta6 - Google Patents

Treatment of acute lung injury and fibrosis with antagonists of avbeta6 Download PDF

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US20020004482A1
US20020004482A1 US09/130,870 US13087098A US2002004482A1 US 20020004482 A1 US20020004482 A1 US 20020004482A1 US 13087098 A US13087098 A US 13087098A US 2002004482 A1 US2002004482 A1 US 2002004482A1
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antagonist
fibrosis
mice
αvβ6
lung
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Xiaozhu Huang
Dean Sheppard
Robert Pytela
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University of California
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Assigned to NATIONAL INSTITUTES OF HEALTH, THE reassignment NATIONAL INSTITUTES OF HEALTH, THE CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: UNIVERSITY OF CALIFORNIA
Assigned to CALIFORNIA, UNIVERSITY OF, REGENTS OF THE, THE reassignment CALIFORNIA, UNIVERSITY OF, REGENTS OF THE, THE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: PYTELA, ROBERT, HUANG, XIAOZHU, SHEPPARD, DEAN
Priority to US09/365,695 priority patent/US6316601B1/en
Priority to US09/818,416 priority patent/US6692741B2/en
Publication of US20020004482A1 publication Critical patent/US20020004482A1/en
Priority to US10/754,435 priority patent/US7150871B2/en
Priority to US11/559,172 priority patent/US7544358B2/en
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839Immunoglobulins [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
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00Drugs for disorders of the respiratory system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00Drugs for disorders of the urinary system
    • A61P13/10Drugs for disorders of the urinary system of the bladder
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00Drugs for disorders of the urinary system
    • A61P13/12Drugs for disorders of the urinary system of the kidneys
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • A61P35/04Antineoplastic agents specific for metastasis
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546Integrin superfamily
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00Genetically modified animals
    • A01K2217/05Animals comprising random inserted nucleic acids (transgenic)
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00Genetically modified animals
    • A01K2217/07Animals genetically altered by homologous recombination
    • A01K2217/075Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • Integrins are heterodimeric cell adhesion receptors composed of two subunits, ⁇ and ⁇ .
  • the integrin ⁇ v ⁇ 6 is a fibronectin and tenascin receptor expressed predominantly by epithelial cells. In healthy adult primate tissues, ⁇ 6 mRNA and protein are rarely detected, although ⁇ 6 is expressed during fetal development, wound healing, and in some epithelial tumors. When the ⁇ 6 subunit is expressed in a colon carcinoma cell line, from which it is normally absent, expression of the subunit confers an enhanced ability to proliferate.
  • ⁇ v ⁇ 6 integrin An 11 amino acid COOH-terminal region, unique to the ⁇ 6 subunit, is required for the proliferation-enhancing activity of the ⁇ v ⁇ 6 integrin (Agrez et al. J. Cell. Biol. 127:547-556 (1994).
  • ⁇ 6 expression is induced in type II aveolar epithelial cells during injury caused by injection of live bacteria, and ⁇ 6 expression is observed at focal sites of subclinical inflammation, as well as in a variety of clinical specimens from patients with chronic or acute inflammation of the lungs or kidneys (Breuss et al. J. Cell Sci, 108:2241-2251 (1995).
  • Huang et al. J. Cell Biol. 133:921-928 (1996) disclosed mice homozygous for a null mutation in the gene encoding the ⁇ 6 subunit had juvenile baldness associated with infiltration of macrophages into the skin, and accumulated activated lymphocytes around conducting airways in the lungs.
  • Pulmonary fibrosis is a common disorder thought to be due to the destructive effects of products released from leukocytes (see, for example, Marshall et al., Int. J. Biochem. Cell Bio. 29:107-120 (1997)).
  • Bleomycin-induced lung injury and pulmonary fibrosis are associated with and may depend upon the recruitment and activation of lymphocytes (Schrier, D. J. et al., Am. J. Pathol. 116:270-278 (1984)).
  • therapies for parenchymal lung injury and pulmonary fibrosis is the use of “anticytokine” therapeutic approaches (Coker et al. Thorax 52 (2): 294-296 (1997)).
  • One aspect of the invention is a method of treating acute lung injury in a patient comprising administering to the patient a therapeutic dose of an antagonist of ⁇ v ⁇ 6.
  • the invention also provides methods of inhibiting lung metastasis comprising administering to the patient a therapeutic dose of an antagonist of ⁇ v ⁇ 6.
  • a further aspect of the invention is a method of treating fibrosis in a patient comprising administering to the patient a therapeutic dose of an antagonist of ⁇ v ⁇ 6.
  • a further aspect of the invention is a monoclonal antibody produced by the hybridoma ATCC HB 12382.
  • a further aspect of the invention is the hybridoma ATCC HB 12382 .
  • FIG. 1A is a graph comparing lung hydroxyproline content in mice expressing a null mutation of the ⁇ 6 integrin subunit gene with control mice in the presence of bleomycin (blm) or saline (sal).
  • FIG. 1B is a photograph of trichrome staining of low power lung sections demonstrating dense accumulation of collagenous extracellular matrix in lungs of bleomycin treated wild type ( ⁇ 6+/+) but not ⁇ 6 ⁇ / ⁇ mice 30 days after treatment.
  • FIG. 2 is a graph comparing the increase in lung water in wild type mice ( ⁇ 6+/+) compared to ⁇ 6 ⁇ / ⁇ mice in the presence of bleomycin (blm) or saline (sal).
  • FIG. 3 is a graph comparing the recruitment of lymphocytes in wild type ( ⁇ 6+/+) and ⁇ 6 ⁇ / ⁇ mice after administration of bleomycin (blm) or saline (sal).
  • the instant invention provides methods and compositions for treatment of acute lung injury, such as but not limited to lung injury resulting from bacterial sepsis, hemorrhagic shock, toxic inhalation, and bleomycin and other drug-induced lung injury. Additionally, the compositions of the invention are useful in the treatment of fibrosis in epithelial organs, such as lung, liver, kidney, bladder, and esophagus.
  • compositions can be provided prophylactically or therapeutically to patients having or at risk of having symptoms of acute lung injury or fibrosis.
  • patients having had exposure to a toxic inhalant would likely be treated after such exposure, whereas a patient receiving bleomycin can be treated prophylactically and/or therapeutically.
  • compositions of the invention are administered on a daily basis for at least a period of 1-5 days, although patients with established pulmonary fibrosis, a progressive disease, may receive therapeutic doses for periods of months to years.
  • therapeutic dose is a dose which prevents, alleviates, abates, or otherwise reduces the severity of symptoms in a patient.
  • antagonists of ⁇ v ⁇ 6 include but are not limited to antibodies which specifically bind to ⁇ 6; antibodies which specifically bind to a ⁇ v ⁇ 6 ligand; ligands for ⁇ v ⁇ 6; antisense nucleic acids; and peptide; non-peptide, and peptidomimetic analogs of such ligands.
  • Antibodies can be synthetic, monoclonal, or polyclonal and can be made by techniques well known in the art.
  • the antagonist is an antibody which specifically recognizes the cytoplasmic region of the ⁇ 6 subunit (for example, see Weinacker et al. J. Cell Bio. 269:1-9 (1994)).
  • “human” monoclonal antibodies having human constant and variable regions are often preferred so as to minimize the immune response of a patient against the antibody.
  • Such antibodies can be generated by immunizing transgenic animals which contain human immunoglobulin genes. See Jakobovits et al. Ann NY Acad Sci 764:525-535 (1995).
  • such terms are intended to cover but are not limited to antibody fragments, isotype switched antibodies, humanized antibodies (e.g., mouse-human, human-mouse, and the like), hybrids, antibodies having plural specificities, fully synthetic antibody-like molecules, and the like.
  • antibodies can be screened for the ability to block the binding of a ligand to the ⁇ v ⁇ 6 and/or for other properties, such as the ability to protect in vivo against bleomycin-induced pulmonary fibrosis.
  • An exemplary anti- ⁇ 6 monoclonal antibody is 10D5 (ATCC deposit no.HB12382, deposited Aug. 6, 1997).
  • antagonists which are peptides, polypeptides, proteins, or peptidomimetics designed as ligands for ⁇ v ⁇ 6 on the basis of the presence of the cell adhesion domain arginine-glycine-aspartic acid (RGD).
  • RGD arginine-glycine-aspartic acid
  • the design of such molecules as ligands for the integrins is exemplified, for example, in Pierschbacher et al., J. Cell. Biochem. 56:150-154 (1994)); Ruoslahti, Ann. Rev. Cell, Dev. Biol. 12:697-715 (1996); Chorev et al.
  • antisense nucleic acid molecules are used as antagonists of ⁇ v ⁇ 6.
  • Antisense nucleic acid molecules are complementary oligonucleotide strands of nucleic acids designed to bind to a specific sequence of nucleotides to inhibit production of a targeted protein.
  • the nucleotide sequence of the ⁇ 6 integrin subunit was disclosed in U.S. Ser. No. 07/728,215, filed Jul. 11, 1991, hereby incorporated by reference in its entirety. These agents may be used alone or in combination with other antagonists.
  • the antisense antagonist may be provided as an antisense oligonucleotide such as RNA (see, for example, Murayama et al.
  • Antisense genes may also be provided in a viral vector, such as, for example, in hepatitis B virus (see, for example, Ji et al., J. Viral Hepat. 4:167-173 (1997)); in adeno-associated virus (see, for example, Xiao et al. Brain Res. 756:76-83 (1997)); or in other systems including but not limited to an HVJ(Sendai virus)-liposome gene delivery system ( see, for example, Kaneda et al. Ann. N.Y. Acad. Sci.
  • a “peptide vector” see, for example, Vidal et al. CR Acad. Sci III 32):279-287 (1997)); as a gene in an episomal or plasmid vector (see, for example, Cooper et al. Proc Natl. Acad. Sci. U.S.A. 94:6450-6455 (1997), Yew et al. Hum Gene Ther. 8:575-584 (1997)); as a gene in a peptide-DNA aggregate (see, for example, Niidome et al. J. Biol. Chem. 272:15307-15312 (1997)); as “naked DNA” (see, for example, U.S. Pat. No. 5,580,859 and U.S. Pat. No. 5,589,466); and in lipidic vector systems (see, for example, Lee et al. Crit Rev Ther Drug Carrier Syst. 14:173-206 (1997))
  • Candidate antagonists of ⁇ v ⁇ 6 can be screened for function by a variety of techniques known in the art and/or disclosed within the instant application, such as protection against bleomycin-induced fibrosis in a mouse model; inhibition of the proliferation of tumor cells (Agrez et al., J. Cell Bio., 127:547-556 (1994)); and inhibition of cell migration and/or inhibition of cell adhesion (see Experimental Examples section).
  • a multitude of appropriate formulations of the antagonists of the invention can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, (15th Edition, Mack Publishing Company, Easton, Pa. (1975)), particularly Chapter 87, by Blaug, Seymour, therein.
  • These formulations include for example, powders, pastes, ointments, jelly, waxes, oils, lipids, anhydrous absorption bases, oil-in-water or water-in-oil emulsions, emulsions carbowax (polyethylene glycols of a variety of molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax.
  • the quantities of active ingredient necessary for effective therapy will depend on many different factors, including means of administration, target site, physiological state of the patient, and other medicaments administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the active ingredients. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, for example, in Goodman and Gilman's the Pharmacological Basis of Therapeutics, 7th Edition (1985), MacMillan Publishing Company, New York, and Remington's Pharmaceutical Sciences 18th Edition, (1990) Mack Publishing Co, Easton Penn. Methods for administration are discussed therein, including oral, intravenous, intraperitoneal, intramuscular, transdermal, nasal, iontophoretic administration, and the like.
  • compositions of the invention may be administered in a variety of unit dosage forms depending on the method of administration.
  • unit dosage forms suitable for oral administration include solid dosage forms such as powder, tablets, pills, capsules, and dragees, and liquid dosage forms, such as elixirs, syrups, and suspensions.
  • the active ingredients may also be administered parenterally in sterile liquid dosage forms.
  • Gelatin capsules contain the active ingredient and as inactive ingredients powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like.
  • inactive ingredients examples include red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink and the like.
  • Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract.
  • Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
  • compositions of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • compositions of the invention may also be administered via liposomes.
  • Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
  • the composition of the invention to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a desired target, such as antibody, or with other therapeutic or immunogenic compositions.
  • liposomes either filled or decorated with a desired composition of the invention of the invention can delivered systemically, or can be directed to a tissue of interest, where the liposomes then deliver the selected therapeutic/immunogenic peptide compositions.
  • Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol.
  • the selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream.
  • a variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al. Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, incorporated herein by reference.
  • a liposome suspension containing a composition of the invention may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the composition of the invention being delivered, and the stage of the disease being treated.
  • nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more compositions of the invention of the invention, and more preferably at a concentration of 25%-75%.
  • compositions of the invention are preferably supplied in finely divided form along with a surfactant and propellant.
  • Typical percentages of compositions of the invention are 0.01%-20% by weight, preferably 1%-10%.
  • the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
  • Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • Mixed esters such as mixed or natural glycerides may be employed.
  • the surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%.
  • the balance of the composition is ordinarily propellant.
  • a carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
  • constructs of the invention can additionally be delivered in a depot-type system, an encapsulated form, or an implant by techniques well-known in the art. Similarly, the constructs can be delivered via a pump to a tissue of interest.
  • any of the foregoing formulations may be appropriate in treatments and therapies in accordance with the present invention, provided that the active agent in the formulation is not inactivated by the formulation and the formulation is physiologically compatible.
  • Pulmonary fibrosis is a common disorder thought to be due to the destructive effects of products released from leukocytes. Although the respiratory epithelium is damaged during the development of fibrosis, epithelial cells themselves have not been previously shown to contribute to this process.
  • bleomycin a drug known to cause pulmonary fibrosis
  • mice expressing a null mutation of a single integrin subunit ( ⁇ 6) gene that is completely restricted to epithelial cells.
  • ⁇ 6 ⁇ / ⁇ mice were dramatically protected from bleomycin-induced fibrosis. Therapies targeting this integrin could therefore provide new approaches for treatment of this largely untreatable disorder.
  • integrin ⁇ v ⁇ 6 is expressed exclusively on epithelial cells, principally during organogenesis and in response to injury. ⁇ 6 ⁇ / ⁇ mice have exaggerated inflammatory responses to cutaneous and airway injury, but develop and reproduce normally (Huang, X.Z. et al., J. Cell Biol 133:921-928 (1996)).
  • CD4+ and CD8+ lymphocytes were counted CD4+ and CD8+ lymphocytes and evaluated lymphocyte activation by measuring expression of the interleukin 2-receptor (CD25) in cells obtained from minced lungs of saline or bleomycin-treated mice at 5 and 15 days after treatment. Consistent with our earlier report, there were more CD4+, CD8+ and CD25+ cells in the lungs of ⁇ 6 ⁇ / ⁇ mice than in wild type animals. Bleomycin induced a large increase in the numbers of CD4 and CD8 expressing lymphocytes, and marked increase in the percentage of lymphocytes expressing CD25 (FIG.
  • the ⁇ 6 subunit has only been reported to form a single integrin heterodimer, ⁇ v ⁇ 6 and is restricted to epithelial cells.
  • the local concentrations of at least two ligands for this integrin fibronectin and tenascin
  • fibronectin and tenascin increase.
  • expression of ⁇ v ⁇ 6 plays a role in terminating mononuclear cell inflammatory responses in the skin and conducting airways of the lung (Huang, X.Z. et al., J. Cell Biol. 133:921-928 (1996)).
  • Respiratory epithelial cells have long been thought of principally as components of a passive barrier, separating other lung cells from the potentially toxic components of inhaled air. At this interface, however, these cells are well positioned to initiate and modulate local responses to injury.
  • respiratory epithelial cells have the capacity to synthesize and secrete a number of proteins that can initiate and modulate responses to injury, including chemokines (e.g. interleukin-8, GRO ⁇ . GRO ⁇ , RANTES, GMCSF, MIP-1 ⁇ , and MCP-1), other cytokines (e.g., IL-6, IL-11, and IL-15) and growth factors (e.g. TGF ⁇ ).
  • chemokines e.g. interleukin-8, GRO ⁇ . GRO ⁇ , RANTES, GMCSF, MIP-1 ⁇ , and MCP-1
  • other cytokines e.g., IL-6, IL-11, and IL-15
  • growth factors e.g. TGF ⁇
  • ⁇ 6 ⁇ / ⁇ mice were immunized with either keratinocytes obtained from wild type mice or with recombinant secreted human ⁇ v ⁇ 6 (Weinacker et al., J. Biol. Chem. 269:6940-6948 (1994)) in Freund's adjuvant.
  • Mouse splenocytes were harvested and fused with SP2/0 mouse myenoma cells according to standard procedures.
  • ⁇ 6- and mock-transfected SW 480 cells were used for screening the resulting supernatant by flow cytometry. Antibodies found to recognize ⁇ 6-transfected but not mock transfected SW480 cells were used for later experiments.
  • This antibody precipitated heterodimers of the appropriate molecular mass to be ⁇ v ⁇ 6 from ⁇ 6+/+ keratinocytes but not from ⁇ 6 ⁇ / ⁇ keratinocytes, indicating that these antibodies are specific for the integrin ⁇ v ⁇ 6.
  • 96-well non-tissue culture treated polystyrene multiwell microtiter plates (Linbro/Titertek, Flow Laboratories, McLean, Va.) were coated with vitronectin, fibronectin or collagen.
  • a 100 ⁇ l solution containing various amounts of matrix was added to the wells and incubated at 37° C. for 1 hour. After incubation, wells were washed with PBS, then blocked with 1% BSA in serum-free DMEM at 37° C. for 30 minutes. Control wells were filled with 1% BSA in DMEM.
  • Cells were harvested in the same was as for the migration assay and resuspended in serum-free KGM, and then added to each protein-coated well in the presence or absence of PMA.
  • For blocking experiments cells were incubated with antibodies for 5 minutes at 4° C. before plating. The plates were centrifuged (top side up) at 10 ⁇ g for 5 minutes before incubation for 1 hour at 34° C. in humidified 7% CO 2 . Non-adherent cells were removed by centrifugation top side down at 48 ⁇ g for 5 minutes. The attached cells were fixed with 1% formaldehyde and stained with 0.5% crystal violet, then the wells were washed with PBS. The relative number of cells in each well was evaluated by measuring the absorbance at 595 nm in a Microplate Reader (Bio-Rad).
  • antibodies were added into upper and lower chambers in the presence of PMA. After a 6 hour incubation, cells were fixed with 2% paraformaldehyde and stained with 0.5% crystal violet in 1% formaldehyde. Cells in the upper chamber were removed and cells on the lower surface were counted with a 10 ⁇ grid at high power magnification (40 ⁇ ). Multiple fields were counted and averaged for each condition studied.
  • a transgenic mouse line that spontaneously develops metastatic breast cancer was cross-bred with beta- 6 knockout mice.
  • Offspring mice that lacked ⁇ 6 developed fast growing primary mammary tumors, but had greatly reduced incidence and extent of lung metastasis compared to littermates that expressed ⁇ 6.
  • Immunohistochemistry showed that ⁇ 6 is strongly expressed around the edge of metastatic lesions. This suggests that ⁇ 6 is required for maximal metastasis in this model.
  • a ⁇ 6 expressing cell line derived from the primary tumor was injected into syngeneic mice that were either wild-type or ⁇ 6 knockout. Lung metastasis rapidly occurred in both groups of mice, suggesting that the presence of inflammation in the knockout mice does not interfere with tumor cell growth in the lung.

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Priority Applications (5)

Application Number Priority Date Filing Date Title
US09/130,870 US20020004482A1 (en) 1997-08-08 1998-08-07 Treatment of acute lung injury and fibrosis with antagonists of avbeta6
US09/365,695 US6316601B1 (en) 1997-08-08 1999-08-02 Antibodies specific for β6 integrins
US09/818,416 US6692741B2 (en) 1997-08-08 2001-03-27 Treatment of acute lung injury and fibrosis with antagonists of β6-specific antibodies
US10/754,435 US7150871B2 (en) 1997-08-08 2004-01-09 Treatment of acute lung injury, fibrosis and metastasis with antagonists of αvβ6
US11/559,172 US7544358B2 (en) 1997-08-08 2006-11-13 Treatment of acute lung injury, fibrosis and metastasis with antagonists of αvβ6

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US09/365,695 Division US6316601B1 (en) 1997-08-08 1999-08-02 Antibodies specific for β6 integrins
US09/818,416 Continuation US6692741B2 (en) 1997-08-08 2001-03-27 Treatment of acute lung injury and fibrosis with antagonists of β6-specific antibodies

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US09/365,695 Expired - Lifetime US6316601B1 (en) 1997-08-08 1999-08-02 Antibodies specific for β6 integrins
US09/818,416 Expired - Lifetime US6692741B2 (en) 1997-08-08 2001-03-27 Treatment of acute lung injury and fibrosis with antagonists of β6-specific antibodies
US10/754,435 Expired - Lifetime US7150871B2 (en) 1997-08-08 2004-01-09 Treatment of acute lung injury, fibrosis and metastasis with antagonists of αvβ6
US11/559,172 Expired - Fee Related US7544358B2 (en) 1997-08-08 2006-11-13 Treatment of acute lung injury, fibrosis and metastasis with antagonists of αvβ6

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US09/818,416 Expired - Lifetime US6692741B2 (en) 1997-08-08 2001-03-27 Treatment of acute lung injury and fibrosis with antagonists of β6-specific antibodies
US10/754,435 Expired - Lifetime US7150871B2 (en) 1997-08-08 2004-01-09 Treatment of acute lung injury, fibrosis and metastasis with antagonists of αvβ6
US11/559,172 Expired - Fee Related US7544358B2 (en) 1997-08-08 2006-11-13 Treatment of acute lung injury, fibrosis and metastasis with antagonists of αvβ6

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US9745376B2 (en) 2002-03-13 2017-08-29 Biogen Ma Inc. Anti-ανβ6 antibodies
US10035860B2 (en) 2013-03-15 2018-07-31 Biogen Ma Inc. Anti-alpha V beta 6 antibodies and uses thereof
US10035859B2 (en) 2013-03-15 2018-07-31 Biogen Ma Inc. Anti-alpha V beta 6 antibodies and uses thereof

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US9745376B2 (en) 2002-03-13 2017-08-29 Biogen Ma Inc. Anti-ανβ6 antibodies
US8992924B2 (en) 2005-07-08 2015-03-31 Biogen Idec Ma Inc. Anti-ανβ6 antibodies and uses thereof
USRE44681E1 (en) 2006-07-10 2013-12-31 Biogen Idec Ma Inc. Compositions and methods for inhibiting growth of SMAD4-deficient cancers
WO2008147434A1 (en) * 2006-10-19 2008-12-04 The Regents Of The University Of California TREATMENT AND PREVENTION OF CHRONIC ASTHMA USING ANTAGONISTS OF INTEGRIN αVβ6
US10035860B2 (en) 2013-03-15 2018-07-31 Biogen Ma Inc. Anti-alpha V beta 6 antibodies and uses thereof
US10035859B2 (en) 2013-03-15 2018-07-31 Biogen Ma Inc. Anti-alpha V beta 6 antibodies and uses thereof

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HUP0003547A3 (en) 2002-05-28
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ES2235350T3 (es) 2005-07-01
IL134288A0 (en) 2001-04-30
DE69828614T2 (de) 2006-02-09
KR20010022740A (ko) 2001-03-26
NZ515955A (en) 2003-04-29
BR9814040A (pt) 2000-10-03
EP1504764A1 (en) 2005-02-09
HU228900B1 (en) 2013-06-28
US7544358B2 (en) 2009-06-09
EP1930022B1 (en) 2011-06-08
US20010056076A1 (en) 2001-12-27
DE69840113D1 (de) 2008-11-20
PL341029A1 (en) 2001-03-26
KR100586202B1 (ko) 2006-06-07
CZ2000413A3 (cs) 2000-11-15
CY1111792T1 (el) 2015-10-07
CN1267224A (zh) 2000-09-20
EE04752B1 (et) 2006-12-15
PT1504764E (pt) 2009-01-14
JP2001513333A (ja) 2001-09-04
US20040208878A1 (en) 2004-10-21
US6316601B1 (en) 2001-11-13
AU739283B2 (en) 2001-10-11
TR200202323T2 (tr) 2002-12-23
US6692741B2 (en) 2004-02-17
DK1930022T3 (da) 2011-07-25
EE200000068A (et) 2000-10-16
WO1999007405A1 (en) 1999-02-18
EP0996460B1 (en) 2005-01-12
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US7150871B2 (en) 2006-12-19
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ATE511850T1 (de) 2011-06-15
IL134288A (en) 2008-07-08
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PT1930022E (pt) 2011-08-01
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US20070148173A1 (en) 2007-06-28
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