EP0977585A1 - Procedes de diagnostic et de traitement de troubles lies au poids corporel chez l'animal - Google Patents

Procedes de diagnostic et de traitement de troubles lies au poids corporel chez l'animal

Info

Publication number
EP0977585A1
EP0977585A1 EP98902655A EP98902655A EP0977585A1 EP 0977585 A1 EP0977585 A1 EP 0977585A1 EP 98902655 A EP98902655 A EP 98902655A EP 98902655 A EP98902655 A EP 98902655A EP 0977585 A1 EP0977585 A1 EP 0977585A1
Authority
EP
European Patent Office
Prior art keywords
integrin
icam receptor
agent
animal
icam
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98902655A
Other languages
German (de)
English (en)
Other versions
EP0977585A4 (fr
Inventor
Denisa D. Wagner
Zhao Ming Dong
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Immune Disease Institute Inc
Original Assignee
Immune Disease Institute Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Immune Disease Institute Inc filed Critical Immune Disease Institute Inc
Publication of EP0977585A1 publication Critical patent/EP0977585A1/fr
Publication of EP0977585A4 publication Critical patent/EP0977585A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70525—ICAM molecules, e.g. CD50, CD54, CD102
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • C07K14/70553—Integrin beta2-subunit-containing molecules, e.g. CD11, CD18
    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • C07K16/2821—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 immunoglobulin superfamily against ICAM molecules, e.g. CD50, CD54, CD102
    • 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
    • C07K16/2845—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 against integrin beta2-subunit-containing molecules, e.g. CD11, CD18
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70525—ICAM molecules, e.g. CD50, CD54, CD102
    • 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
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/02—Nutritional disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • This invention relates generally to treatments, diagnoses, and drug screens for body weight related disorders in animals.
  • Body weight disorders including obesity and severe weight loss, afflict a large number of people.
  • Obesity is a condition in which there is an excess of body fat. It is often associated with other disease conditions including diabetes, high blood pressure and high cholesterol levels. Based on the Body Mass Index as an indicator for obesity, it has recently been reported that 59% of American adults exceed a healthy weight. Severe weight loss is a condition which can result from a variety of sources including cancer, AIDS, tissue wasting, anorexia nervosa, chronic infection or gastrointestinal disease.
  • Several genes have been previously reported to be implicated in contributing to obesity in mice (ob, db, tub, A y and fat).
  • the invention features a method for determining if an animal is at risk for a body weight disorder.
  • An animal is provided.
  • An aspect of ICAM receptor, e.g., ICAM-1 , or ⁇ 2 integrin, e.g., Mac-1 , metabolism or structure is evaluated in the animal.
  • An abnormality in the aspect of ICAM receptor or ⁇ 2 integrin metabolism or structure is diagnostic of being at risk for a body weight disorder.
  • Another aspect of the invention is a method for detecting the presence of a disease affecting body weight associated with elevated or decreased levels of ICAM receptor or ⁇ 2 integrin polypeptide in an animal.
  • the level of ICAM receptor or ⁇ 2 integrin polypeptide in a biological sample from a first animal is evaluated.
  • the level obtained in the evaluating step is compared to a level of ICAM receptor or ⁇ 2 integrin polypeptide present in a normal second animal or in the first animal at an earlier time.
  • An increase in the level of ICAM receptor or ⁇ 2 integrin as compared to a normal level is indicative of a disease affecting body weight associated with elevated levels of ICAM receptor or ⁇ 2 integrin polypeptide, and a decreased level of ICAM receptor or ⁇ 2 integrin polypeptide as compared to a normal level is indicative of a disease effecting body weight associated with decreased levels of ICAM receptor or ⁇ 2 integrin.
  • the evaluating step comprises contacting the biological sample having ICAM receptor or ⁇ 2 integrin polypeptide with an antibody that specifically binds to ICAM receptor or ⁇ 2 integrin polypeptide under conditions which allow the formation of reaction complexes comprising the antibody and the ICAM receptor or ⁇ 2 integrin polypeptide.
  • the formation of the reaction complexes comprising the antibody and the ICAM receptor or ⁇ 2 integrin polypeptide is detected.
  • the amount of the reaction complexes formed is evaluated, the amount corresponding to the level of ICAM receptor or ⁇ 2 integrin polypeptide in the biological sample.
  • Another aspect of the invention is a method for evaluating an agent for use in modulating body weight in an animal.
  • a test cell, cell-free system or animal having a non-wild-type pattern of ICAM receptor or ⁇ 2 integrin metabolism is provided.
  • An agent is provided.
  • the agent is administered to the test cell, cell-free system or animal in a therapeutically effective amount.
  • the effect of the agent on an aspect of ICAM receptor or ⁇ 2 integrin metabolism or on a parameter related to body weight is evaluated.
  • a change in the aspect of ICAM receptor or ⁇ 2 integrin metabolism or the parameter related to body weight is indicative of the usefulness of the agent in modulating body weight in the animal.
  • the method employs two phases for evaluating an agent for use in modulating body weight, an initial in vitro phase and then an in vivo phase.
  • the agent is administered to a test cell or cell-free system in vitro. If a change in the aspect of ICAM receptor or ⁇ 2 integrin metabolism occurs, then the agent is further administered to a test animal in a therapeutically effective amount.
  • the in vivo effect of the agent on an aspect of ICAM receptor or ⁇ 2 integrin metabolism or a parameter related to body weight is evaluated.
  • a change in the aspect of ICAM receptor or ⁇ 2 integrin metabolism or the parameter related to body weight is indicative of the usefulness of the agent in modulating body weight.
  • the test animal can have the same genotype or a different genotype from the test cell or cell-free system.
  • Another aspect of the invention is a method for evaluating an agent for the ability to modulate body weight in an animal.
  • An agent is provided.
  • ICAM receptor, an extracellular portion of ICAM receptor, ⁇ 2 integrin or an extracellular portion of ⁇ 2 integrin is provided.
  • the agent is contacted with ICAM receptor, the extracellular portion of ICAM receptor, ⁇ 2 integrin or the extracellular portion of ⁇ 2 integrin. It is determined if the agent interacts with ICAM receptor, the extracellular portion of ICAM receptor, ⁇ 2 integrin or the extracellular portion of ⁇ 2 integrin.
  • Another aspect of the invention is a method for evaluating an agent for the ability to modulate body weight in an animal by determining an alteration in the binding of ICAM receptor or ⁇ 2 integrin or extracellular portions thereof to a binding molecule.
  • An agent is provided.
  • ICAM receptor or an extracellular portion thereof, or ⁇ , integrin or an extracellular portion thereof, is provided.
  • a binding molecule or an extracellular portion thereof is provided.
  • the agent, the ICAM receptor or extracellular portion thereof or ⁇ 2 integrin or extracellular portion thereof, and the binding molecule or extracellular portion thereof, are combined.
  • the formation of a complex is detected.
  • An alteration in the formation of the complex in the presence of the agent as compared to in the absence of the agent is indicative of the agent altering the binding of the ICAM receptor or extracellular portion thereof or the ⁇ 2 integrin or extracellular portion thereof to the binding molecule.
  • a test animal is further provided, and the agent is further administered to the test animal in a therapeutically effective amount. The in vivo effect of the agent on the body weight of the test animal is evaluated.
  • Another aspect of the invention is a method for treating a body weight related disorder in an animal.
  • An animal in need of treatment for a body weight related disorder is provided.
  • An agent capable of altering an aspect of ICAM receptor or ⁇ 2 integrin metabolism or structure is provided.
  • the agent is administered to the animal in a therapeutically effective amount such that treatment of the body weight related disorder occurs.
  • Another aspect of the invention is a method for treating an animal at risk for a body weight related disorder.
  • An animal at risk for a body weight related disorder is provided.
  • An agent capable of altering an aspect of ICAM receptor or ⁇ 2 integrin structure or metabolism is provided.
  • the agent is administered to the animal in a therapeutically effective amount such that treatment of the animal occurs.
  • Another aspect of the invention is a method for monitoring a therapeutic treatment of a disease affecting body weight associated with elevated or decreased levels of ICAM receptor or ⁇ 2 integrin polypeptide in an animal.
  • the levels of ICAM receptor or ⁇ 2 integrin polypeptide in a plurality of biological samples obtained at different time points from an animal undergoing a therapeutic treatment for a disease affecting body weight associated with elevated or decreased levels of ICAM receptor or ⁇ 2 integrin polypeptide is evaluated.
  • Another aspect of the invention is a pharmaceutical composition for treating a body weight related disorder in an animal comprising a therapeutically effective amount of an agent, the agent being capable of altering an aspect of ICAM receptor or ⁇ 2 integrin metabolism or structure in the animal so as to result in treatment of the body weight related disorder in the animal, and a pharmaceutically acceptable carrier.
  • Another aspect of the invention is a method of making an ICAM receptor or ⁇ 2 integrin polypeptide having an antagonist or agonist activity so as to modulate body weight of an animal.
  • An ICAM receptor or ⁇ 2 integrin polypeptide is provided.
  • the amino acid sequence of the polypeptide is altered.
  • the altered polypeptide is tested for an effect on an aspect of ICAM receptor or ⁇ 2 integrin metabolism, a change in the aspect of ICAM receptor or ⁇ 2 integrin metabolism being indicative of an ICAM receptor or ⁇ 2 integrin polypeptide having an antagonist or agonist activity so as to modulate body weight of an animal.
  • Another aspect of the invention is a method for increasing the fat content of an animal liver, e.g., a goose liver.
  • An animal is provided. Soluble ICAM receptor or soluble ⁇ 2 integrin obtained from the animal is administered into the animal so as to increase the fat content of the liver of the animal.
  • Yet another aspect of the invention is a method for increasing the fat content in milk secreted by an animal.
  • An animal capable of secreting milk is provided.
  • An agent capable of altering an aspect of ICAM receptor or ⁇ 2 integrin structure or metabolism is provided.
  • the agent is administered to the animal in an amount so as to increase the fat content in the milk of the animal.
  • Fig. 1 depicts growth curves of wild-type and ICAM-1 -/- mice on a normal chow diet (5% fat).
  • Fig. 2 (a, b, c and d) depicts growth curves of wild-type and ICAM-1 -/- mice on a Western-type diet (21 % fat).
  • Fig. 3 depicts growth curves of wild-type and Mac-1 -/- mice on a Western-type diet (21% fat).
  • This invention provides a method for determining if an animal is at risk for a body weight disorder.
  • An animal is provided.
  • An aspect of ICAM receptor or ⁇ 2 integrin metabolism or structure is evaluated in the animal.
  • An abnormality in the aspect of ICAM receptor or ⁇ 2 integrin metabolism or structure is diagnostic of being at risk for a body weight disorder.
  • Non-human animals include, e.g., mammals, e.g., monkeys, chimpanzees, apes, rodents, pigs, rabbits, goats, cows or geese.
  • An animal also includes transgenic non-human animals.
  • transgenic animal is meant to include an animal that has gained new genetic information from the introduction of foreign DNA, i.e., partly or entirely heterologous DNA, into the DNA of its cells; or introduction of a lesion, e.g., an in vitro induced mutation, e.g., a deletion or other chromosomal rearrangement into the DNA of its cells; or introduction of homologous DNA into the DNA of its cells in such a way as to alter the genome of the cell into which the DNA is inserted, e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout.
  • the animal may include a transgene in all of its cells including germ line cells, or in only one or some of its cells.
  • Transgenic animals of the invention can serve as a model for studying body weight related disorders. In certain embodiments, the determination for being at risk for body weight related disorders is done in a prenatal animal.
  • a body weight disorder is meant to include, e.g., obesity or weight loss, e.g., severe weight loss.
  • Obesity is a condition in which there is an excess of body fat.
  • the most common indicator for obesity is Body Mass Index (BMI), which is the individual's weight in kilograms divided by his height in meters squared. BMI is highly correlated with body fat.
  • BMI Body Mass Index
  • the 1995 Guidelines from the National Institutes of Health and the American Health Foundation define healthy weight as a BMI below 25.
  • Obesity is often associated with other disease conditions, e.g., diabetes, high blood pressure and high cholesterol levels.
  • Weight loss is a condition which can occur as a result of diminished food intake, or in the absence of dietary restriction, e.g., with normal or excessive food intake. Weight loss occurring with normal or excessive food intake can result, e.g., from insulin-dependent diabetes mellitus, thyrotoxicosis or malabsorption of food. Weight loss resulting from diminished food intake can result, e.g., from cancer, AIDS, tissue wasting, anorexia nervosa, chronic infection or gastrointestinal disease. For example, cancer cachexia is a clinical syndrome that develops as a consequence of the nutritional and metabolic abnormalities of the tumor-bearing host, generally with advanced malignancies.
  • This syndrome includes anorexia, body weight loss, severe tissue wasting, asthenia and organ dysfunction.
  • Individuals with malignancies generally have aberrations in lipid metabolism, including an increased rate of lipolysis with accelerated catabolism of body fat stores.
  • lipid metabolism including an increased rate of lipolysis with accelerated catabolism of body fat stores.
  • glucose administration normal individuals suppress lipid mobilization and preferentially oxidize glucose.
  • Individuals with cancer fail to suppress endogenous lipid mobilization, and persistent oxidation of free fatty acids occurs.
  • Relative changes in the proportions of specific lipids contribute to further body compositional and metabolic derangements.
  • Conventional nutrition support generally does not reduce morbidity in cancer patients.
  • ICAM receptor intercellular adhesion molecule
  • ICAM receptors mediate leukocyte adhesion.
  • ICAM receptors include, e.g., ICAM-1, ICAM-2 and ICAM-3.
  • a preferred ICAM receptor is ICAM-1.
  • ICAM-1 is expressed, e.g., on leukocytes, endothelium, epithelium, hepatocytes, adipocytes, myocytes and fibroblasts.
  • ICAM-1 is a major cell-cell adhesion molecule in inflammatory and immune systems.
  • the primary counterreceptors for ICAM-1 are the ⁇ 2 integrins, e.g., Mac-1 and LFA-1.
  • ⁇ 2 integrin is meant any ⁇ 2 leukocyte integrin.
  • ⁇ 2 integrins include, e.g., Mac-1 and
  • Mac-1 also referred to as ⁇ M ⁇ 2 or CD1 l b /CD18. Mac-1 is involved, e.g., in several neutrophil functions including adhesion to the endothelium, phagocytosis and neutrophil apoptosis.
  • ICAM receptor and ⁇ , integrin are also involved in the regulation of body weight, e.g., adipose tissue mass.
  • Animals deficient in ICAM receptor or ⁇ 2 integrin become obese.
  • ICAM receptor and ⁇ 2 integrin play a role in preventing excessive body fat deposition, by regulating lipid metabolism and/or energy expenditure.
  • ICAM receptor or ⁇ 2 integrin metabolism is meant any aspect of the production, release, expression, function, action, interaction, e.g., cell/cell adhesion, or regulation of ICAM receptor or ⁇ 2 integrin.
  • the metabolism of ICAM receptor or ⁇ 2 integrin includes modifications, e.g., covalent or non-covalent modifications, of ICAM receptor or ⁇ 2 integrin polypeptide.
  • the terms peptides, proteins and polypeptides are used interchangeably herein.
  • the metabolism of ICAM receptor or ⁇ 2 integrin includes modifications, e.g., covalent or non-covalent modifications, that ICAM receptor or ⁇ 2 integrin induces in other substances.
  • the metabolism of ICAM receptor or ⁇ , integrin also includes changes in the distribution, concentration, activation or phosphorylation of ICAM receptor or ⁇ 2 integrin polypeptide, and changes ICAM receptor or ⁇ 2 integrin induces in the distribution, concentration, activation or phosphorylation of other substances.
  • ICAM receptor or ⁇ 2 integrin metabolism can be evaluated.
  • the methods used are standard techniques known to those skilled in the art and can be found in standard references, e.g., Ausubel et al., ed., Current Protocols in Mol. Biology, New York: John Wiley & Sons, 1990; Drewes et al, Mol. and Cell Biol. 16:925-931 (1996).
  • ICAM receptor or ⁇ 2 integrin metabolism that can be evaluated include the binding activity of ICAM receptor or ⁇ 2 integrin polypeptide to a binding molecule; the transactivation activity of ICAM receptor or ⁇ 2 integrin polypeptide on a target gene; the level of ICAM receptor or ⁇ 2 integrin polypeptide; the level of ICAM receptor or ⁇ 2 integrin mRNA; or the level of ICAM receptor or ⁇ 2 integrin phosphorylation.
  • binding molecule any molecule to which ICAM receptor or ⁇ 2 integrin can bind, e.g., a nucleic acid, e.g., a DNA regulatory region, a protein, a metabolite, a peptide mimetic, a non-peptide mimetic, an antibody, or any other type of ligand.
  • the binding molecule itself is ICAM receptor or ⁇ , integrin, i.e., ICAM receptor, e.g., ICAM-1, can bind to ⁇ 2 integrin, e.g., Mac-1. Binding can be shown, e.g., by electrophoretic mobility shift analysis (EMSA).
  • ESA electrophoretic mobility shift analysis
  • Transactivation of a target gene by ICAM receptor or ⁇ 2 integrin can be determined, e.g., in a transient transfection assay in which the promoter of the target gene is linked to a reporter gene, e.g., ⁇ -galactosidase or luciferase, and co-transfected with an ICAM receptor or ⁇ 2 integrin expression vector.
  • a reporter gene e.g., ⁇ -galactosidase or luciferase
  • levels of ICAM receptor or ⁇ 2 integrin protein, mRNA or phosphorylation can, e.g., be measured in a sample, e.g., a tissue sample, e.g., blood.
  • an aspect of ICAM receptor or ⁇ 2 integrin structure is evaluated, e.g., ICAM receptor or ⁇ 2 integrin gene structure or ICAM receptor or ⁇ 2 integrin protein WO 98/32460 _g_ PCT/US98/01110
  • the DNA sequence of the gene is determined and/or the amino acid sequence of the protein is determined. Standard cloning and sequencing methods can be used as are known to those skilled in the art.
  • the binding activity of an antisense nucleic acid with the cellular ICAM receptor or ⁇ 2 integrin mRNA and/or genomic DNA is determined using standard methods known to those skilled in the art so as to detect the presence or absence of the target mRNA or DNA sequences to which the antisense nucleic acid would normally specifically bind.
  • the invention also includes a method for detecting the presence of a disease affecting body weight associated with elevated or decreased levels of ICAM receptor or ⁇ 2 integrin polypeptide in an animal.
  • the level of ICAM receptor or ⁇ 2 integrin polypeptide in a biological sample from a first animal is evaluated.
  • the level obtained in the evaluating step is compared to a level of ICAM receptor or ⁇ 2 integrin polypeptide present in a normal second animal or in the first animal at an earlier time.
  • An increase in the level of ICAM receptor or ⁇ 2 integrin as compared to a normal level is indicative of a disease affecting body weight associated with elevated levels of ICAM receptor or ⁇ 2 integrin polypeptide, and a decreased level of ICAM receptor or ⁇ 2 integrin polypeptide as compared to a normal level is indicative of a disease effecting body weight associated with decreased levels of ICAM receptor or ⁇ 2 integrin.
  • the evaluating step comprises contacting the biological sample having ICAM receptor or ⁇ 2 integrin polypeptide with an antibody that specifically binds to ICAM receptor or ⁇ 2 integrin polypeptide under conditions which allow the formation of reaction complexes comprising the antibody and the ICAM receptor or ⁇ 2 integrin polypeptide.
  • the formation of the reaction complexes comprising the antibody and the ICAM receptor or ⁇ 2 integrin polypeptide is detected.
  • the amount of the reaction complexes formed is evaluated, the amount corresponding to the level of ICAM receptor or ⁇ 2 integrin polypeptide in the biological sample.
  • Biological sample is meant to include, e.g., blood, leukocytes, endothelium, epithelium, hepatocytes, myocytes, fibroblasts, adipose tissue and lymph.
  • a biological sample is also meant to include samples or portions of the biological sample that have been resuspended in other media or pelleted.
  • a normal animal is meant an animal with unimpaired ICAM receptor or ⁇ 2 integrin.
  • a normal level of ICAM receptor or ⁇ 2 integrin is meant the level of ICAM receptor or ⁇ 2 integrin in a normal animal.
  • the normal level can be ascertained from a separate normal animal other than the animal being tested, or it can be ascertained from the tested animal from earlier obtained samples when the tested animal was normal.
  • a disease affecting body weight that is associated with elevated levels of ICAM receptor or ⁇ 2 integrin is meant to include, e.g., weight loss related diseases.
  • a disease affecting body weight that is associated with decreased levels of ICAM receptor or ⁇ 2 integrin is meant to include, e.g., obesity.
  • the antibodies can be labeled, e.g., with a radioactive label or a non-radioactive label, e.g., fluorescent labels.
  • the invention also includes a method for evaluating an agent for use in modulating body weight in an animal.
  • a test cell, cell-free system or animal having a non-wild-type pattern of ICAM receptor or ⁇ 2 integrin metabolism is provided.
  • An agent is provided.
  • the agent is administered to the test cell, cell-free system or animal in a therapeutically effective amount.
  • the effect of the agent on an aspect of ICAM receptor or ⁇ 2 integrin metabolism or on a parameter related to body weight is evaluated.
  • a change in the aspect of ICAM receptor or ⁇ 2 integrin metabolism or the parameter related to body weight is indicative of the usefulness of the agent in modulating body weight in the animal.
  • the method employs two phases for evaluating an agent for use in modulating body weight, an initial in vitro phase and then an in vivo phase.
  • the agent is administered to a test cell or cell-free system in vitro. If a change in the aspect of ICAM receptor or ⁇ 2 integrin metabolism occurs, then the agent is further administered to a test animal in a therapeutically effective amount.
  • the in vivo effect of the agent on an aspect of ICAM receptor or ⁇ 2 integrin metabolism or a parameter related to body weight is evaluated.
  • a change in the aspect of ICAM receptor or ⁇ 2 integrin metabolism or the parameter related to body weight is indicative of the usefulness of the agent in modulating body weight.
  • the test animal can have the same genotype or a different genotype from the test cell or cell-free system.
  • Modulating body weight is meant to include increasing body weight or decreasing body weight.
  • cell is meant a cell or a group of cells, or a cell that is part of an animal.
  • the cell can be a human or non-human cell.
  • Cell is also meant to include a transgenic cell.
  • the cell can be obtained, e.g., from a culture or from an animal.
  • the animal can be a natural animal or non- human transgenic animal.
  • the transgenic cell or non-human transgenic animal has an ICAM receptor or ⁇ 2 integrin transgene, or fragment or analog thereof.
  • the transgenic cell or non-human transgenic animal has a deletion, e.g., a knockout, or an addition of a gene coding for ICAM receptor or ⁇ 2 integrin.
  • a non-wild-type pattern of ICAM receptor or ⁇ 2 integrin metabolism can result, e.g., from underexpression, overexpression, no expression, or a temporal, site or distribution change in expression.
  • Such a non-wild-type pattern can result, e.g., from one or more mutations in the ICAM receptor or ⁇ 2 integrin gene, in a binding molecule gene, or in any other gene which directly or indirectly affects ICAM receptor or ⁇ 2 integrin metabolism.
  • a mutation is meant to include, e.g., an alteration, e.g., in gross or fine structure, in a nucleic acid.
  • Examples include single base pair alterations, e.g., missense or nonsense mutations, frameshifts, deletions, insertions and translocations.
  • Mutations can be dominant or recessive. Mutations can be homozygous or heterozygous.
  • any aspect of ICAM receptor or ⁇ 2 integrin metabolism can be evaluated.
  • the aspect of metabolism is the binding of ICAM receptor to ⁇ 2 integrin.
  • Any parameter related to body weight can be evaluated.
  • the parameter is lipid metabolism, e.g., regulation of fatty acid oxidation.
  • An agent is meant to include, e.g., any substance, e.g., a drug for weight gain or a drug for weight loss.
  • the agent of this invention preferably can change an aspect of ICAM receptor or ⁇ 2 integrin metabolism. Such change can be the result of any of a variety of events, including, e.g., preventing or reducing interaction between ICAM receptor or ⁇ 2 integrin and a binding molecule; inactivating ICAM receptor or ⁇ 2 integrin and/or the binding molecule, e.g., by cleavage or other modification; altering the affinity of ICAM receptor or ⁇ 2 integrin and the binding molecule for each other; diluting out ICAM receptor or ⁇ 2 integrin and/or the binding molecule; preventing expression of ICAM receptor or ⁇ 2 integrin and/or the binding molecule; reducing synthesis of ICAM receptor or ⁇ 2 integrin and/or the binding molecule; synthesizing an abnormal ICAM receptor or ⁇ 2 integrin and/
  • the binding molecule can be on the same or different cell as ICAM or ⁇ 2 integrin.
  • agents include ICAM receptor or ⁇ 2 integrin polypeptide or a biologically active fragment or analog thereof; a nucleic acid encoding ICAM receptor or ⁇ 2 integrin polypeptide or a biologically active fragment thereof; a nucleic acid encoding an ICAM receptor or ⁇ 2 integrin regulatory sequence or a biologically active fragment thereof; a binding molecule for ICAM receptor or ⁇ 2 integrin polypeptide; a binding molecule for ICAM receptor or ⁇ 2 integrin nucleic acid, the ICAM receptor or ⁇ 2 integrin nucleic acid being, e.g., a nucleic acid comprising a regulatory region for ICAM receptor or ⁇ 2 integrin or a nucleic acid comprising a structural region for ICAM receptor or ⁇ 2 integrin or a biologically active fragment of ICAM receptor or ⁇ 2 integrin; an antisense nucleic acid; a
  • ICAM receptor or ⁇ 2 integrin allows a search for natural or artificial ligands to regulate fat metabolism in the treatment of body weight disorders.
  • the agent is a natural ligand for ICAM receptor or ⁇ 2 integrin.
  • the agent is an artificial ligand for ICAM receptor or ⁇ 2 integrin.
  • analog is meant a compound that differs from naturally occurring ICAM receptor or ⁇ 2 integrin in amino acid sequence or in ways that do not involve sequence, or both.
  • Analogs of the invention generally exhibit at least about 90% homology, preferably at least about 95% homology, and most preferably at least about 99% homology, with a segment of 20 amino acid residues, preferably with more than 40 amino acid residues, or more preferably yet with substantially the entire sequence of a naturally occurring ICAM receptor or ⁇ 2 integrin sequence.
  • Non-sequence modifications include, e.g., in vivo or m vitro chemical derivatizations of ICAM receptor or ⁇ 2 integrin.
  • Non-sequence modifications include, e.g., changes in phosphorylation, acetylation, methylation, carboxylation, or glycosylation. Methods for making such modifications are known to those skilled in the art. For example, phosphorylation can be modified by exposing ICAM receptor or ⁇ 2 integrin to phosphorylation-altering enzymes, e.g., kinases or phosphatases.
  • Preferred analogs include ICAM receptor or ⁇ 2 integrin or biologically active fragments thereof, whose sequences differ from the wild-type sequence by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not abolish ICAM receptor or ⁇ 2 integrin biological activity.
  • Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative substitutions are shown in
  • Amino acid sequence variants of a protein can be prepared by any of a variety of methods known to those skilled in the art. For example, random mutagenesis of DNA which encodes a protein or a particular domain or region of a protein can be used, e.g., PCR mutagenesis (using, e.g., reduced Taq polymerase fidelity to introduce random mutations into a cloned fragment of DNA; Leung et al., Technique 1 :11-15 (1989)), or saturation mutagenesis (by, e.g., chemical treatment or irradiation of single-stranded DNA in vitro, and synthesis of a complementary DNA strand; Myers et al., Science 229:242-247 (1985)).
  • PCR mutagenesis using, e.g., reduced Taq polymerase fidelity to introduce random mutations into a cloned fragment of DNA; Leung et al., Technique 1 :11-15 (1989)
  • saturation mutagenesis
  • Random mutagenesis can also be accomplished by, e.g., degenerate oligonucleotide generation (using, e.g., an automatic DNA synthesizer to chemically synthesize degenerate sequences; Narang, Tetrahedron 39:3 (1983); Itakura et al., Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. A.G.
  • Non-random or directed mutagenesis can be used to provide specific sequences or mutations in specific regions. These techniques can be used to create variants which include, e.g., deletions, insertions, or substitutions, of residues of the known amino acid sequence of a protein.
  • the sites for mutation can be modified individually or in series, e.g., by (i) substituting first with conserved amino acids and then with more radical choices depending upon results achieved, (ii) deleting the target residue, (iii) inserting residues of the same or a different class adjacent to the located site, or (iv) combinations of the above.
  • Methods for identifying desirable mutations include, e.g., alanine scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)), oligonucleotide-mediated mutagenesis (Adelman et al., DNA 2:183-193 (1983)); cassette mutagenesis (Wells et al., Gene 34:315-323 (1985)), combinatorial mutagenesis, and phage display libraries (Ladner et al.. WO88/06630).
  • analogs within the invention include, e.g., those with modifications which increase peptide stability. Such analogs may contain, e.g., one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are, e.g.: analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., ⁇ or ⁇ amino acids; and cyclic analogs. Analogs can be made by methods known to those skilled in the art.
  • fragment By fragment is meant some portion of the naturally occurring ICAM receptor or ⁇ 2 integrin polypeptide. Preferably, the fragment is at least about 3 amino acid residues, more preferably at least about 10 to at least about 20 amino acid residues. In certain embodiments, entire domains containing at least about 50 amino acids are used. Fragments include, e.g., truncated secreted forms, proteolytic fragments, splicing fragments, other fragments, and chimeric constructs between at least a portion of the relevant gene, e.g., ICAM receptor or ⁇ 2 integrin, and another molecule. Fragments of ICAM receptor or ⁇ 2 integrin can be generated by methods known to those skilled in the art.
  • ICAM receptor or ⁇ 2 integrin The ability of a candidate fragment to exhibit a biological activity of ICAM receptor or ⁇ 2 integrin can be assessed by methods known to those skilled in the art. Also included are ICAM receptor or ⁇ 2 integrin fragments containing residues that are not required for biological activity of the fragment or that result from alternative mRNA splicing or alternative protein processing events.
  • Fragments of a protein can be produced in several ways, e.g., recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide. Expression of the mutagenized DNA produces polypeptide fragments. Digestion with "end-nibbling" endonucleases can thus generate DNAs which encode an array of fragments. DNAs which encode fragments of a protein can also be generated, e.g., by random shearing, restriction digestion or a combination of the above-discussed methods.
  • fragments of ICAM receptor or ⁇ 2 integrin can be made by expressing ICAM receptor or ⁇ 2 integrin DNA which has been manipulated in vitro to encode the desired fragment, e.g., by restriction digestion of the DNA sequence for the gene.
  • Fragments can also be chemically synthesized using techniques known in the art, e.g., conventional Merrifield solid phase f-Moc or t-Boc chemistry.
  • peptides of the present invention can be arbitrarily divided into fragments of desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length.
  • ICAM receptor or ⁇ 2 integrin or a biologically active fragment or analog thereof, or a binding molecule or a biologically active fragment or analog thereof can, e.g., compete with its cognate molecule for the binding site on the complementary molecule, and thereby reduce or eliminate binding between ICAM receptor or ⁇ 2 integrin and the binding molecule.
  • ICAM receptor or ⁇ 2 integrin or a binding molecule can be obtained, e.g., from purification or secretion of naturally occurring ICAM receptor or ⁇ 2 integrin or a binding molecule, from recombinant ICAM receptor or ⁇ 2 integrin or a binding molecule, or from synthesized ICAM receptor or ⁇ 2 integrin or a binding molecule.
  • An agent can also be a nucleic acid used as an antisense molecule.
  • Antisense therapy is meant to include, e.g., administration or in situ generation of oligonucleotides or their derivatives which specifically hybridize, e.g., bind, under cellular conditions, with the cellular mRNA and/or genomic DNA encoding an ICAM receptor or ⁇ 2 integrin polypeptide, or mutant thereof, so as to inhibit expression of the encoded protein, e.g., by inhibiting transcription and/or translation.
  • the binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.
  • the antisense construct binds to a naturally-occurring sequence of an ICAM receptor or ⁇ 2 integrin gene which, e.g., is involved in expression of the gene. These sequences include, e.g., start codons and stop codons.
  • the antisense construct binds to a nucleotide sequence which is not present in the wild-type gene.
  • the antisense construct can bind to a region of an ICAM receptor or ⁇ 2 integrin gene which contains an insertion of an exogenous, non-wild-type sequence.
  • the antisense construct can bind to a region of an ICAM receptor or ⁇ 2 integrin gene which has undergone a deletion, thereby bringing two regions of the gene together which are not normally positioned together and which, together, create a non-wild-type sequence.
  • antisense constructs which bind to non-wild- type sequences provide the advantage of inhibiting the expression of a mutant ICAM receptor or ⁇ 2 integrin gene, without inhibiting expression of any wild-type ICAM receptor or ⁇ 2 integrin gene.
  • an antisense construct of the present invention can be delivered, e.g., as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes an ICAM receptor or ⁇ 2 integrin polypeptide.
  • the antisense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences of an ICAM receptor or ⁇ 2 integrin gene.
  • Such oligonucleotide probes are preferably modified oligonucleotides which are resistant to endogenous nucleases, e.g.
  • nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphorothioate and methylphosphonate analogs of DNA.
  • phosphoramidate, phosphorothioate and methylphosphonate analogs of DNA See also U.S. Patents 5,176,996; 5,264,564; and 5,256,775. Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed. (See, e.g.. van der Krol and Molo, Biotechniques 6:958- 976, (1988); Stein and Cohen, Cancer Res. 48:2659-2668 (1988)).
  • antisense oligonucleotides include phosphorothioate antisense oligonucleotides which target the AUG translation initiation codon or sequences in the 3'- untranslated region of ICAM-1 message (Chiang et al., J. Biol. Chem. 266:18162-18171 (1991); antisense oligonucleotides to ICAM-1 3'- untranslated region (Kumaska et al., J. Clin. Invest. 97:2362-2369 (1996)).
  • mimetic is meant a molecule which resembles in shape and/or charge distribution ICAM receptor or ⁇ 2 integrin or a binding molecule.
  • the mimetic can be a peptide or a non- peptide.
  • Mimetics can act as therapeutic agents because they can, e.g., competitively inhibit binding of ICAM receptor or ⁇ 2 integrin to a binding molecule.
  • scanning mutagenesis e.g., alanine scanning mutagenesis, linker scanning mutagenesis or saturation mutagenesis
  • peptide mimetics e.g., diazopine or isoquinoline derivatives
  • non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (see, e.g.. Freidinger et al., in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands (1988)); azepine (see, e.g.. Huffman et al., in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands (1988)); substituted gamma lactam rings (see, e.g.. Garvey et al., in Peptides: Chemistry and Biology, G.R.
  • keto-methylene pseudopeptides see, e.g.. Ewenson et al., J. Med. Chem. 29:295 (1986); Ewenson et al., in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, IL (1985)); ⁇ -turn dipeptide cores (see, e.g.. Nagai et al., Tetrahedron Lett. 26:647 (1985); Sato et al., J. Chem. Soc. Perkin Trans.
  • NIF Neutrophil Adhesion Inhibitor is a naturally occurring 41kDa glycoprotein derived from hookworm.
  • peptides and mimetics of the active epitope of this protein can be administered orally.
  • Peptides derived from fibrinogen specifically ⁇ chain have been shown to bind to the I-domain of Mac-1 and inhibit its function (Wright et al., Proc. Natl. Acad. Sci. USA 85:7734-7738 (1988); Altieri et al., J. Biol.
  • Non-peptide inhibitors of Mac-1 have been described, including leumedins (N-[9H-(2,7- dimethylfluorenyl-9-methoxy)carbonyl]-leucine, NPC 15669) (Bator et al., Immunopharmacology 23:139 (1992); Burch et al., J. Immunology 150:3397-3403 (1993)).
  • Antibodies are meant to include antibodies against any moiety that directly or indirectly affects ICAM receptor or ⁇ 2 integrin metabolism.
  • the antibodies can be directed against, e.g., ICAM receptor or ⁇ 2 integrin or a binding molecule, or a subunit or fragment thereof.
  • antibodies include anti-ICAM receptor or anti- ⁇ 2 integrin antibodies; anti-binding molecule antibodies; and Fab 2 ' fragments of the inhibitory antibody generated through, e.g., enzymatic cleavage.
  • Both polyclonal and monoclonal antibodies can be used in this invention. Preferably, monoclonal antibodies are used.
  • the antibodies have a constant region derived from a human antibody and a variable region derived from an inhibitory mouse monoclonal antibody.
  • Examples of antibodies to Mac-1 include monoclonal antibodies to ⁇ M (CDl lb, part of Mac-1) (Simpson et al., J. Clin. Invest. 81:624-629 (1988); Hill et al, Surgery 1 12:166-172 (1992); and anti- ⁇ 2 monoclonal antibodies (Vedder et al., Surgery 106:509-516 (1989); Vedder et al., Proc. Natl. Acad. Sci.
  • LFA-1 examples include antibodies to the related integrin subunit ⁇ L (CDl la, part of LFA-1 (Fisher et al., Blood 77:249-256 (1991)).
  • monoclonal antibodies to ICAM-1 are described in Bowes et al., Exp. Neurol. 119:215-219 (1993); Seecamp et al., Am. J. Pathol. 143:464-472 (1993).
  • Human monoclonal antibodies for ICAM-1 and Mac-1 are commercially available, e.g., from R&D Systems, Minneapolis, MN.
  • Agents also include inhibitors of a molecule that are required for synthesis, post-translational modification, or functioning of ICAM receptor or ⁇ 2 integrin and/or a binding molecule, or activators of a molecule that inhibits the synthesis or functioning of ICAM receptor or ⁇ 2 integrin and/or the binding molecule.
  • Agents include, e.g., cytokines, growth factors, hormones, signaling components, kinases, phosphatases, homeobox proteins, transcription factors, translation factors and post-translation factors or enzymes.
  • Agents are also meant to include ionizing radiation, non-ionizing radiation, ultrasound and toxic agents which can, e.g., at least partially inactivate or destroy ICAM-1 or Mac-1 and/or the binding molecule.
  • agent is also meant to include agents which are not entirely ICAM receptor or ⁇ 2 integrin specific.
  • an agent may alter other lipid metabolism related genes or proteins. Such overlapping specificity may provide additional therapeutic advantage.
  • the invention also includes the agent so identified as being useful in modulating body weight.
  • the invention also includes a method for evaluating an agent for the ability to modulate body weight in an animal.
  • An agent is provided.
  • ICAM receptor, an extracellular portion of ICAM receptor, ⁇ 2 integrin or an extracellular portion of ⁇ 2 integrin is provided.
  • the agent is contacted with ICAM receptor, the extracellular portion of ICAM receptor, ⁇ 2 integrin or the extracellular portion of ⁇ 2 integrin. It is determined if the agent interacts with ICAM receptor, the extracellular portion of ICAM receptor, ⁇ 2 integrin or the extracellular portion of ⁇ 2 integrin. If interaction is found, then the agent is further administered to a test animal in a therapeutically effective amount. The in vivo effect of the agent on the body weight of the test animal is evaluated.
  • determining whether interaction has occurred comprises determining whether binding has occurred between the agent and the compound.
  • the test agent is administered more than one time to the test animal.
  • the invention also includes a method for evaluating an agent for the ability to modulate body weight in an animal by determining an alteration in the binding of ICAM receptor or ⁇ 2 integrin or extracellular portions thereof to a binding molecule.
  • An agent is provided.
  • ICAM receptor or an extracellular portion thereof or ⁇ 2 integrin or an extracellular portion thereof is provided.
  • a binding molecule or an extracellular portion thereof is provided.
  • the agent, the ICAM receptor or extracellular portion thereof or ⁇ 2 integrin or an extracellular portion thereof, and the binding molecule or extracellular portion thereof, are combined.
  • the binding includes, e.g., inhibiting or promoting the binding.
  • the binding molecule is ⁇ 2 integrin.
  • the binding molecule is ICAM receptor.
  • the efficacy of the agent can be assessed, e.g., by generating dose response curves from data obtained using various concentrations of the agent. Methods for determining formation of a complex are standard and are known to those skilled in the art.
  • the invention also includes the agent so identified as being able to alter the binding of ICAM receptor or ⁇ 2 integrin polypeptide to a binding molecule.
  • the invention also includes a method for treating a body weight related disorder in an animal.
  • An animal in need of treatment for a body weight related disorder is provided.
  • An agent capable of altering an aspect of ICAM receptor or ⁇ 2 integrin metabolism or structure is provided.
  • the agent is administered to the animal in a therapeutically effective amount such that treatment of the body weight related disorder occurs.
  • the body weight related disorder can be, e.g., obesity or weight loss.
  • the obesity is associated with diabetes, high blood pressure or high cholesterol levels.
  • the weight loss results from cancer, AIDS, tissue wasting, anorexia nervosa, chronic infection, gastrointestinal disease, insulin-dependent diabetes mellitus, thyrotoxicosis or malabsorption of food.
  • Treating is meant to include, e.g., preventing, treating, reducing the symptoms of, or curing the body weight related disorder.
  • the agent can be, e.g., ICAM receptor or ⁇ 2 integrin polypeptide or a biologically active fragment or analog thereof; a nucleic acid encoding ICAM receptor or ⁇ 2 integrin polypeptide or a biologically active fragment thereof; a nucleic acid encoding an ICAM receptor or ⁇ 2 integrin regulatory sequence or a biologically active fragment thereof; a binding molecule for ICAM receptor or ⁇ 2 integrin polypeptide; a binding molecule for ICAM receptor or ⁇ 2 integrin nucleic acid, the ICAM receptor or ⁇ 2 integrin nucleic acid being, e.g., a nucleic acid comprising a regulatory region for ICAM receptor or ⁇ 2 integrin or a nucleic acid comprising a structural region for ICAM receptor or ⁇ 2 integrin or a biologically active fragment of ICAM receptor
  • ICAM receptor or ⁇ 2 integrin For body weight disorders involving underexpression or no expression of ICAM receptor or ⁇ 2 integrin, that result in obesity, it is preferred to add more ICAM receptor or ⁇ 2 integrin, either directly or indirectly.
  • addition of ICAM receptor or ⁇ 2 integrin can result from addition of ICAM receptor or ⁇ 2 integrin polypeptide, or addition of the ICAM receptor or ⁇ 2 integrin gene, or from up-regulation of the ICAM receptor or ⁇ 2 integrin gene, or from addition of an agonist of ICAM receptor or ⁇ 2 integrin.
  • the ICAM receptor or ⁇ 2 integrin genes can be modified so as to give higher expression. Such modification can be done in vitro or in vivo by standard techniques known to those skilled in the art.
  • ICAM receptor or ⁇ 2 integrin polypeptide For body weight disorders involving overexpression of ICAM receptor or ⁇ 2 integrin, that result in a weight loss disorder, it is preferred to inhibit the ICAM receptor or ⁇ 2 integrin polypeptide, either directly or indirectly.
  • an inhibitor of ICAM receptor or ⁇ 2 integrin e.g., an antagonist, e.g., an antibody for ICAM receptor or ⁇ 2 integrin, an ICAM receptor or ⁇ 2 integrin polypeptide fragment or analog, a mimetic of ICAM receptor or ⁇ 2 integrin or an antisense molecule for ICAM receptor or ⁇ 2 integrin, or an inhibitor of a molecule, e.g., a cytokine, that induces the expression, e.g., the overexpression of ICAM receptor or ⁇ 2 integrin.
  • an antagonist e.g., an antibody for ICAM receptor or ⁇ 2 integrin, an ICAM receptor or ⁇ 2 integrin polypeptide fragment or analog
  • certain fragments or analogs of ICAM receptor or ⁇ 2 integrin can compete with the cognate molecule for the binding site on a complementary molecule, and thereby reduce or eliminate binding between ICAM receptor or ⁇ 2 integrin and their binding molecules.
  • Administration of the agent can be accomplished by any method which allows the agent to reach the target cells. These methods include, e.g., injection, deposition, implantation, suppositories, oral ingestion, inhalation, topical administration, or any other method of administration where access to the target cells by the agent is obtained. Injections can be, e.g., intravenous, intradermal, subcutaneous, intramuscular or intraperitoneal.
  • Implantation includes inserting implantable drug delivery systems, e.g., microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, polymeric systems, e.g., matrix erosion and/or diffusion systems and non-polymeric systems, e.g., compressed, fused or partially fused pellets.
  • Suppositories include glycerin suppositories.
  • Oral ingestion doses can be enterically coated.
  • Inhalation includes administering the agent with an aerosol in an inhalator, either alone or attached to a carrier that can be absorbed.
  • Administration of the agent can be alone or in combination with other therapeutic agents.
  • the agent can be combined with a suitable carrier, incorporated into a liposome, or incorporated into a polymer release system.
  • the administration can be designed so as to result in sequential exposures to the agent over some time period, e.g., hours, days, weeks, months or years. This can be accomplished by repeated administrations of the agent by one of the methods described above, or alternatively, by a controlled release delivery system in which the agent is delivered to the animal over a prolonged period without repeated administrations.
  • a controlled release delivery system is meant that total release of the agent does not occur immediately upon administration, but rather is delayed for some time period. Release can occur in bursts or it can occur gradually and continuously.
  • Administration of such a system can be, e.g., by long acting oral dosage forms, bolus injections, transdermal patches or sub-cutaneous implants.
  • Examples of systems in which release occurs in bursts include, e.g., systems in which the agent is entrapped in liposomes which are encapsulated in a polymer matrix, the liposomes being sensitive to a specific stimuli, e.g., temperature, pH, light or a degrading enzyme, and systems in which the agent is encapsulated by an ionically-coated microcapsule with a microcapsule core-degrading enzyme.
  • Examples of systems in which release of the agent is gradual and continuous include, e.g., erosional systems in which the agent is contained in a form within a matrix, and diffusional systems in which the agent permeates at a controlled rate, e.g., through a polymer.
  • Such sustained release systems can be, e.g., in the form of pellets or capsules.
  • the agent can be suspended in a liquid, e.g., in dissolved form or colloidal form.
  • the liquid can be a solvent, partial solvent or non-solvent. In many cases water or an organic liquid can be used.
  • the agent can be administered prior to or subsequent to the appearance of body weight related disorder symptoms.
  • the agent is administered to patients with familial histories of body weight related disorders, or who have phenotypes that may indicate a predisposition to a body weight related disorder, or who have been diagnosed as having a genotype which predisposes the patient to a body weight related disorder, or who have a disease which is associated with a body weight related disorder.
  • the agent is administered to the animal in a therapeutically effective amount.
  • therapeutically effective amount is meant that amount which is capable of at least partially preventing or reversing the body weight related disorder.
  • a therapeutically effective amount can be determined on an individual basis and will be based, at least in part, on consideration of the species of animal, the animal's size, the animal's age, the agent used, the type of delivery system used, the time of administration relative to the onset of body weight related disorder symptoms, and whether a single, multiple, or controlled release dose regimen is employed.
  • a therapeutically effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.
  • the concentration of the agent is at a dose of about 0.1 to about 1000 mg/kg body weight, more preferably at about 0.1 to about 500 mg/kg, more preferably yet at about 0.1 to about 100 mg/kg, and most preferably at about 0.1 to about 5 mg/kg.
  • the specific concentration partially depends upon the particular agent used, as some are more effective than others.
  • the dosage concentration of the agent that is actually administered is dependent at least in part upon the final concentration that is desired at the site of action, the method of administration, the efficacy of the particular agent, the longevity of the particular agent, and the timing of administration relative to the onset of the body weight related disorder symptoms.
  • the dosage form is such that it does not substantially deleteriously affect the animal.
  • the dosage can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.
  • various gene constructs can be used as part of a gene therapy protocol to deliver nucleic acids encoding, e.g., either an agonistic or antagonistic form of an ICAM receptor or ⁇ 2 integrin polypeptide or their binding molecules.
  • Expression vectors can be used for in vivo transfection and expression of an ICAM receptor or ⁇ 2 integrin polypeptide in particular cell types so as to reconstitute the function of, or alternatively, abrogate the function of ICAM receptor or ⁇ 2 integrin polypeptide in a cell in which non- wild-type ICAM receptor or ⁇ 2 integrin is expressed.
  • Expression constructs of the ICAM receptor or ⁇ 2 integrin polypeptide, and mutants thereof, may be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively delivering the ICAM receptor or ⁇ 2 integrin gene to cells in vivo.
  • Approaches include, e.g., insertion of the subject gene in viral vectors including, e.g., recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus- 1, or recombinant bacterial or eukaryotic plasmids.
  • Viral vectors transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g. antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO 4 precipitation carried out in vivo or in vitro.
  • lipofectin cationic liposomes
  • derivatized e.g. antibody conjugated
  • polylysine conjugates e.g. antibody conjugated
  • gramacidin S e.g. antibody conjugated
  • artificial viral envelopes e.g., artificial viral envelopes or other such intracellular carriers
  • CaPO 4 precipitation carried out in vivo or in vitro.
  • Administration can be directed to one or more cell types, and to one or more cells within a cell type, so as to be therapeutically effective, by methods that are known to those skilled in the art.
  • the agent is administered to the liver or adipose tissue of the animal.
  • the agent is administered to a stem cell of the animal.
  • a genetically engineered ICAM receptor or ⁇ 2 integrin is administered to the liver or to hematopoietic stem cells by a viral vector.
  • administration is done in a prenatal animal or embryonic cell. It will be recognized that the particular gene construct provided for in in vivo transduction of ICAM receptor or ⁇ 2 integrin expression are also useful for in vitro transduction of cells, such as for use in the diagnostic assays described above.
  • the invention also includes a method for treating an animal at risk for a body weight related disorder.
  • An animal at risk for a body weight related disorder is provided.
  • An agent capable of altering an aspect of ICAM receptor or ⁇ 2 integrin structure or metabolism is provided.
  • the agent is administered to the animal in a therapeutically effective amount such that treatment of the animal occurs.
  • Being at risk for a body weight related disorder can result from, e.g., a familial history of a body weight related disorder, phenotypic symptoms which predispose to a body weight related disorder, a genotype which predisposes to a body weight related disorder, or having a disease which is associated with a body weight related disorder.
  • the invention also includes a method for monitoring a therapeutic treatment of a disease affecting body weight associated with elevated or decreased levels of ICAM receptor or ⁇ 2 integrin polypeptide in an animal.
  • the levels of ICAM receptor or ⁇ 2 integrin polypeptide in a plurality of biological samples obtained at different time points from an animal undergoing a therapeutic treatment for a disease affecting body weight associated with elevated or decreased levels of ICAM receptor or ⁇ 2 integrin polypeptide is evaluated.
  • the invention also includes a pharmaceutical composition for treating a body weight related disorder in an animal comprising a therapeutically effective amount of an agent, the agent being capable of altering an aspect of ICAM receptor or ⁇ 2 integrin metabolism or structure in the animal so as to result in treatment of the body weight related disorder in the animal, and a pharmaceutically acceptable carrier.
  • the agent is an antagonist of ICAM receptor or ⁇ 2 integrin, e.g., an antibody for ICAM receptor or ⁇ 2 integrin, a fragment or analog of ICAM receptor or ⁇ 2 integrin, a small molecule antagonist of ICAM receptor or ⁇ , integrin, a mimetic of ICAM receptor or ⁇ 2 integrin, an antisense molecule for ICAM receptor or ⁇ 2 integrin, or a binding molecule for ICAM receptor or ⁇ 2 integrin.
  • an antagonist of ICAM receptor or ⁇ 2 integrin e.g., an antibody for ICAM receptor or ⁇ 2 integrin, a fragment or analog of ICAM receptor or ⁇ 2 integrin, a small molecule antagonist of ICAM receptor or ⁇ , integrin, a mimetic of ICAM receptor or ⁇ 2 integrin, an antisense molecule for ICAM receptor or ⁇ 2 integrin, or a binding molecule for ICAM receptor or ⁇ 2 integrin.
  • the agent is an agonist or super agonist of ICAM receptor or ⁇ 2 integrin.
  • the invention also includes a method of making an ICAM receptor or ⁇ 2 integrin polypeptide having an antagonist or agonist activity so as to modulate body weight of an animal.
  • An ICAM receptor or ⁇ 2 integrin polypeptide is provided.
  • the amino acid sequence of the polypeptide is altered.
  • the altered polypeptide is tested for an effect on an aspect of ICAM receptor or ⁇ 2 integrin metabolism, a change in the aspect of ICAM receptor or ⁇ 2 integrin metabolism being indicative of an ICAM receptor or ⁇ 2 integrin polypeptide having an antagonist or agonist activity so as to modulate body weight of an animal.
  • polypeptides can be generated and tested for an effect on an aspect of ICAM receptor or ⁇ 2 integrin metabolism by methods known to those skilled in the art, e.g., as described herein.
  • the altered polypeptide is further tested in an animal to determine if the altered polypeptide modulates the body weight of the animal.
  • the invention also includes a method for increasing the fat content of an animal liver.
  • An animal is provided. Soluble ICAM receptor or soluble ⁇ 2 integrin obtained from the animal is administered into the animal so as to increase the fat content of the liver in the animal.
  • the animal is a goose.
  • the animal is engineered to lack ICAM-1.
  • Such a method can be used in the food industry to produce, e.g., fatty goose livers for pate.
  • the invention also includes a method for increasing the fat content in milk secreted by an animal.
  • An animal capable of secreting milk is provided.
  • An agent capable of altering an aspect of ICAM receptor or ⁇ 2 integrin structure or metabolism is provided.
  • the agent is administered to the animal in an amount so as to increase the fat content in the milk of the animal.
  • the animal is a livestock milk-producing animal.
  • the animal is a non- human transgenic lactating animal.
  • a preferred animal is a goat. Since certain membrane proteins are found on fat mass envelopes in secreted milk, by increasing fat production in milk, the amount of the membrane protein on the secreted fat is increased. Such a method is therefore useful, e.g., for increasing the yield per animal of a desired membrane protein.
  • Example 1 Growth of wild-type and ICAM-1 -/- mice on a normal chow diet (5% fat
  • ICAM-1 -/- mice gained more weight than wild-type mice on a normal chow diet (5% fat), and thus became spontaneously obese.
  • the animals used were ICAM-1 -/- mice on a C57BL/6 background. The mice were maintained on 12-h dark and 12-h light cycles. Water and food were available ad libitum. The mouse food was mouse chow (Prolab 3000; PMI Feeds, Inc. St. Louis, MO) which contained 5.0%) (wt/wt) fat, 5% (wt/wt) fiber and 22% (wt/wt) protein. Growth curves of wild-type and ICAM-1 -/- mice on a normal chow diet were determined. See Fig. 1.
  • ICAM-1 -/- mice On normal chow diet, ICAM-1 -/- mice maintained a body weight comparable to wild- type animals until 16 weeks of age. Thereafter, it was observed that ICAM-1 -/- mice gained more weight than control mice. Compared with other genetically obese mice, the growth rate of ICAM-1 -/- mice was similar to that of Tubby mice, except that the weight gain of Tubby mice occurs earlier (at 9-12 week-old). At 24 weeks of age, the average body weight of ICAM-1 -/- and Tubby mice were similar (46 g in Tubby males vs. 44 g in ICAM-1 -/- males; compared to 36 g in wild-type controls). However, unlike Tubby mice which gradually ate more food than controls, it was found that consumption of chow food by ICAM-1 -/- and control mice were comparable (20-25 g/two weeks, at 16 to 24 weeks of age).
  • Body-mass-index was calculated as body weight (g) divided by the square of body length (anal-nasal length, cm).
  • White fat-pad included the subcutaneous, inguinal, omental and retro-peritoneal fat-pad. Brown fat-pad was taken from the interscapular site.
  • the 5.2 g excess in white fat (8.81 ⁇ 0.47 g in ICAM-1 -/- mice vs. 3.66 ⁇ 0.36 g in wild- type mice, p ⁇ 0.0001) was composed predominantly of subcutaneous fat (54%).
  • the weight of interscapular brown fat-pad was also significantly increased in ICAM-1 -/- mice.
  • Brown adipose tissue is an important site of facultative energy expenditure and functions to prevent obesity. Therefore, the increase of brown adipose tissue in obese ICAM-1 -/- mice may be secondary to increased white adipose tissue weight as a compensatory factor protecting against further white fat deposition, or it may reflect impairment in function of brown fat tissue resulting in cellular enlargement with accumulation of more lipid vacuoles.
  • Example 2 Growth of wild-type and ICAM-1 -/- mice on a Western-type diet (21% fat)
  • ICAM-1 -/- mice rapidly gained more weight than wild-type mice when fed a Western-type diet containing 21% fat, and thus are sensitive to diet-induced obesity.
  • Seven week old ICAM-1 -/- and wild-type control mice were given a Western-type diet containing 21 > fat.
  • the Western-type diet (Harlan Teklad Adjusted Calories Western-Type Diet No. 88137, Madison, WI) contained 21% (wt/wt) fat (42% of calories), 49.2% (wt/wt) carbohydrate and 19.8% (wt/wt) protein. See Fig. 2.
  • ICAM-1 -/- mice are susceptible to diet-induced obesity.
  • Example 3 Growth of wild-type and Mac-1 -/- mice on a Western-type diet (21% fat)
  • Mac-1 -/- mice gained more weight than wild-type mice when fed a Western-type diet containing 21% fat, and thus are sensitive to diet-induced obesity. Growth curves of wild-type and Mac-1 -/- mice on a Western-type diet were determined.
  • Mac-1 -/- mice had a significant increase in white fat (56% subcutaneous fat) and brown fat (see Table 4), while they did not consume more food than the controls (40-45 g/two week periods, shown in Fig. 4).
  • mice on C57BL/6 X 129 mixed background in each group were fed a Western-type diet for 20 weeks starting at 9 weeks of age.
  • White fat-pad included the subcutaneous, inguinal, omental and retroperitoneal fat-pad. Brown fat-pad was taken from the interscapular site.
  • Example 4 Treating an individual having a body weight disorder with the gene for Mac-1 subunit ⁇ M
  • This example illustrates a method for treating an individual having a body weight disorder by delivering the Mac-1 subunit ⁇ M gene to hematopoietic stem cells of the individual.
  • Human Mac-1 subunit ⁇ M cDNA is subcloned into a restriction enzyme site of Moloney murine retrovirus so that the Mac-1 subunit ⁇ M gene is driven by the Moloney murine retrovirus long terminal repeat element. (See Walsh et al., Blood 84:453-459 (1994)).
  • Stem cells are collected from the bone marrow of an individual having a body weight disorder.
  • the bone marrow cells are processed on the Ceprate Stem cell Concentrator (CellPro, Inc., Bothell, WA) according to manufacturer's instructions so as to obtain a CD34-enriched population of stem cells.
  • Transduction of the CD34-enriched stem cells is performed by culturing the CD34- enriched stem cells in fresh retroviral supernatant. The supernatant is removed and the cells are cryopreserved.
  • the stem cells having a high level expression of Mac-1 are injected intravenously into the individual from whom the stem cells were originally collected. (See Dunbar et al., Blood 85:3048-3057 (1995)). This treatment results in alleviation of the body weight disorder.
  • Example 5 Treating an individual having a body weight disorder with the gene for ICAM-1
  • This example illustrates a method for treating an individual having a body weight disorder by delivering the ICAM-1 gene to the liver of the individual.
  • the liver is one of the most important organs involved in lipid metabolism.
  • a high level of expression of ICAM-1 in hepatocytes can enhance degradation of fat in the liver resulting in resistance to obesity.
  • Liver- directed gene therapy has been successfully carried out in the treatment of hypercholesterolaemia. See Grossman et al., Nature Medicine 1 :1148-1154 (1995).
  • An expression vector for ICAM-1 is prepared from a replication defective adenovirus (see Finkel and Epstein, FASEB J. 9:843-851 (1995)) containing an expressible cDNA copy of human ICAM-1 driven by the cytomegalovirus promotor, by cotransfection of a plasmid encoding ICAM-1 into 293 cells. Adenoviruses that are modified so as to produce less antigenicity are preferred. The viral vectors are then purified and titered. The viral vectors are delivered to the liver by portal injection or through insertion of a fine tube to the hepatic artery. Expression of ICAM-1 on the surface of the hepatocytes is verified. A fine needle aspiration of a liver sample is prepared and ICAM-1 expression is identified by a standard immunohistochemic method. This treatment results in alleviation of the body weight disorder.

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Abstract

On décrit un procédé permettant de déterminer si un animal présente un risque de développer un trouble lié au poids corporel. On choisit un animal et on évalue chez cet animal un aspect du récepteur ICAM ou du métabolisme ou de la structure de l'intégrine β2. Un aspect anormal du récepteur ICAM ou bien du métabolisme ou de la structure de l'intégrine β2 déclenche un diagnostic de risque de développer un trouble lié au poids corporel. On décrit également des procédés de détection de la présence d'une maladie affectant le poids corporel, des procédés d'évaluation d'un agent destiné à être utilisé pour moduler le poids corporel, des procédés de traitement d'un trouble lié au poids corporel, des procédés de surveillance d'un traitement thérapeutique d'une maladie affectant le poids corporel et des procédés permettant d'augmenter la teneur en graisse du foie ou du lait d'un animal. Des compositions pharmaceutiques sont également décrites.
EP98902655A 1997-01-24 1998-01-22 Procedes de diagnostic et de traitement de troubles lies au poids corporel chez l'animal Withdrawn EP0977585A4 (fr)

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WO1994011400A1 (fr) * 1992-11-18 1994-05-26 Helsinki University Licensing Ltd. Oy Peptides de icam-2 et icam-1 chez l'homme et leurs analogues s'utilisant en therapie et diagnostic

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