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WO1999053953A2 - Methodes et produits concernant les interactions metaboliques dans les maladies - Google Patents

Methodes et produits concernant les interactions metaboliques dans les maladies Download PDF

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Publication number
WO1999053953A2
WO1999053953A2 PCT/US1999/006874 US9906874W WO9953953A2 WO 1999053953 A2 WO1999053953 A2 WO 1999053953A2 US 9906874 W US9906874 W US 9906874W WO 9953953 A2 WO9953953 A2 WO 9953953A2
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cell
hla
mhc class
cells
agent
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PCT/US1999/006874
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WO1999053953A3 (fr
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Martha K. Newell
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University Of Vermont
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Priority to CA002324995A priority Critical patent/CA2324995A1/fr
Priority to AU33705/99A priority patent/AU768656B2/en
Priority to EP99915109A priority patent/EP1077724A2/fr
Priority to JP2000544356A priority patent/JP2002512203A/ja
Publication of WO1999053953A2 publication Critical patent/WO1999053953A2/fr
Publication of WO1999053953A3 publication Critical patent/WO1999053953A3/fr
Priority to AU2004201144A priority patent/AU2004201144B2/en

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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56977—HLA or MHC typing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/217—IFN-gamma
    • 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
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • 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
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells

Definitions

  • Uncoupling proteins reversibly uncouple oxidative phosphorylation from electron transport and thereby can decrease mitochondrial membrane potential (Harper, M.E. 1997. Obesity research continues to spring leaks. Clinical Investigations in Medicine 20, no. 4:239-244). Elevating glucose concentrations can increase mitochondrial membrane potential (Harper, M.E. 1997. Obesity research continues to spring leaks. Clinical Investigations in Medicine 20, no. 4:239-244). Cell death is a physiologic process that ensures homeostasis is maintained between cell production and cell turnover in self-renewing tissues and is essential to the proper functioning of the immune system.
  • PC 12 cells a cell line derived from rat pheochromocytoma (Greene and Tischler, 1976) have been extensively used as a model for the study of nerve growth factor (NGF)-induced neuronal differentiation and dependency (Mills et al., 1997), and of neuronal cell apoptosis resulting from serum and/or trophic factor withdrawal (Mesner et al., 1995, Fulle et al., 1997), oxidative stress (Vinard et al., 1996) and, the addition of calcium ionophores (Fulle et al, 1997).
  • NGF nerve growth factor
  • EGF Epidermal growth factor
  • the invention involves the finding that mitochondrial metabolism plays an essential role in regulating cellular division and cell death occurring in various diseases. It was found according to the invention that the status of the cellular proton motor force which can be assessed by the coupling relationship between electron transport and oxidative phosphorylation plays an important role in the signal which determines whether a cell will undergo cellular division, cellular differentiation or cellular death. This finding has important implications for treating diseases associated with excessive cellular division, aberrant differentiation, and premature cellular death, e.g., for the treatment of cancers, autoimmune disease, neurodegenerative diseases, etc.
  • the expression of immune recognition molecules on the surface of cells is important in regulating the processes of cell division, differentiation and apoptosis occurring in various diseases. It was discovered, for instance, according to the invention that the expression of immune recognition molecules on the surface of a cell correlates with the ability of the cell to undergo differentiation. For instance, upon removal of NGF from a nerve cell, the surface expression of B7 molecules is down regulated and the nerve cell undergoes apoptosis.
  • the induction kinetics and expression of Fas, B7.1 and B7.2 molecules on the membrane surface of differentiated PC 12 cells and its mutants, and TrkA cells have been examined and are described in the Examples below.
  • the invention is a method for decreasing mitochondrial membrane potential in a mammalian cell.
  • the method involves the step of administering an MHC class II HLA-DR ligand to the mammalian cell to selectively engage MHC class II HLA-DR on the surface of the cell in an amount effective to decrease mitochondrial membrane potential in the mammalian cell, wherein the mammalian cell is not an antigen presenting cell.
  • MHC class II HLA-DR is expressed on the surface of the mammalian cell.
  • the method involves the step of contacting the mammalian cell with an amount of an MHC class II HLA-DR inducing agent effective to induce the expression of MHC class II HLA-DR on the surface of the mammalian cell.
  • the methods of the invention are useful for inducing cell division, cell lysis, cell differentiation and cell apoptosis, depending on the metabolic condition of the cell.
  • the invention is a method for inducing lysis of a mammalian cell.
  • the method includes the steps of contacting the mammalian cell with an amount of an MHC class II HLA-DR inducing agent effective to induce the expression of MHC class II HLA-DR on the surface of the mammalian cell, and contacting the MHC class II HLA-DR on the surface of the mammalian cell with an amount of an MHC class II HLA-DR ligand effective for causing lysis of the mammalian cell.
  • the MHC class II HLA-DR inducing agent does not include adriamycin and gamma interferon.
  • the invention is a method for inducing cell lysis in a tumor cell. The method involves the steps of contacting a tumor cell with an amount of an MHC class II HLA-DR inducing agent effective to induce the expression of MHC class II HLA-DR on the surface of the tumor cell, and contacting the MHC class II HLA-DR on the surface of the tumor cell with an amount of an MHC class II HLA-DR ligand effective for causing cell lysis of the tumor cell.
  • the MHC class II HLA-DR inducing agent is any agent which induces expression of
  • the MHC class II HLA-DR ligand is selected from the group consisting of an anti- MHC class II HLA-DR antibody, CD4 molecules, ⁇ T cell receptor molecules, ⁇ T cell receptor molecules and a MHC class II HLA-DR binding peptide.
  • the MHC class II HLA-DR inducing agent and the MHC class II HLA-DR ligand are administered simultaneously. In another embodiment the MHC class II HLA-DR inducing agent and the MHC class II HLA-DR ligand are administered orally. In yet another embodiment the MHC class II HLA-DR inducing agent and the MHC class II HLA-DR ligand are administered locally.
  • the inducing agent is selected from the group consisting of adriamycin, gamma interferon, bacterial byproducts such as lipopolysaccharides, mycobacterial antigens such as BCG, a UCP expression vector, a TCR ⁇ engagement molecule and a fatty acid.
  • HLA-DR ligand are administered orally.
  • MHC class II HLA-DR inducing agent and the MHC class II HLA-DR ligand are administered locally.
  • a method for inducing apoptosis in a tumor cell involves the steps of contacting a tumor cell with an amount of a metabolic modifying agent, which when exposed to a cell causes coupling of electron transport and oxidative phosphorylation, effective to increase the mitochondrial membrane potential in the tumor cell, and contacting the tumor cell with an amount of an apoptotic chemotherapeutic agent effective for inducing apoptosis in the tumor cell.
  • the metabolic modifying agent is added to the tumor cell to induce coupling of electron transport and oxidative phosphorylation.
  • the metabolic modifying agent is selected from the group consisting of glucose, phorbol myristate acetate in combination with ionomycin, MHC class II HLA-DP/DQ ligand, GDP, CD40 binding peptide, UCP antisense, dominant negative UCP, sodium acetate, and staurosporine.
  • Fas expression is induced on the cell surface and a apoptotic chemotherapeutic agent can be added to induce apoptosis of the tumor cell.
  • the apoptotic chemotherapeutic agent is selected from the group consisting of adriamycin, cytarabine, doxorubicin, and methotrexate.
  • the metabolic modifying agent and the apoptotic chemotherapeutic agent are administered simultaneously. In another embodiment the metabolic modifying agent and the apoptotic chemotherapeutic agent are administered orally. In yet another embodiment the metabolic modifying agent and the apoptotic chemotherapeutic agent are administered locally.
  • the tumor cell is sensitive to the apoptotic chemotherapeutic agent, and wherein the amount of metabolic modifying agent is effective to increase mitochondrial membrane potential and the amount of apoptotic chemotherapeutic agent is effective to inhibit the proliferation of the tumor cell when the mitochondrial membrane potential is increased.
  • a method for decreasing mitochondrial membrane potential in a cell of a subject includes the step of administering an MHC class II HLA-DR ligand to the subject to selectively engage MHC class II HLA-DR on the surface of the cell in an amount effective to decrease mitochondrial membrane potential in the mammalian cell.
  • the method is performed in vivo.
  • the method is performed ex vivo.
  • mammalian cells include but are not limited to antigen presenting cells, T cells, and tumor cells.
  • the mammalian cell is a pancreatic ⁇ cell of a type I diabetic and wherein the MHC class II HLA-DR ligand is administered to the pancreatic ⁇ cell in vivo.
  • the mammalian cell is a tumor cell and wherein the MHC class II HLA-DR ligand is administered to the tumor cell in vivo.
  • the invention in another aspect is a method for treating a subject having a tumor sensitive to treatment with a combination of an apoptotic chemotherapeutic agent and a metabolic modifying agent.
  • the method includes the steps of administering to a subject in need of such treatment an apoptotic chemotherapeutic agent and a metabolic modifying agent in a combined amount effective to inhibit growth of the tumor, said combined amount being an amount of apoptotic chemotherapeutic agent and an amount of metabolic modifying agent, wherein the amount of metabolic modifying agent is effective to increase mitochondrial membrane potential and the amount of apoptotic chemotherapeutic agent is effective to inhibit the proliferation of the tumor cell when the mitochondrial membrane potential is increased.
  • the invention in another aspect is a method for inhibiting pancreatic ⁇ cell death in a Type I diabetic.
  • the progression of pancreatic ⁇ cell death in type I diabetes involves two steps.
  • the first phase of type I diabetes is the insulitis phase which results when membrane potential is increased, the ⁇ cells become cell surface Fas positive, but Fas-death insensitive, .
  • the first phase it is desirable to decrease the membrane potential and cause the cells to use fatty acids for fuel and become cell surface Fas negative.
  • the diabetes is not treated during the first phase then it progresses to a second phase.
  • the membrane potential is decreased and the ⁇ cell is induced to die if it remains cell surface Fas positive.
  • the invention contemplates a two phase approach to the treatment of type I diabetes.
  • the method involves the step of contacting a pancreatic ⁇ cell of a Type I diabetic with an amount of a Fas binding agent effective to inhibit selective engagement of Fas on the surface of the pancreatic ⁇ cell.
  • a method for inducing pancreatic ⁇ cell death in a Type II diabetic is provided.
  • the method includes the steps of contacting a pancreatic ⁇ cell of a Type II diabetic with an amount of an MHC class II HLA-DR inducing agent effective to induce the expression of the MHC class II HLA-DR on the surface of the pancreatic ⁇ cell, and selectively engaging the MHC class II HLA-DR by contacting the cell with an MHC class II HLA-DR ligand effective to induce pancreatic ⁇ cell death.
  • the MHC class II HLA-DR inducing agent is selected from the group consisting of adriamycin, gamma interferon, bacterial byproducts such as lipopolysaccharides, mycobacterial antigens such as BCG, a UCP expression vector, a TCR ⁇ engagement molecule and a fatty acid in one embodiment.
  • the method includes the step of contacting the a Fas ligand bearing tumor cell with acetate in an amount effective to induce Fas associated cell death.
  • the a Fas ligand bearing tumor cell is contacted with the acetate in an amount effective to sensitize the cell to a chemotherapeutic agent and further comprising the step of contacting the cell with a chemotherapeutic agent.
  • a preferred chemotherapeutic agent is methotrexate.
  • the method may also involve the step of administering a Fas ligand to the a Fas ligand bearing tumor cell.
  • the Fas ligand bearing tumor cell is selected from the group consisting of a melanoma cell and a colon carcinoma cell.
  • the assay includes at least the following steps: isolating a tumor cell from a subject; exposing the tumor cell to a chemotherapeutic agent; and, detecting the presence of a cell death marker selected from the group consisting of a Fas molecule on the surface of the tumor cell, a B7 molecule on the surface of the tumor cell, an MHC class II HLA-DR on the surface of the tumor cell, and a mitochondrial membrane potential indicative of cellular coupling wherein the presence of the cell death marker indicates that the cell is susceptible to treatment with a chemotherapeutic agent.
  • a cell death marker selected from the group consisting of a Fas molecule on the surface of the tumor cell, a B7 molecule on the surface of the tumor cell, an MHC class II HLA-DR on the surface of the tumor cell, and a mitochondrial membrane potential indicative of cellular coupling wherein the presence of the cell death marker indicates that the cell is susceptible to treatment with a chemotherapeutic agent.
  • Yet another assay of the invention is a method for screening a subject for susceptibility to disease.
  • This method involves the steps of isolating a cell selected from the group consisting of peripheral blood lymphocyte and skin from a subject; and, detecting the presence of an MHC marker selected from the group consisting of an MHC class II HLA-DP/DQ, B7-2, B7-1 and MHC class II HLA-DR on the surface of the cell, wherein the presence of MHC class II HLA- DP/DQ is indicative of susceptibility to atherosclerosis and resistance to autoimmune disease and the presence of MHC class II HLA-DR, B7-2, or B7-1 is indicative of resistance to atherosclerosis and susceptibility to autoimmune disease.
  • the MHC class II HLA-DR ligand is selected from the group consisting of an anti-MHC class II HLA-DR antibody, CD4 molecules, ⁇ T cell receptor molecules, ⁇ T cell receptor molecules and a MHC class II HLA-DR binding peptide.
  • the MHC class II HLA-DR inducing agent and the MHC class II HLA-DR ligand are present in an amount effective to lyse a tumor cell.
  • the composition may be formulated in a pharmaceutically acceptable carrier.
  • the method includes the steps of contacting a nerve cell with an amount of a B7 inducing agent effective to induce the expression of B7 on the surface of the nerve cell, and exposing the nerve cell to a neural activating cell to cause differentiation of the nerve cell.
  • the invention is a method for inducing nerve cell differentiation. The method involves the step of contacting a nerve cell with an amount of a B7 inducing agent effective to induce the expression of B7 on the surface of the nerve cell in the presence of an endogenous neural activating cell.
  • the method also includes the step of contacting the nerve cell with an amount of a metabolic modifying agent, which when exposed to a cell causes increased coupling of electron transport and oxidative phosphorylation, effective to prevent dissipation of proton motor force in the nerve cell prior to contacting the nerve cell with the B7 inducing agent.
  • the metabolic modifying agent is glucose, phorbol myristate acetate in combination with ionomycin, MHC class II HLA-DP/DQ ligand, GDP, CD40 binding peptide, sodium acetate, UCP antisense, dominant negative UCP,, and staurosporine.
  • the neural activating cell is a T cell, a macrophage, or a dendritic cell.
  • the invention is a method for inducing apoptosis in a nerve cell.
  • the method includes the steps of contacting a nerve cell with an amount of a metabolic modifying agent, which when exposed to a nerve cell causes an increase in coupling of electron transport and oxidative phosphorylation, effective to prevent dissipation of proton motor force in the nerve cell, and contacting a neural activating cell with an amount of a B7 receptor blocking agent effective for inducing apoptosis in the nerve cell.
  • the metabolic modifying agent in various embodiments, is glucose, phorbol myristate acetate in combination with ionomycin, MHC class II HLA-DP/DQ ligand, GDP, CD40 binding peptide, sodium acetate, UCP antisense, dominant negative UCP pen or staurosporine.
  • the B7 receptor blocking agent is an anti-CD28 antibody, CD28 binding peptide, CTLA4 analog, anti-CTLA4 antibody, or CTLA4 binding peptide.
  • the B7 expressing nerve cell constitutively expresses B7.
  • the B7 expressing nerve cell is a nerve cell which constitutively expresses a UCP gene.
  • the B7 expressing nerve cell is a nerve cell which constitutively expresses a B7 gene.
  • the method in another embodiment includes the step of administering a B7 inducing agent effective to induce endogenous B7 expression on the surface of the nerve cell.
  • the injured tissue may be any tissue in which a nerve is damaged. In one embodiment the injured tissue is a spinal chord. In another embodiment the injured tissue is a severed limb.
  • a method for selectively killing a tumor cell includes the step of contacting the tumor cell with a compound selected from the group consisting of acetate, GDP and an apoptopic chemotherapeutic agent in an amount effective to kill the tumor cell.
  • B7 (B7.1) cDNA (SEQ ID NO:l).
  • SEQ ID NO: 5 is the nucleotide sequence of the human uncoupling (UCP-1) cDNA with GenBank Ace. no.U28480.
  • SEQ ID NO: 6 is the predicted amino acid sequence of the translation product of human uncoupling cDNA (UCP-1) (SEQ ID NO:5).
  • SEQ ID NO: 7 is the nucleotide sequence of the human uncoupling (UCP-2) cDNA with GenBank Ace. no.U82819.
  • SEQ ID NO: 8 is the predicted amino acid sequence of the translation product of human uncoupling cDNA (UCP-2) (SEQ ID NO:7).
  • SEQ ID NO: 9 is the nucleotide sequence of the human uncoupling (UCP-3S) cDNA with GenBank Ace. no.U82818.
  • SEQ ID NO: 10 is the predicted amino acid sequence of the translation product of human uncoupling cDNA (UCP-3S) (SEQ ID NO:9).
  • the invention relates to methods and products involving the control of cell division, differentiation, death, and apoptosis by the regulation of cell surface immune recognition molecules. It was discovered according to one aspect of the invention that proton motor force (assessed as mitochondrial metabolism) is integrally related to the regulation of cellular division and cellular apoptosis.
  • proton motor force asserts as mitochondrial metabolism
  • the ability to manipulate mitochondrial metabolic processes has led to the development of methods for treating diseases associated with excessive cellular proliferation or premature cellular death.
  • the ability to manipulate the expression of cell surface immune recognition molecules such that a nerve cell can stimulate local NGF production from an NGF producing cell has led to the development of methods for treating neurodegenerative diseases associated with premature cellular death.
  • the regulation of proton motor force is directly related to the expression of these cell surface immune recognition molecules involved in the signaling process of cell death and in immune response signaling, and thus can be manipulated as one method for regulating the expression of the immune recognition molecules.
  • the ability to control the expression of these cell surface molecules is a useful and powerful technique for therapeutically manipulating the processes of cellular death, apoptosis, differentiation and proliferation. Monitoring the expression of these proteins is also useful for screening assays to assess disease states as well as the mitochondrial metabolic status of cells.
  • the cell When a cell has been induced to express MHC class II HLA-DPDQ on the cell surface such that the electron transport is relatively coupled and the cell is using glucose for fuel and the cell is contacted with a MHC class II HLA-DPDQ ligand, then the cell generally will interpret that signal as a cell division signal, and cause cellular division.
  • the methods of the invention have broad utility in regulating mammalian cell growth and death in vitro, in vivo and ex vivo. Because mammalian cells utilize the basic process of mitochondrial metabolism in regulating their own growth and differentiation, any type of mammalian cell can be manipulated according to the methods of the invention.
  • the methods for increasing or decreasing mitochondrial metabolism are performed in vitro by contacting an MHC class II HLA-DPDQ or -DR expressing cell with an MHC class II HLA- DPDQ or -D ligand, respectively, the methods are not performed on antigen presenting cells. When the same methods are performed ex vivo or in vivo they may however, be performed on antigen presenting cells as well as any other type of mammalian cell.
  • an "antigen presenting cell” is used herein consistently with its well known meaning in the art and includes, for instance, dendritic cells, macrophage, etc.
  • the in vitro methods of the invention are useful for a variety of purposes.
  • the methods of the invention may be useful for identifying drugs which have an effect, such as a preventative effect, on cellular division or death by contacting cells which are caused by the manipulations of the invention to undergo cellular division or death.
  • the manipulated cell When returned to the subject, the manipulated cell will be programed for cell death or division, depending on the treatment to which it was exposed.
  • Ex vivo manipulation of cells has been described in several references in the art, including Engleman, E.G., 1997, Cytotechnology, 25:1 ; Van Schooten, W., et al., 1997, Molecular Medicine Today, June, 255; Steinman, R.M., 1996, Experimental Hematology, 24, 849; and Gluckman, J.C., 1997, Cytokines, Cellular and Molecular Therapy, 3:187.
  • the ex vivo activation of cells of the invention may be performed by routine ex vivo manipulation steps known in the art. In vivo methods are also well known in the art.
  • a subject as used herein means humans, primates, horses, cows, pigs, sheep, goats, dogs, cats and rodents.
  • the invention thus is useful for therapeutic purposes and also is useful for research purposes such as testing in animal or in vitro models of medical, physiological or metabolic pathways or conditions.
  • the mammalian cell is a tumor cell
  • the method is useful for inducing cell lysis in many types of mammalian cells but is particularly useful for inducing cell lysis in a tumor cell.
  • a "tumor cell” as used herein is a cell which is undergoing unwanted mitotic proliferation.
  • a tumor cell when used in the in vitro aspects of the invention can be isolated from a tumor within a subject or may be part of an established cell line.
  • a tumor cell in a subject may be part of any type of cancer.
  • Cancers include but are not limited to biliary tract cancer; brain cancer, including glioblastomas and medulloblastomas; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms, including acute lymphocytic and myelogenous leukemia; multiple myeloma; AIDS associated leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms, including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas, including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer, including squamous cell carcinoma; ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreas cancer; prostate cancer; rectal cancer; sarcomas, including leiomyosar
  • the mammalian cell is a tumor cell and the cell is only treated with an MHC class II HLA-DR inducing agent but not an MHC class II HLA-DR ligand the MHC class II HLA-DR inducing agent does not include adriamycin and gamma interferon.
  • the MHC class II HLA-DR inducing agent is adriamycin or gamma interferon
  • the method of lysing the tumor cell requires the additional step of contacting the tumor cell with an MHC class II HLA-DR ligand to cause cell lysis.
  • Cell lysis is the necrotic death of a cell which occurs by osmotic rupture.
  • MHC class II HLA-DR inducing agent is an isolated molecule.
  • An isolated molecule is one which has been removed from its natural surroundings and formulated for administration to an organism.
  • Adriamycin, gamma interferon, bacterial byproducts such as lipopolysaccharides, mycobacterial antigens such as BCG are all well known compounds which can be purchased from a variety of commercial sources.
  • UCP expression vector can be prepared by methods well known in the art, such methods are described in detail below.
  • Fatty acids are also well known compounds that can be purchased commercially from many sources.
  • Preferred fatty acids include but are not limited to oleic acid, palmitate, and myristic acid.
  • a "TCR ⁇ engagement molecule" as used herein refers to any compound that can bind to and cause cell surface crosslinking of CD4 and the ⁇ T cell receptor ( ⁇ TCR).
  • ⁇ TCR ⁇ T cell receptor
  • Such compounds are known in the art.
  • heterobifunctional antibodies are capable of crosslinking CD4 and ⁇ TCR by interacting with both molecules on the surface of the cell.
  • Other CD4/ ⁇ TCR binding molecules can be identified with routine experimentation and are also encompassed by the term TCR ⁇ engagement molecule. Routine screening methods for identifying such binding molecules are set forth below.
  • MHC class II HLA-DR refers to a subregion of the human major histocompatibility class II genetic locus.
  • MHC class II HLA-DR is the protein expressed on the surface of a cell which corresponds to the MHC class II HLA-DR genetic locus.
  • HLA-DR refers to the human subclass of MHC, the invention is intended to encompass the corresponding subclass of MHC in other species, which have different nomenclature, such as the IE region in the corresponding subclass in the mouse.
  • MHC class II HLA-DR binding peptides can be identified with routine experimentation and are also encompassed by the term MHC class II HLA-DR ligand. Routine screening methods for identifying such binding molecules are set forth below.
  • the methods of the invention in some aspects may also be performed using endogenous MHC class II HLA-DR ligand.
  • An "endogenous MHC class II HLA-DR ligand" is different than an "MHC class II HLA-DR ligand" used above which is an isolated composition.
  • the endogenous MHC class II HLA-DR ligand may be a cell having a cell surface MHC class II HLA-DR binding peptide.
  • the method would only include the step of contacting a tumor cell with an amount of an MHC class II HLA-DR inducing agent effective to induce the expression of MHC class II HLA-DR on the surface of the tumor cell in the presence of an endogenous MHC class II HLA-DR ligand.
  • the endogenous MHC class II HLA-DR ligand is a cell having a cell surface MHC class II HLA-DR binding peptide which is already present in interactive proximity to the MHC class II HLA-DR, the cell does not have to be manually brought into contact with the MHC class II HLA-DR.
  • Another aspect of the invention involves the induction of apoptosis in a tumor cell rather than cell lysis. In both apoptosis and cell lysis the cell dies but the processes occur through different mechanisms and when the cell is in a different metabolic state. As described above, when the methods of the invention are performed to induce cell lysis in a tumor cell the cell is in an uncoupled state.
  • Metabolic modifying agent is an agent which when exposed to a cell causes coupling of electron transport and oxidative phosphorylation, resulting in increased mitochondrial membrane potential within the cell.
  • Metabolic modifying agents include but are not limited to glucose, sodium acetate, phorbol myristate acetate in combination with ionomycin, MHC class II HLA-DP/DQ ligand, guanosine diphosphate (GDP), CD40 binding peptide, sodium acetate, UCP antisense, dominant negative UCP,, and staurosporine.
  • CD40 binding peptides are any peptide molecules which interact with CD40, causing CD40 crosslinking on a cell surface. These molecules include, for example, CD40 ligand, which is a well known molecule. CD40 binding peptides are not limited to CD40 ligand, however, but include other molecules which can be identified with routine experimentation. Routine screening methods for identifying such binding molecules are set forth below. UCP antisense molecules and dominant negative UCP molecules are also known in the art and are described in more detail below.
  • MHC class II HLA-DP/DQ refers to another subregion of the human major histocompatibility class II genetic locus.
  • MHC class II HLA-DP/DQ is the protein expressed on the surface of a cell which corresponds to the MHC class II HLA-DP/DQ genetic locus.
  • HLA-DP/DQ refers to the human subclass of MHC, the invention is intended to encompass the corresponding subclass of MHC in other species, which have different nomenclature, such as the IA region in the subclass in the mouse.
  • the mitochondrial membrane potential is maintained by the energy generating system of the mitochondria.
  • electron transport is coupled to oxidative phosphorylation resulting in the production of ATP from glucose.
  • Uncoupling proteins UCPs
  • Other tissue often referred to as the immuno-privileged tissue such as the brain, testis, ovary, eye, and pancreatic ⁇ cells, express UCPs which cause electron transport to be uncoupled to oxidative phosphorylation under normal conditions.
  • glucose cannot be converted to ATP while the UCP is active because of the uncoupling and the energy produced is converted into other energy forms such as heat and released. If the metabolic processing systems in these tissues are caused to undergo coupling the membrane potential would increase.
  • a "decrease in mitochondrial membrane potential” is a decrease relative to the normal status of the cell being examined and results from the dissipation of proton motor force. If electron transport and oxidative phosphorylation are normally coupled within the cell then the baseline potential will be relatively high and when the ATP generating systems are uncoupled a decrease in mitochondrial membrane potential from that baseline level is observed.
  • Fas is expressed on the cell surface and when a cell is uncoupled Fas generally is transported to intracellular stores.
  • Fas When a cell is coupled and Fas is on the surface engagement of Fas sends a signal to the cell instructing the cell to undergo cellular division.
  • a chemotherapeutic agent is added then the signal is changed to a signal which instructs the cell to undergo apoptosis.
  • a chemotherapeutic agent When a cell is uncoupled and ordinarily Fas is not expressed on the cell surface. Under certain disease conditions such as Type I diabetes (discussed in more detail below), or when the cell has been irradiated Fas can be expressed on the surface of uncoupled cells. When this occurs engagement of Fas sends a signal to the cell to die.
  • An "apoptotic chemotherapeutic agent" as used herein is a group of molecules which function by a variety of mechanisms to induce apoptosis in rapidly dividing cells.
  • methotrexate methotrexate
  • pyrimidine analogs e.g. 5-fluorouracil floxuridine, cytarabine, and azauridine and its prodrug form azaribine
  • purine analogs and related materials e.g. 6- mercaptopurine, 6-thioguanine, pentostatin
  • Natural Products such as the vinca alkaloids (e.g. vinblastine, Vincristine), epipodophylotoxins (e.g.
  • hydroxyurea methylhydiazine derivatives (e.g. procarbazine), adreocortical suppressants (e.g. mitotane, aminoglutethimide) taxol;
  • Hormones and Antagonists such as adrenocorticosteroids (e.g. prednisone or the like), progestins (e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate), estrogens (e.g. diethyestilbestrol, ethinyl estradiol, and the like), antiestrogens (e.g. tamoxifen), androgens (e.g.
  • adrenocorticosteroids e.g. prednisone or the like
  • progestins e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol
  • testosterone propionate, fluoxymesterone, and the like testosterone propionate, fluoxymesterone, and the like
  • antiandrogens e.g. flutamide
  • gonadotropin-releasing hormone analogs e.g. leuprolide
  • DNA damaging compounds such as adriamycin.
  • the combined amount of metabolic modifying agent and apoptotic chemotherapeutic agent effective to inhibit growth of the tumor cell is that amount is effective to inhibit the proliferation of the tumor cell when the mitochondrial membrane potential is increased.
  • An effective amount means that amount necessary to delay the onset of, inhibit the progression of, halt altogether the onset or progression of or diagnose the particular condition being treated.
  • an effective amount for treating a tumor cell is that amount necessary to halt the proliferation of the cell.
  • the effective amount is that amount necessary to kill the cell.
  • an effective amount for treating cancer will be that amount necessary to favorably affect mammalian cancer cell proliferation in-situ.
  • a maximum dose be used, that is, the highest safe dose according to sound medical judgment.
  • the screening assay may indicate that the tumor is mostly resistant to a chemotherapeutic agent.
  • Resistant tumors may also be treated by the methods of the invention.
  • One aspect of the invention involves the discovery that resistant tumors cells have a mitochondrial metabolic state in which electron transport is uncoupled from oxidative phosphorylation. It was discovered according to the invention that by altering the metabolic state of the tumor cell and thereby causing electron transport to be coupled to oxidative phosphorylation it is possible to cause the resistant cell to revert such that it becomes sensitive to chemotherapy.
  • the method is performed by administering to the subject an amount of a chemotherapeutic agent, and substantially simultaneously therewith an amount of a metabolic modifying agent which together are effective for inhibiting growth of the tumor.
  • the metabolic modifying agent causes electron transport in the cell to be coupled to oxidative phosphorylation. As discussed above once these processes are coupled Fas is expressed on the surface and the cell becomes susceptible to apoptosis induced by the chemotherapeutic agent.
  • the assay can be performed using routine equipment known in the art. For instance the change in the presence of the cell death marker can be detected by contacting the tumor cell with a cell death ligand attached to a solid support.
  • the invention also encompasses kits for screening a subject for susceptibility to disease.
  • This kit includes at least a container housing a first binding compound that selectively binds to a protein selected from the group consisting of B7-2, B7-1 and MHC class II HLA-DR; a container housing a second binding compound that selectively binds to a MHC class II HLA- DP/DQ protein; and instructions for determining whether an isolated cell of a subject selectively interacts with the first or second binding compound, wherein the presence of MHC class II HLA- DP/DQ on the cell surface which interacts with the second compound is indicative of susceptibility to atherosclerosis and resistance to autoimmune disease and the presence of MHC class II HLA-DR on the cell surface which interacts with the first compound is indicative of resistance to atherosclerosis and susceptibility to autoimmune disease.
  • kits for screening a tumor cell of a subject for susceptibility to treatment with a chemotherapeutic agent include a container housing a cell death marker detection reagent; and instructions for using the cell death marker detection reagent for detecting the presence of a cell death marker selected from the group consisting of a Fas molecule on the surface of the tumor cell, an MHC class II HLA-DR on the surface of the tumor cell, and a mitochondrial membrane potential indicative of cellular coupling wherein the presence of the cell death marker indicates that the cell is susceptible to treatment with a chemotherapeutic agent.
  • the kit may also include a container housing a chemotherapeutic agent.
  • the kit may include a panel of chemotherapeutic agents, housed in separate compartments.
  • the invention also involves the discovery that mitochondrial metabolic regulation is directly related to the expression of immune recognition molecules on a cell surface.
  • immune recognition molecules are cell surface proteins which mark a cell for identification by immune cells. Immune recognition molecules include but are not limited to MHC, and in particular MHC class II HLA-DR, B7-1, B7-2 and CD-40.
  • MHC mitochondrial metabolic status of the cell
  • MHC class II HLA-DP/DQ is actually increased.
  • MHC class II HLA-DP/DQ is not defined as an immune recognition molecule.
  • the invention encompasses a method for inducing the expression of immune recognition molecules on a cell surface.
  • the method involves contacting the cell with an amount of a metabolic inhibition agent effective to decrease mitochondrial membrane potential, wherein a decrease in mitochondrial membrane potential causes induction of the expression of immune recognition molecules on the cell surface.
  • a “metabolic inhibition agent” as used herein is an agent that causes electron transport to become uncoupled from oxidative phosphorylation, and includes for example apoptotic chemotherapeutic agents, bacterial byproducts, mycobacterial antigens, UCP expression vectors, and fatty acids.
  • Diabetes mellitus which encompasses both Type I (i.e., Insulin Dependent Diabetes Mellitus (IDDM)) and Type II (i.e., Non-Insulin Dependent Diabetes Mellitus (NIDDM)), is known to affect more than one hundred million individuals worldwide. Although the exact cause of diabetes is unclear it is believed that diabetes may arise from any of a variety of physiological conditions such as genetic syndromes, viral infections, age related deterioration of structures responsible for maintaining the glycemic response, pancreatic disease, hormonal abnormalities, certain drugs or chemicals, insulin receptor abnormalities, etc.
  • a "type I diabetic” is a subject who has diabetes mellitus caused by a destruction of beta cells in the pancreas. Type I diabetics require daily insulin administration which may be reduced but not altogether eliminated by careful restriction of diet.
  • Fas can induce mitosis or apoptosis depending on the cell and the experimental circumstances.
  • a ⁇ cell compensatory hypersecretion of insulin occurs and this process is accompanied by cell surface expression of the molecule Fas.
  • NOD mice an animal model for Type 1 diabetes, are crossed with mice having the Ipr mutation (Fas deficient), the animals are resistant to disease.
  • destruction of ⁇ cells in the NOD accelerates when Fas Ligand is placed on the insulin promotor.
  • pancreatic ⁇ cells become coupled and express Fas on the cell surface.
  • the disease then progresses to the stage when pancreatic ⁇ cell begin to be killed.
  • the metabolic state changes again to uncoupled and Fas is still expressed on the surface.
  • the cell is killed as soon as Fas is engaged without the need for any other agents.
  • the methods of the invention include a method for inhibiting pancreatic ⁇ cell death in a Type I diabetic by altering the mitochondrial metabolic state.
  • Another method for inhibiting the death of a pancreatic ⁇ cell in a Type I diabetic can be accomplished by contacting a pancreatic ⁇ cell of a Type I diabetic with an amount of a Fas binding agent effective to inhibit selective engagement of Fas on the surface of the pancreatic ⁇ cell.
  • a Fas binding agent effective to inhibit selective engagement of Fas on the surface of the pancreatic ⁇ cell.
  • Fas binding agents then can easily be tested without undue experimentation in vitro for their ability to bind Fas but not induce cell death in uncoupled cells.
  • Uncoupled cells can be prepared according to the methods described above. Fas can be induced to be expressed on the surface of cells using irradiation as has previously been identified in the prior art. Once uncoupled cells expressing Fas have been developed potential Fas binding agents can be incubated with these cells and cell lysis can be assayed by the methods described herein or by other methods known in the art.
  • the invention is also useful for treating type II diabetics.
  • a "type II diabetic" is a subject who has diabetes mellitus caused by abnormal insulin secretion and/or resistance to insulin action in target tissues. The physiological problem which occurs in a Type II diabetic is very different than that which occurs in a type I diabetic. In type II diabetes the pancreatic ⁇ cells undergo excessive proliferation. It is desirable to inhibit proliferation of these cells.
  • mitochondrial metabolism and the related expression of MHC class II on the surface of a cell is indicative of the susceptibility of the host of that cell to developing atherosclerosis, autoimmune disease or multiple sclerosis.
  • MHC class II HLA-DP/DQ When electron transport and oxidative phosphorylation are in a coupled state in a cell the cell expresses MHC class II HLA-DP/DQ on the surface.
  • electron transport and oxidative phosphorylation are in an uncoupled state in a cell the cell expresses MHC class II HLA-DR on the surface.
  • a cell in a coupled state that has MHC class II HLA-DP/DQ on the surface will be stimulated to divide when the MHC class II HLA-DP/DQ is engaged.
  • a cell in an uncoupled state that has MHC class II HLA-DR on the surface will be stimulated to lyse when the MHC class II HLA-DR is engaged.
  • Atherosclerosis is a group of diseases affecting the cardiovascular system and includes myocardial infarction, stroke, angina pectoris and peripheral cardiovascular disease. Despite significant advices in therapy, cardiovascular disease remains the single most common cause of morbidity and mortality in the developed world. Many individuals are susceptible to developing future cardiovascular disorders, and this susceptibility has usually been defined in terms of risk factors such as family history of premature ischemic heart disease, hyperlipidemia, cigarette smoking, hypertension, low HDL cholesterol, diabetes mellitus, hyperinsulinemia, abdominal obesity, and high lipoprotein.
  • the invention includes a new method for determining an individuals susceptibility to developing atherosclerosis. As used herein susceptibility to atherosclerosis indicates a likelihood of 10% greater than the average of developing atherosclerosis.
  • Autoimmune disease is a class of diseases in which an individuals own antibodies react with host tissue or in which immune effector T cells are autoreactive to endogenous self peptides and cause destruction of tissue. It is well established that MHC class II alleles act as major genetic elements in susceptibility to a variety of autoimmune diseases. These include rheumatoid arthritis, celiac disease, pemphigus vulgaris, and the prototype for autoimmune disease, systemic lupus erythematosus (SLE). The invention includes a new method for determining an individuals susceptibility to developing autoimmune disease. As used herein susceptibility to Autoimmune disease indicates a likelihood of 10% greater than the average of developing autoimmune disease.
  • damaging agents such as diabetogenic viruses, inflammation, ischemia, age, or oxidative stress, may damage mitochondrial metabolism, increase cell surface Fas expression, and render the cells susceptible to Fas-induced apoptosis or oncosis, respectively.
  • apoptosis (on the right of the panel), which is thought to occur "silently” without additional inflammation, occurs to some of the ⁇ cells and that oncosis occurs in later stages of disease resulting from T cell mediated (FasL dependent) ⁇ cell destruction.
  • the invention in other aspects relates to methods for selectively killing a Fas ligand bearing tumor cell.
  • the method involves the steps of contacting the Fas ligand bearing tumor cell with acetate in an amount effective to induce Fas associated cell death.
  • a Fas ligand bearing tumor cell is any tumor cell which inducibly or constitutively expressed a Fas ligand on the cell surface. Such cells can easily be identified by those of skill in the art since the Fas ligand is a well known molecule. These cells include but are not limited to melanoma cells and colon carcinoma cells.
  • acetate alone is sufficient to kill a Fas ligand bearing tumor cell
  • the cell can also be treated with a chemotherapeutic agent and/or a Fas ligand to promotes killing.
  • a chemotherapeutic agent and/or a Fas ligand to promotes killing.
  • the use of these secondary compounds allows the use of less of the acetate to be used to accomplish the cell killing.
  • the combination of acetate and chemotherapeutic agents and or Fas ligands allows less of all three reagents to be used than would otherwise be required to kill the cell.
  • Other methods for selectively killing a cell include contacting the cell with a nucleic acid selected form the group consisting of a UCP anti-sense nucleic acid and a UCP dominant- negative nucleic acid in an amount effect to inhibit UCP function.
  • a cell can also be killed according to the invention by contacting the cell with a compound selected from the group consisting of acetate and GDP and an apoptopic chemotherapeutic agent in an amount effective to kill the cell.
  • the invention also encompasses methods for promoting a Thl immune response. The method is performed by administering to a subject who has been exposed to an antigen an effective amount for inducing a Thl immune response of a MHC class II HLA-DR inducing agent to induce DR on a T cell.
  • the CTLA-4/CD28/B7 system is a group of proteins involved in regulating T-cell proliferation through this secondary signaling pathway.
  • the T-cell proliferative response is controlled by the interaction of the B7 family of proteins, which are expressed on the surface of APCs, with CTLA-4 (cytotoxic T lymphocyte antigen #4) and CD28.
  • CTLA-4 cytotoxic T lymphocyte antigen #4
  • CD28 CD28.
  • the B7 family of proteins is composed of structurally related glycoproteins including B7-
  • B7-2, and B7-3 (Galea-Lauri et al., Cancer Gene Therapy, v. 3, p. 202-213 (1996); Boussiotis, et al., Proc. Nat. Acad. Sci. USA, v. 90, p.l 1059-11063 (1993)).
  • the different B7 proteins appear to have different expression patterns on the surface of antigen presenting cells.
  • B7-2 is constitutively expressed on the surface of monocytes, whereas B7-1 is not, although B7-1 expression is induced in these cells when the cells are stimulated with interferon gamma (IFN- ⁇ ).
  • IFN- ⁇ interferon gamma
  • the different expression patterns may indicate a different role for each of the B7 family members.
  • the B7 proteins are believed to be involved in the events relating to stimulation of an immune response by its ability to interact with various immune cell surface receptors. It is believed, for example, that B7 plays a role in augmenting T-cell proliferation and cytokine production through its interaction with the CD28 receptor.
  • CD28 a homodimeric glycoprotein having two disulfide linked 44-kd subunits, is found on 95% of CD4 + and 50% of CD8 + cells.
  • Studies using monoclonal antibodies reactive with CD28 have demonstrated that CD28 is involved in a secondary signal pathway in the activation of T-cell proliferation.
  • Antibodies which block the interaction of CD28 with its ligand have been found to inhibit T-cell proliferation in vitro resulting in antigen specific T cell anergy. (Harding et al., Nature, v. 356, p. 607 (1991)).
  • CTLA-4 T-cell surface receptor protein
  • nerve cells can be induced to express B7 and can interact with T- and other immune cells through B7/CD28/CTLA4 molecules.
  • the B7 on the nerve cell surface can engage the CD28/CTLA4 on the immune cell surface to co-stimulate the immune cell, leading to activation of the immune cell.
  • the activated immune cell then releases nerve growth factor which stimulates the nerve cell.
  • B7 inducing agent is an agent which causes B7 ( and other related family members retaining sequence homology with B7) to be expressed on a nerve cell surface.
  • the B7 inducing agent is a pharmacological agent that causes dissipation of proton motor force such as by uncoupling of electron transport and oxidative phosphorylation, resulting in reduced mitochondrial membrane potential within the cell.
  • B7 inducing agents which cause dissipation of the proton motor force include but are not limited to adriamycin, gamma interferon, bacterial byproducts such as lipopolysaccharides, lipoproteins BCG, fatty acids, cAMP inducing agents and a UCP expression vector.
  • a "cAMP inducing agent” as used herein is any compound which elevates intracellular levels of cAMP. Such compounds include but are not limited to isoproterenol, epinephrine, norepinephrine, phosphodiester inhibitors, theophylline, and caffeine.
  • the B7 inducing agent is a B7 expression vector. Such a vector can be stably expressed in the nerve cell to produce B7 which can be expressed on the cell surface.
  • the B7 inducing agent is an isolated molecule. An isolated molecule is one which has been removed from its natural surroundings and formulated for administration to an organism.
  • an “amount of a B7 inducing agent effective to induce the expression of B7 on the surface of the nerve cell” as used herein, refers to an amount which is effective to cause dissipation of a proton motor force and thus to decrease the mitochondrial membrane potential in the nerve cell.
  • the amount is that amount which is necessary to induce the expression of at least a single B7 molecule on the cell surface.
  • the nerve cell is contacted with the B7 inducing agent to cause expression of B7 on the surface.
  • the step of contacting the cell with B7 inducing agent can be performed by any means known in the art. For instance, if the B7 inducing agent is applied in vitro, it may simply be added as part of the cellular medium to a tissue culture dish of nerve cells. If the method is performed in vivo, then the step of contacting may be performed by administering the B7 inducing agent by commonly used therapeutic techniques, such as parenteral administration, oral administration, or local administration. Other methods are well known to those of ordinary skill in the art. According to a method of the invention the B7 expressing nerve cell is exposed to a neural activating cell.
  • a “neural activating cell” as used herein, is a cell which is capable of producing nerve growth factor when activated and which includes a cell surface B7 receptor.
  • B7 receptors include CD28 and CTLA-4.
  • Many cells which are the neural activating cells of the invention have been described in the prior art. These cells include, for example, T cells (including both gamma, delta and alpha-beta T cells), macrophage, dendritic cells, CTLA-4 or CD-28 expressing B cells.
  • the neural activating cell may also be a cell which produces nerve growth factor upon activation and which is engineered to express a B7 receptor on its surface, e.g. by transfection with an inducible or constitutively expressed B7 receptor gene, such as by the methods described above.
  • the methods of the invention in some aspects may also be performed using an endogenous neural activating cell.
  • the endogenous neural activating cell may be a cell having a cell surface B7 receptor, such as CD28 and CTLA-4.
  • the method would only include the step of contacting a nerve cell with an amount of a B7 inducing agent effective to induce the expression of B7 on the surface of the nerve cell in the presence of a neural activating cell.
  • the neural activating cell is a cell having a cell surface B7 receptor which is already present in interactive proximity to the B7, the cell does not have to be manually brought into contact with the B7 on the nerve cell.
  • the cell surface B7 can interact with the B7 receptor to activate the neural activating cell. Once activated, the neural activating cell produces and releases nerve growth factor into the local environment. This locally produced nerve growth factor is capable of causing the nerve cell to become differentiated.
  • the mechanism through which neuro-differentiation occurs is that the nerve growth factor interacts with the nerve cell surface nerve growth factor receptor such as Trk. It is also believed that engagement of the B7 on the cell surface or the induction thereof causes the expression of nerve growth factor receptors on the surface of the nerve.
  • the receptors for nerve growth factor may be induced to be expressed on the surface of the nerve cell.
  • Two known nerve growth factors are tyrosine, kinase A (TrkA) and p75NGRF. When these receptors interact with nerve growth factor on the surface of a nerve cell, it stimulates the cell to undergo neuronal differentiation. Expression of these receptors on the surface of the nerve cell may be performed by any method known in the art. For instance, the nerve cell may be recombinantly engineered to constitutively or inducibly express the DNA for these receptors, such as by the methods described above.
  • Nerve growth factor originally described by Levi-Montalcini and Hamburger in 1953 (Levi-Montalcini and Hamburger, 1953), contains two copies of three types of polypeptides designated ⁇ , ⁇ and ⁇ and exhibits approximately 50% of homology with other neurotrophins i.e., brain-derived neurotrophic factor (BDNF), NT-3, NT-4 and NT-5 (Siegel et al., 1994). It binds to tyrosine kinase A (TrkA) and p75NGF receptors in a synergistic manner (Canossa et al., 1996).
  • BDNF brain-derived neurotrophic factor
  • TrkA tyrosine kinase A
  • TrkA tyrosine kinase A
  • p75NGF receptors in a synergistic manner
  • nerve cells express molecules which are requisite for T cell activation, indicating that there is a neuro-immunological intercellular interactive component that occurs during neuronal differentiation.
  • NGF and EGF have profound effects on the differentiation process in utero and early life and on the regeneration process after pathologic damage.
  • the data provided in the examples is relevant since it not only demonstrates the existence of inducible surface molecules on post-mitotic neurons, but their ability to be kinetically modified by the presence or absence of specific trophic factors is also highlighted.
  • the presence of Fas on the neuronal cell surface suggests that PC 12 cells and their variants are vulnerable to apoptosis or that the molecule is capable of transmitting a mitotic signal if required.
  • Another aspect of the invention involves a method for inducing apoptosis in a nerve cell.
  • the method involves the step of contacting a nerve cell with an amount of a metabolic modifying agent which when exposed to a nerve cell causes coupling of electron transport and oxidative phosphorylation effective to increase the mitochondrial membrane potential in the nerve cell and contacting a neural activating cell with an amount of a B7 receptor blocking agent effective for inducing apoptosis in the nerve cell.
  • a metabolic modifying agent which when exposed to a nerve cell causes coupling of electron transport and oxidative phosphorylation effective to increase the mitochondrial membrane potential in the nerve cell
  • a neural activating cell with an amount of a B7 receptor blocking agent effective for inducing apoptosis in the nerve cell.
  • B7 receptor blocking agent is any agent which interacts with a B7 receptor but does not cause activation of the cell and prevents that receptor from binding to B7.
  • These agents include, for example, but are not limited to anti-CD28 antibodies, CD28 binding peptides, anti-CTLA-4 antibodies, CTLA-4 analogs and CTLA-4 binding peptides which do not cause activation of the receptor.
  • Other B7 receptor blocking agents can be identified by those of skill in the art by routine experimentation using immune cell activation assays such as a T cell activation assay. This method is useful whenever it is desirable to induce apoptosis of a nerve cell. For instance, it may be useful to induce apoptosis of a nerve cell in vitro in order to screen molecules for their ability to prevent apoptosis of nerve cells. Other uses will be apparent to those of ordinary skill in the art.
  • Fas when a cell is coupled, Fas is expressed on the cell surface and when a cell is uncoupled Fas generally is transported to intracellular stores.
  • Fas When a cell is coupled and Fas is on the surface engagement of Fas sends a signal to the cell instructing the cell to undergo cellular division.
  • Fas When a cell is uncoupled ordinarily Fas is not expressed on the cell surface.
  • Fas In the presence of NGF, however, Fas is down regulated and is no longer expressed on the cell surface.
  • a damaged tissue if a nerve cell is in an uncoupled state, and expresses both Fas and B7 on the surface, then the presence or absence of NGF will determine the fate of the cell.
  • an NGF producing cell according to the invention will stimulate production of NGF by interacting with that cell.
  • the local NGF produced will cause the down regulation of Fas and the cell will undergo differentiation. If an NGF producing cell is not available or if B7 is not expressed on the surface of the nerve cell, then environmental factors can stimulate Fas to cause apoptosis.
  • Another aspect of the invention is a method for reinnervating an injured tissue.
  • the method involves the step of implanting a B7 expressing nerve cell in the injured tissue, wherein the implanted B7 expressing nerve cell will undergo neuronal differentiation in the presence of a neural activating cell in the injured tissue to reinnervate the injured tissue.
  • Methods are known in the art implanting nerve cells into living tissue.
  • nerves can be implanted directly into exposed tissue or may be implanted in biodegradable tubes which will guide the extension of the nerve into surrounding tissue where it can be differentiated.
  • An injured tissue is a tissue in which nerve damage has been sustained.
  • An injured tissue may include for example, a spinal chord injury, a severed or severely damaged limb or any other tissue which can be innervated and in which the nerve has been damaged.
  • Neural activating cells are generally found in skin and muscle surrounding the nerves of an injured tissue. These neural activating cells can stimulate the differentiation of the nerve cell once they are activated by interaction with the B7 on the surface of the nerve cell.
  • the invention also includes a method for treating a neurodegenerative disorder by administering an amount of a B7 inducing agent effective to induce the expression of B7 on the surface of a nerve cell.
  • An amount that is effective to induce the expression on the surface is an amount which is effective to cause dissipation of a proton motor force and thus to decrease the mitochondrial membrane potential in the nerve cell.
  • a "neurodegenerative disorder” as used herein is a disorder associated with the death or injury of neuronal cells. For example, the loss of dopaminergic neurons in the substantia nigra ultimately leads to Parkinson's Disease. The deposition of ⁇ -amyloid protein in the brain generally causes neural damage leading to Alzheimer's Disease.
  • Conditions involving injuries such as brain ischemia, spinal chord damage, and severance of limbs often causes extensive neuronal cell death.
  • a nerve When a nerve is severed, the regions of the nerve cells which are distal to the severance become separated from the nerve cell body and degenerate. After such a severance, it is possible for the nerve cell body to regenerate by re-extension of the severed axons. This process of nerve regeneration does not occur naturally in the absence of certain environmental conditions. In some cases in the prior art, various factors such as nerve growth factor have been added to the nerve to attempt to stimulate the regeneration.
  • the methods of the invention describe a different system in which the nerve cell is manipulated to express an immune recognition molecule on its surface which can then cause the local expression of nerve growth factor leading to differentiation. This method more closely simulates the natural processes of neuronal regeneration.
  • Other neurodegenerative diseases include for example but are not limited to epileptic seizures and amyotrophic lateral sclerosis.
  • the invention also includes compositions of the above described agents.
  • One composition of the invention includes a metabolic modifying agent and an apoptotic chemotherapeutic agent.
  • the pharmaceutical preparations of the invention are administered to subjects in effective amounts.
  • An effective amount means that amount necessary to delay the onset of, inhibit the progression of, halt altogether the onset or progression of or diagnose the particular condition being treated.
  • the metabolic modifying agent and the apoptotic chemotherapeutic agent are present in an effective dose for treating a tumor.
  • the metabolic modifying agent and the apoptotic chemotherapeutic agent are present in an effective dose for treating type II diabetes.
  • an effective amount for treating cancer and type I diabetes will be that amount necessary to favorably affect mammalian cell proliferation in-situ.
  • One composition of the invention is a B7 inducing agent and a B7 receptor inducing agent.
  • the B7 inducing agent and B7 receptor inducing agent present in an effective dose for treating neurodegenerative disease.
  • an effective amount for neurodegenerative disease will be that amount necessary to favorably affect nerve cell differentiation in-situ.
  • effective amounts will depend, of course, on the particular condition being treated; the severity of the condition; individual patient parameters including age, physical condition, size and weight; concurrent treatment; frequency of treatment; and the mode of administration. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment.
  • doses of active compounds will be from about O.Olmg/kg per day to 1000 mg/kg per day. It is expected that doses range of 50-500 mg/kg will be suitable, in one or several administrations per day. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate levels of compounds.
  • the invention involves the use of several different types of binding peptides or molecuels,
  • binding peptides of the invention can be identified using routine assays, such as the binding and activation assays described in the Examples and elsewhere throughout this patent application.
  • the binding peptides of the invention are isolated peptides.
  • isolated peptides means that the peptides are substantially pure and are essentially free of other substances with which they may be found in nature or in vivo systems to an extent practical and appropriate for their intended use.
  • the peptides are sufficiently pure and are sufficiently free from other biological constituents of their hosts cells so as to be useful in, for example, producing pharmaceutical preparations or sequencing.
  • an isolated peptide of the invention may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the peptide may comprise only a small percentage by weight of the preparation.
  • the peptide is nonetheless substantially pure in that it has been substantially separated from the substances with which it may be associated in living systems.
  • the binding peptides also may easily be synthesized or produced by recombinant means by those of skill in the art. Methods for preparing or identifying peptides which bind to a particular target are well known in the art.
  • Molecular imprinting for instance, may be used for the de novo construction of macromolecular structures such as peptides which bind to a particular molecule. See for example Kenneth J. Shea, Molecular Imprinting of Synthetic Network Polymers: The De Novo synthesis of Macromolecular Binding and Catalytic Sites, TRIP Vol. 2, No. 5, May 1994; Klaus Mosbach, Molecular Imprinting, Trends in Biochem.
  • One method for preparing mimics of the known binding peptides involves the steps of: (i) polymerization of functional monomers around a known binding peptide or the binding region of an antibody which also binds to the targets (the template) that exhibits a desired activity; (ii) removal of the template molecule; and then (iii) polymerization of a second class of monomers in the void left by the template, to provide a new molecule which exhibits one or more desired properties which are similar to that of the template.
  • the binding peptides may also be identified by conventional screening methods such as phage display procedures (e.g., methods described in Hart, et al., J. Biol. Chem. 269:12468 (1994)).
  • Hart et al. report a filamentous phage display library for identifying novel peptide ligands for mammalian cell receptors.
  • phage display libraries using, e.g., Ml 3 or fd phage are prepared using conventional procedures such as those described in the foregoing reference.
  • the libraries display inserts containing from 4 to 80 amino acid residues.
  • the inserts optionally represent a completely degenerate or a biased array of peptides.
  • Ligands having the appropriate binding properties are obtained by selecting those phages which express on their surface a ligand that binds to the target molecule. These phages then are subjected to several cycles of reselection to identify the peptide ligand-expressing phages that have the most useful binding characteristics. Typically, phages that exhibit the best binding characteristics (e.g., highest affinity) are further characterized by nucleic acid analysis to identify the particular amino acid sequences of the peptides expressed on the phage surface and the optimum length of the expressed peptide to achieve optimum binding. Alternatively, such peptide ligands can be selected from combinatorial libraries of peptides containing one or more amino acids. Such libraries can further be synthesized which contain non-peptide synthetic moieties which are less subject to enzymatic degradation compared to their naturally-occurring counte ⁇ arts.
  • Screening of peptides of the invention also can be carried out utilizing a competition assay. If the peptide being tested competes with the known monoclonal antibody, as shown by a decrease in binding of the known monoclonal antibody, then it is likely that the peptide and the known monoclonal antibody bind to the same, or a closely related, epitope. Still another way to determine whether a peptide has the specificity of the known monoclonal antibody is to pre- incubate the known monoclonal antibody with the target with which it is normally reactive, and then add the peptide being tested to determine if the peptide being tested is inhibited in its ability to bind the target. If the peptide being tested is inhibited then, in all likelihood, it has the same, or a functionally equivalent, epitope and specificity as the known monoclonal antibody.
  • An anti-idiotypic antibody can be prepared by immunizing an animal with the known monoclonal antibodies.
  • the immunized animal will recognize and respond to the idiotypic determinants of the immunizing known monoclonal antibodies and produce an antibody to these idiotypic determinants.
  • the anti-idiotypic antibodies of the immunized animal which are specific for the known monoclonal antibodies of the invention, it is possible to identify other clones with the same idiotype as the known monoclonal antibody used for immunization.
  • Idiotypic identity between monoclonal antibodies of two cell lines demonstrates that the two monoclonal antibodies are the same with respect to their recognition of the same epitopic determinant.
  • anti-idiotypic antibodies it is possible to identify other hybridomas expressing monoclonal antibodies having the same epitopic specificity.
  • an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the image of the epitope bound by the first monoclonal antibody.
  • the binding peptides useful according to the invention are antibodies or functionally active antibody fragments. Antibodies are well known to those of ordinary skill in the science of immunology. Many of the binding peptides described herein are available from commercial sources as intact functional antibodies. As used herein, the term "antibody” means not only intact antibody molecules but also fragments of antibody molecules retaining specific binding ability.
  • antibody means not only intact immunoglobulin molecules but also the well-known active fragments F(ab') 2 , and Fab. F(ab') 2 , and Fab fragments which lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).
  • the peptide of the invention is an intact soluble monoclonal antibody in an isolated form or in a pharmaceutical preparation.
  • An intact soluble monoclonal antibody as is well known in the art, is an assembly of polypeptide chains linked by disulfide bridges. Two principle polypeptide chains, referred to as the light chain and heavy chain, make up all major structural classes (isotypes) of antibody. Both heavy chains and light chains are further divided into subregions referred to as variable regions and constant regions.
  • the term "monoclonal antibody” refers to a homogenous population of immunoglobulins which specifically bind to an epitope (i.e. antigenic determinant) , e.g., of MHC class II HLA-DR.
  • the peptide useful according to the methods of the present invention may be an intact humanized a monoclonal antibody.
  • a "humanized monoclonal antibody” as used herein is a human monoclonal antibody or functionally active fragment thereof having human constant regions and a binding CDR3 region from a mammal of a species other than a human.
  • Humanized monoclonal antibodies may be made by any method known in the art. Humanized monoclonal antibodies, for example, may be constructed by replacing the non-CDR regions of a non-human mammalian antibody with similar regions of human antibodies while retaining the epitopic specificity of the original antibody.
  • non-human CDRs and optionally some of the framework regions may be covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody.
  • FR and/or Fc/pFc' regions there are entities in the United States which will synthesize humanized antibodies from specific murine antibody regions commercially, such as Protein Design Labs (Mountain View California).
  • Protein Design Labs Manton View California
  • a humanized form of the Pharmingen anti-Fas antibody used in the attached Examples could be easily prepared and used according to the methods of the invention.
  • the DNA sequence encoding the domain may be prepared by oligonucleotide synthesis.
  • Another method involves the preparation of the DNA sequence encoding the variable CDR containing domain by oligonucleotide site-directed mutagenesis. Each of these methods is well known in the art. Therefore, those skilled in the art may construct humanized antibodies containing a murine CDR region without destroying the specificity of the antibody for its epitope.
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd (heavy chain variable region).
  • the Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
  • the B7 and UCP expression vectors and other relevant expression vectors described herein can be prepared and inserted into cells using routine procedures known in the art. These procedures are set forth below in more detail.
  • the term "IRM" (immune recognition molecule) nucleic acid is used herein to refer to each of the nucleic acids encompassed by the expression vectors described herein. Although UCP is not an immune molecule the term IRM is used to encompass UCP nucleic acids to simplify the discussion.
  • IRM nucleic acid refers to a nucleic acid molecule which: (1) hybridizes under stringent conditions to a nucleic acid having the sequence of SEQ ID NO:l, 3, 5, 7, 9, and 11 and (2) codes for a IRM polypeptide (i.e., the respective immune recognition polypeptide).
  • the preferred IRM nucleic acid has the nucleic acid sequence of SEQ ID NO:l, 3, 5, 7, 9, and 11 (the nucleic acids encoding the human B7.1, B7.2, UCP-1, UCP-2, UCP-3S, and CD28 polypeptides respectively).
  • the IRM nucleic acids may be intact IRM nucleic acids which include the nucleic acid sequence of Sequence ID No.
  • IRM nucleic acids further embrace nucleic acid molecules which differ from the sequence of SEQ ID NO:l, 3, 5, 7, 9, and 11 in codon sequence due to the degeneracy of the genetic code.
  • the IRM nucleic acids of the invention may also be functionally equivalent variants, analogs and fragments of the foregoing nucleic acids. "Functionally equivalent", in reference to a IRM nucleic acid variant, analog or fragment, refers to a nucleic acid that codes for a IRM polypeptide that is capable of functioning as an immune recognition molecule or an uncoupling protein.
  • the IRM nucleic acid molecules can be identified by conventional techniques, e.g., by identifying nucleic acid sequences which code for IRM polypeptides and which hybridize to a nucleic acid molecule having the sequence of SEQ ID NO:l, 3, 5, 7, 9, and 11 under stringent conditions.
  • stringent conditions refers to parameters with which the art is familiar. More specifically, stringent conditions, as used herein, refer to hybridization at 65 °C in hybridization buffer (3.5 x SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone, 0.02% bovine serum albumin, 2.5mM NaH 2 PO 4 (pH 7), 0.5% SDS, 2mM EDTA).
  • SSC 0.15M sodium chloride/0.15M sodium citrate, pH 7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetraacetic acid.
  • SSC 0.15M sodium chloride/0.15M sodium citrate, pH 7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetraacetic acid.
  • homologs and alleles typically will share at least 40% nucleotide identity with SEQ ID NO:l, 3, 5, 7, 9, and 11; in some instances, will share at least 50% nucleotide identity; and in still other instances, will share at least 60% nucleotide identity.
  • the preferred homologs have at least 70% sequence homology to SEQ ID NO: 1 , 3, 5, 7, 9, and 11. More preferably the preferred homologs have at least 80% and, most preferably, at least 90% sequence homology to SEQ ID NO:l, 3, 5, 7, 9, and 11 -5.
  • the invention also includes degenerate nucleic acids which include alternative codons to those present in the naturally occurring nucleic acid that codes for the human IRM polypeptide.
  • serine residues are encoded by the codons TCA, AGT, TCC, TCG, TCT and AGC.
  • Each of the six codons is equivalent for the pu ⁇ oses of encoding a serine residue.
  • any of the serine-encoding nucleotide codons may be employed to direct the protein synthesis apparatus, in vitro or in vivo, to inco ⁇ orate a serine residue.
  • nucleotide sequence triplets which encode other amino acid residues include, but are not limited to, CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine codons).
  • Other amino acid residues may be encoded similarly by multiple nucleotide sequences.
  • the invention embraces degenerate nucleic acids that differ from the naturally occurring nucleic acids in codon sequence due to the degeneracy of the genetic code.
  • the IRM nucleic acid in one embodiment, is operably linked to a gene expression sequence which directs the expression of the IRM nucleic acid within a eukaryotic cell.
  • the "gene expression sequence” is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the IRM nucleic acid to which it is operably linked.
  • the gene expression sequence may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter.
  • Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPTR), adenosine deaminase, pyruvate kinase, and ⁇ -actin.
  • HPTR hypoxanthine phosphoribosyl transferase
  • adenosine deaminase pyruvate kinase
  • ⁇ -actin ⁇ -actin
  • the gene expression sequence shall include, as necessary, 5' non-transcribing and 5' non-translating sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • 5' non-transcribing sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined IRM nucleic acid.
  • the gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired.
  • the IRM nucleic acid of the invention is linked to a gene expression sequence which permits expression of the IRM nucleic acid in the local environment of a cell, e.g. a damaged nerve cell.
  • the gene expression sequence permits expression of the IRM nucleic acid in a human nerve cell or a neural activating cell.
  • a sequence which permits expression of the IRM nucleic acid in a nerve cell or a neural activating cell is one which is selectively active in nerve cell or a neural activating cell and thereby causes the expression of the IRM nucleic acid in these cells.
  • promoters that are capable of expressing a IRM nucleic acid in a nerve cell or a neural activating cell, as well as other known cells.
  • the IRM nucleic acid sequence and the gene expression sequence are said to be "operably linked” when they are covalently linked in such a way as to place the transcription and/or translation of the IRM coding sequence under the influence or control of the gene expression sequence.
  • two DNA sequences are said to be operably linked if induction of a promoter in the 5' gene expression sequence results in the transcription of the IRM sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the IRM sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
  • a gene expression sequence would be operably linked to a IRM nucleic acid sequence if the gene expression sequence were capable of effecting transcription of that IRM nucleic acid sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
  • the IRM nucleic acid of the invention can be delivered to the cell alone or in association with a vector.
  • a "vector” is any vehicle capable of facilitating: (1) delivery of a IRM molecule to a target cell or (2) uptake of a IRM molecule by a target cell.
  • the vectors transport the IRM molecule into the target cell with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • a "targeting ligand" can be attached to the vector to selectively deliver the vector to a cell which expresses on its surface the cognate receptor for the targeting ligand.
  • the vector (containing a IRM nucleic acid) can be selectively delivered to a cell in, e.g., an injured nerve tissue.
  • the vectors useful in the invention are divided into two classes: biological vectors and chemical/physical vectors.
  • biological vectors are useful for delivery/uptake of IRM nucleic acids to/by a target cell.
  • chemical/physical vectors are also useful for delivery/uptake of IRM nucleic acids to/by a target cell.
  • Bio vectors include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the nucleic acid sequences of the invention, and free nucleic acid fragments which can be attached to the nucleic acid sequences of the invention.
  • the adeno-associated virus can be engineered to be replication -deficient and is capable of infecting a wide range of cell types and species. It further has advantages, such as heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression.
  • adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also fvmction in an extrachromosomal fashion.
  • chemical/physical vectors may be used to deliver a IRM molecule to a target cell and facilitate uptake thereby.
  • a "chemical/physical vector” refers to a natural or synthetic molecule, other than those derived from bacteriological or viral sources, capable of delivering the IRM molecule to a cell.
  • a preferred chemical/physical vector of the invention is a colloidal dispersion system.
  • Colloidal dispersion systems include lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • a preferred colloidal system of the invention is a liposome.
  • Liposomes are artificial membrane vessels which are useful as a delivery vector in vivo or in vitro. It has been shown that large unilamellar vessels (LUV), which range in size from 0.2 - 4.0 ⁇ m can encapsulate large macromolecules. RNA, DNA, and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem.
  • LUV large unilamellar vessels
  • a liposome In order for a liposome to be an efficient gene transfer vector, one or more of the following characteristics should be present: (1) encapsulation of the gene of interest at high efficiency with retention of biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information.
  • Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTINTM and LIPOFECTACETM, which are formed of cationic lipids such as N-[l-(2, 3 dioleyloxy)- propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB).
  • LIPOFECTINTM and LIPOFECTACETM are formed of cationic lipids such as N-[l-(2, 3 dioleyloxy)- propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB).
  • DOTMA N-[l-(2, 3 dioleyloxy)- propyl]-N, N, N-trimethylammonium chloride
  • DDAB dimethyl dioctadecylammonium bromide
  • PCT International application no. PCT/US/03307 Publication No. WO 95/24929, entitled “Polymeric Gene Delivery System", claiming priority to U.S. patent application serial no. 213,668, filed March 15, 1994.
  • PCT/US/0307 describes a biocompatible, preferably biodegradable polymeric matrix for containing an exogenous gene under the control of an appropriate promotor. The polymeric matrix is used to achieve sustained release of the exogenous gene in the patient.
  • the IRM nucleic acids described herein are encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in PCT/US/03307.
  • the polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the IRM molecule is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the IRM molecule is stored in the core of a polymeric shell).
  • Other forms of the polymeric matrix for containing the IRM molecule include films, coatings, gels, implants, and stents.
  • the size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix is introduced.
  • the size of the polymeric matrix further is selected according to the method of delivery which is to be used, typically injection into a tissue or administration of a suspension by aerosol into the nasal and/or pulmonary areas.
  • the polymeric matrix and IRM molecule are encompassed in a surfactant vehicle.
  • the polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the matrix is administered to a nasal and/or pulmonary surface that has sustained an injury.
  • the matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time.
  • the chemical/physical vector is a biocompatible microsphere that is suitable for oral delivery.
  • microspheres are disclosed in Chickering et al., Biotech. And Bioeng, (1996) 52:96-101 and Mathiowitz et al., Nature, (1997) 386:.410-414.
  • Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the IRM nucleic acids of the invention to the subject.
  • Biodegradable matrices are preferred.
  • Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred.
  • the polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable.
  • the polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers.
  • the IRM nucleic acids are delivered using a bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix.
  • exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate
  • non-biodegradable polymers examples include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.
  • Compaction agents also can be used alone, or in combination with, a biological or chemical/physical vector of the invention.
  • a "compaction agent”, as used herein, refers to an agent, such as a histone, that neutralizes the negative charges on the nucleic acid and thereby permits compaction of the nucleic acid into a fine granule. Compaction of the nucleic acid facilitates the uptake of the nucleic acid by the target cell.
  • the compaction agents can be used alone, i.e., to deliver the IRM molecule in a form that is more efficiently taken up by the cell or, more preferably, in combination with one or more of the above-described vectors.
  • the invention also encompasses the use of antisense oligonucleotides that selectively bind to a IRM nucleic acid molecule, and dominant negative IRM to reduce the expression of IRM.
  • Antisense oligonucleotides are useful, for example, for preparing an animal model of a subject having a neurodegenerative disorder. Such animal models can be used in screening assays for identifying therapeutic drugs for treating neurodegenerative disorders..
  • antisense oligonucleotide or “antisense” describes an oligonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an RNA transcript of that gene and, thereby, inhibits the transcription of that gene and/or the translation of the mRNA.
  • the antisense molecules are designed so as to hybridize with the target gene or target gene product and thereby, interfere with transcription or translation of the target mammalian cell gene.
  • the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence.
  • the antisense must be a unique fragment.
  • a unique fragment is one that is a 'signature' for the larger nucleic acid. It, for example, is long enough to assure that its precise sequence is not found in molecules outside of the IRM gene. As will be recognized by those skilled in the art, the size of the unique fragment will depend upon its conservancy in the genetic code. Thus, some regions of SEQ ID NO:l, 3, 5, 7, 9, and 11, will require longer segments to be unique while others will require only short segments, typically between 12 and 32 base pairs (e.g.
  • the antisense oligonucleotide be constructed and arranged so as to bind selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions.
  • the antisense oligonucleotides should comprise at least 7 and, more preferably, at least 15 consecutive bases which are complementary to the target.
  • the antisense oligonucleotides comprise a complementary sequence of 20-30 bases.
  • oligonucleotides may be chosen which are antisense to any region of the gene or RNA (e.g., mRNA) transcripts
  • the antisense oligonucleotides are complementary to 5' sites, such as translation initiation, transcription initiation or promoter sites, that are upstream of the gene that is targeted for inhibition by the antisense oligonucleotides.
  • 5' or 3' enhancers may be targeted. Targeting to mRNA splice sites has also been used in the art but may be less preferred if alternative mRNA splicing occurs.
  • the antisense is targeted, preferably, to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al., Cell Mol. Neurobiol., (1994) 14(5):439-457) and at which proteins are not expected to bind.
  • the selective binding of the antisense oligonucleotide to a mammalian target cell nucleic acid effectively decreases or eliminates the transcription or translation of the mammalian target cell nucleic acid molecule. Reduction in transcription or translation of the nucleic acid molecule is desirable in preparing an animal model for further defining the role played by the mammalian target cell nucleic acid in modulating an adverse medical condition.
  • the invention also includes the use of a "dominant negative UCP" polypeptide.
  • a dominant negative polypeptide is an inactive variant of a protein, which, by interacting with the cellular machinery, displaces an active protein from its interaction with the cellular machinery or competes with the active protein, thereby reducing the effect of the active protein.
  • a dominant negative receptor which binds a ligand but does not transmit a signal in response to binding of the ligand can reduce the biological effect of expression of the ligand.
  • a dominant negative catalytically-inactive kinase which interacts normally with target proteins but does not phosphorylate the target proteins can reduce phosphorylation of the target proteins in response to a cellular signal.
  • a dominant negative transcription factor which binds to a promoter site in the control region of a gene but does not increase gene transcription can reduce the effect of a normal transcription factor by occupying promoter binding sites without increasing transcription.
  • the end result of the expression of a dominant negative polypeptide as used herein in a cell is a reduction in function of active UCP.
  • One of ordinary skill in the art can assess the potential for a dominant negative variant of a protein, and using standard mutagenesis techniques to create one or more dominant negative variant polypeptides. For example, one of ordinary skill in the art can modify the sequence of the UCP by site-specific mutagenesis, scanning mutagenesis, partial gene deletion or truncation, and the like. See, e.g., U.S. Patent No. 5,580,723 and Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. The skilled artisan then can test the population of mutagenized polypeptides for diminution in a selected and/or for retention of such an activity. Other similar methods for creating and testing dominant negative variants of a protein will be apparent to one of ordinary skill in the art.
  • the invention includes transgenic animals and cells transfected with the IRM's.
  • complements of the IRM nucleic acids described above can be useful as anti-sense oligonucleotides, e.g., by delivering the anti-sense oligonucleotide to an animal to induce a "knockout" phenotype.
  • the administration of anti-sense RNA probes to block gene expression is discussed in Lichtenstein, C, Nature 333:801-802 (1988).
  • the IRM nucleic acids can be used to prepare a non-human transgenic animal.
  • a "transgenic animal” is an animal having cells that contain DNA which has been artificially inserted into a cell, which DNA becomes part of the genome of the animal which develops from that cell.
  • Preferred transgenic animals are primates, mice, rats, cows, pigs, horses, goats, sheep, dogs and cats. Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Charles River (Wilmington, MA), Taconic (Germantown, NY), Harlan Sprague Dawley (Indianapolis, IN), etc.
  • Transgenic animals having a particular property associated with a particular disease can be used to study the affects of a variety of drugs and treatment methods on the disease, and thus serve as genetic models for the study of a number of human diseases.
  • the invention therefore, contemplates the use of IRM knockout and transgenic animals as models for the study of neurodegenerative disorders.
  • Pluripotent stem cells derived from the inner cell mass of the embryo and stabilized in culture can be manipulated in culture to incorporate nucleotide sequences of the invention.
  • a transgenic animal can be produced from such cells through implantation into a blastocyst that is implanted into a foster mother and allowed to come to term. See e.g., Robertson et al., Cold Spring Harbor Conference Cell Proliferation 10: 647 (1983); Bradley et al., Nature 309: 255 (1984); Wagner et al., Cold Spring Harbor Symposium Quantitative Biology 50: 691 (1985).
  • a clone containing the sequence(s) of the invention is co-transfected with a gene encoding resistance.
  • the gene encoding neomycin resistance is physically linked to the sequence(s) of the invention.
  • DNA molecules introduced into ES cells can also be integrated into the chromosome through the process of homologous recombination.
  • Capecchi Science. 244: 1288-1292 (1989).
  • Methods for positive selection of the recombination event (e.g., neo resistance) and dual positive-negative selection (e.g., neo resistance and gangcyclovir resistance) and the subsequent identification of the desired clones by PCR have been described by Capecchi, supra and Joyner et al., Nature. 338: 153-156 (1989).
  • the final phase of the procedure is to inject targeted ES cells into blastocysts and to transfer the blastocysts into pseudopregnant females.
  • the resulting chimeric animals are bred and the offspring are analyzed by Southern blotting to identify individuals that carry the transgene.
  • Inactivation or replacement of the endogenous IRM genes can be achieved by a homologous recombination system using embryonic stem cells.
  • the resultant transgenic non- human mammals having a knockout characteristic may be used as a model for neurodegenerative disorders.
  • Nerve cells which do not express IRMs may be predisposed to apoptosis and unable to differentiate and thus, produce a neurodegenerative phenotype.
  • a variety of therapeutic drugs can be administered to the phenotypically neurodegenerative animals to determine the affect of the therapeutic drugs on nerve cell differentiation. In this manner, therapeutic drugs which are useful for preventing or reducing neurodegenerative disorders can be identified. Such agents are useful for, e.g., treating spinal chord injuries or Parkinson's disease.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being co- mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the compositions of the invention, which is preferably isotonic with the blood of the recipient.
  • This aqueous preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example, as a solution in 1 ,3-butane diol.
  • Suitable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • fatty acids such as oleic acid may be used in the preparation of injectables.
  • Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the compositions of the invention into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the compositions of the invention into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • Long-term sustained release means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days.
  • Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
  • Example 1 Metabolic state of a cell is indicative of cell surface Fas expression and sensitivity/resistance to cell death.
  • the cell lines utilized herein include L1210, a leukemic cell line; HL60, a human pro-myelocytic cell line; and PC 12, a pheochromocytoma cell line which can be induced to differentiate into a neuronal cell line in the presence of NGF (Lindenboim, L, et al., Cancer Res, 1995, 55:1242-7).
  • NGF Neuronal growth factor
  • L1210 DDP are resistant to cisplatin and methotrexate; HL60 MDR are resistant to adriamycin induced apoptosis; PC 12 TrkA, which have been transfected with TrkA which results in constitutively expression the NGF receptors, are not susceptible to alcohol and NGF withdrawal as are the PC 12 cells.
  • the apoptosis sensitive cells from each tissue origin were mo ⁇ hologically round, non-adherent, rapidly dividing cells, with the exception of the PC 12 cell line.
  • the apoptosis resistant cells from all tissue origins were mo ⁇ hologically large, adherent, and slowly dividing cells.
  • Fas CD95
  • Fas Ligand CD95L
  • Flow cytometric analysis of Fas expression was performed using an isotype versus FITC-anti-Fas(Pharmingen) on L1210; PC 12; and HL60 cells and resistant cell lines L1210DDP, PC12Trk; and HL60MDR.
  • Rate of glucose utilization was measured by the method of Ashcroft et al. Briefly, cells were incubated 90 min at 37°C in 100 ⁇ l KRB, glucose (5.5 mM), 1.3 ⁇ Ci D-[5- 3 H] glucose (Amersham, Arlington Heights, IL). The reaction was carried out in a 1 ml cup contained in a rubber stoppered 20 ml scintillation vial that had 500 ⁇ l of distilled water surrounding the cup. Glucose metabolism was stopped by injecting 100 ⁇ l 1 M Hcl through the stopper into the cup. An overnight incubation at 37°C was carried out to allow equilibration of the [ 3 H]-H 2 O in the reaction cup and the distilled water, followed by liquid scintillation counting of the distilled water.
  • Rate of glucose oxidation was measured by incubating cells for 90 min at 37 °C in 100 ml of reaction buffer, glucose (2.8, 8.3, 27.7 mmol/1), 1.7 mCi (U-14C glucose).
  • the reaction was carried out in a 1 ml cup in a 20 ml scintillation vial capped by a rubber stopper with a center well that contains filter paper. Metabolism was stopped and CO 2 liberated with 300 ml 1 mol/1 HC1 injected through the stopper into the cup containing the cells. CO 2 was trapped in the filter paper by injecting 10 ml 1 mol/1 KOH into the center well, followed 2 hours later by liquid scintillation counting. Tubes containing NaHCO 3 and no cells were used to estimate the recovery of M CO 2 in the filter paper, routinely close to 100%.
  • L1210 and L1210DDP cells are tumor cell lines and are likely to have increased ratios of glucose oxidation to utilization (Warburg, O, et al., Klin Woch, 1926, 5:829-832).
  • the rate of glucose utilization and oxidation of the Fas deficient and the FasL deficient lymphocytes are demonstrated in Table 2.
  • the ratio of glucose utilization to oxidation is highest in Ipr lymphocytes and lowest in wild type normal, quiescent lymphocytes.
  • Fas Expression Increases as a Function of Glucose We investigated the effect of increasing concentrations of glucose on cell surface Fas expression. L1210 and L1210/DDP cells were cultured in glucose free RPMI media or in media supplemented with insulin and glucose for 16 hours. Intra- and extracellular Fas expression was determined by labelling the cells with FITC-conjugated anti-Fas antibodies (Pharmingen), or FITC-conjugated isotype control, then subtracting the fluorescence intensity of the isotype staining from Fas staining for each treatment group.
  • Fas expression increases as a function of glucose concentration and that as a result the cell surface Fas negative L1210/DDP begin to express cell surface Fas.
  • L1210 DDP cells undergo apoptotic cell death.
  • L1210 or L1210 DDP cells with the staurosporin, which inhibits protein kinase C and increases mitochondrial membrane potential, or an anti-cancer agent to which both cells are sensitive, adriamycin. Fas expression was increased or induced on both L1210 and L1210 DDP, respectively, in the presence of staurosporin or adriamycin.
  • the L1210 DDP changed morphologically and began to divide rapidly, changes which appeared to correspond with a reversion back to the phenotype of the L1210 cells.
  • Fas-deficient lymphocytes express intra-fbut not extra-) cellular Fas molecules: We isolated lymphocytes from spleens of C57BL/6 mice and from C57BL6 transgenics having the lpr mutation (loss of Fas sensitivity). Cells were stained with fluorescein conjugated hamster anti-Fas and examined by confocal microscopy.
  • results demonstrate that unstimulated, non-permeabilized splenocytes from C57BL/6 animals express Fas at low levels relative to isotype controls. Interestingly, significant levels of Fas expression were detected in permeabilized normal lymphocytes. As expected, non-permeabilized cells from C57BL6. pr animals express no detectable cell surface Fas relative to isotype control. Interestingly, intracellular Fas staining of permeabilized splenocytes from C57Bl/6./pr animals reveals intracellular expression of Fas. These results demonstrate that mutations affecting susceptibility to Fas-induced death prevent cell surface, but not intracellular expression of the Fas molecule.
  • Anti-cancer agents induce susceptibility to Fas-induced cell death: To determine if the anti-cancer agent methotrexate sensitizes L 1210 or L1210/DDP cells to Fas induced cell death, we cultured L1210 cells in the presence or absence of 10 "8 M methotrexate for 72 hours. Each group of cells was cultured on uncoated plates or plates coated with lOg/ml anti-Fas (Jo.2.2, Pharmingen). We analyzed cell death using flow cytometry. Forward angle and 90 degree light scatter were used to distinguish between live and dead cells. Dead cells were gated as forward angle light scatter low/high ethidium bromide retaining cells. Percent death was calculated over the total number of cells acquired. In Table 3 below, values indicate % dead cells over background of untreated cells.
  • L 1210 and L 1210/DDP cells were treated with 10-8 M methotrexate for 24 hours.
  • Flow cytometric analysis revealed two populations based on forward side scatter.
  • the forward scatter high populations did not take ethidium bromide and were therefore viable.
  • the forward scatter low populations took up ethidium bromide differentially.
  • the L1210 cells took up a moderate amount.
  • Analysis of DNA fragments reveals that L1210 produced a ladder of nucleosome sized fragments indicative of apoptosis, whereas L1210/DDP cells did not. This latter phenotype - loss in forward scatter and membrane permeability with no "DNA laddering" - is the hall mark of oncosis.
  • Fas Deficient Lymphocytes are also drug resistant to methotrexate:
  • Splenocytes from C57BL/6 lpr or gld animals were isolated, red cells depleted, and single cell suspensions prepared. Cells were cultured in the absence or presence of 5 x 10 "8 M methotrexate for 18 or 32 hours. Cells were harvested and viability was determined by flow cytometric analysis and confirmed with trypan blue exclusion.
  • Drug resistant cells express intracellular fas.
  • UCP and bcl-2 We determined if wild type and/or drug resistant cells express intracellular and surface fas, UCP and bcl-2.
  • the drug resistant cells are cell surface Fas negative and protected from death resulting from changes in mitochondrial membrane permeability transitions.
  • pancreatic ⁇ -cells Response of pancreatic ⁇ -cells to glucose: The experiments described below were designed to test the hypothesis that ⁇ cell metabolism may be linked to immune recognition and destruction. Glucose utilization was measured as [ 3 H]H 2 O production from 5-[ 3 H]glucose in normal rat islets. Glucose oxidation was measured as [ 14 C]CO 2 production from U-[' 4 C]glucose. The data show increasing glucose utilization and oxidation in ⁇ -cells as a function of increasing glucose concentration.
  • the cells were treated overnight with either physiological 11.1 mM glucose or high glucose 55.5 mM glucose. Fas expression was determined by staining with a FITC conjugated antibody. Mean fluorescence of staining with isotype control antibody was subtracted. Measurement of mitochondrial membrane potential was measured using JC-1 as a fluorescence probe. The relative membrane potential was read by taking the red mean fluorescence (aggregated JC-1 labeled) divided by mean green fluorescent (monomeric JC-1) labeled fluorescence.
  • Our data suggest that as glucose concentration increases, the large ⁇ cell subset of gated cells have increased Fas expression and concomitant increased mitochondrial membrane potential, while the smaller (possibly alpha, glucagon producing cells) do not.
  • Mitochondrial membrane potential is assessed flow cytometrically using mitotracker red.
  • the amount of membrane potential was measured in the four strains of animals AB-, AB-Ea,
  • Example 3 Relationship between the metabolic state of a cell and expression of MHC class II molecules
  • Anti-class II mAb induce an increase in apoptotic cell death in resting B cells.
  • Apoptotic indices were generated by comparison to maximum apoptotic death as stimulated by isoproterenol.
  • the data shows that ligation of class II molecules on resting, but not activated B cells, results in apoptotic death.
  • B cells were treated with the stimuli indicated for 10 min at 37 °C, cultured overnight, harvested and assayed.
  • mice that have the lpr mutation, or the gld mutation, which have defects in CD95 and CD95L, respectively (Watanabe-Fukunaga, R, et al., Nature (London), 1992, 356, 314-317; Suda, T et al., Cell, 1993, 75:1169-1178).
  • Total splenic B-cells were isolated from C3H, AKR, C3H.//?r, and MRL.gt ⁇ mice. All of these strains are H-2 k .
  • the B-cells were then combined with either an autoreactive I-Ak-specific T cell hybridoma (Kal-68.4) or with a hen egg lysozyme (HEL) peptide-specific, I-Ak-restricted T cell hybridoma (A6.A2) either with or without a tryptic digest of lysozyme as the source of the required peptide.
  • Kal-68.4 an autoreactive I-Ak-specific T cell hybridoma
  • HEL hen egg lysozyme
  • A6.A2 hen egg lysozyme
  • Apoptotic cells were scored based on their mo ⁇ hology and on their uptake of Hoechst Dye 33342 at 5 ⁇ g/ml final concentration (Cohen, J.J, et al., Ann Rev Immun, l992, 10, 267-293). B and T cells were distinguished by mo ⁇ hology.
  • Kal-68.4 " 30 160 22 320
  • Equal numbers (5 x 10 5 ) of B-cells and T cells were incubated for 16 hr at 37°C in a 24 well microtiter plate.
  • h A6.A2 is I-Ak-restricted T cell hybridoma specific for the hen egg lysozyme peptide HEL(aa34-45).
  • IL-2 titers were determined using HT-2 cells as previously described (36).
  • ⁇ Kal-68.4 is an autoreactive I-Ak-specific T cell hybridoma.
  • a two-color flow cytometric analysis of apoptotic resting B-cells was performed. AKR resting B-cells and Kal-68.4 T hybridomas cells were incubated overnight. Cells were stained with biotin-conjugated mAb directed against B7- 1 , B7-2, or Fas followed by PE-streptavidin, and apoptotic cells were detected using TdT/dUTP-FITC. The contour plots generated showed labeling with dUTP-FITC (as a measure of apoptotic death) versus counter-staining with the indicated mAb for B-cells harvested from culture with T cells. The percentages indicate the relative number of cells in the viable (dUTP-FITC dull) and apoptotic (dUTP-FITC bright) populations.
  • Two-color analysis reveals that the B-cells from these cultures may be divided into viable (deoxyuridine-FITC low) and apoptotic (deoxyuridine-FITC high) populations with apparent differential expression of the counter-stained receptors on the two populations. Histograms of fluorescence intensity of the stained receptors show that Fas and especially B7-1 are upregulated on the apoptotic population whereas B7-2 is expressed at higher levels on the viable population.
  • UCP expression in L 1210 and L 1210/DDP cells was measured in response to staurosporin and PMA.
  • LI 210 cells expressed the lowest levels of UCP (approximately 1 10 Geo MFI over background).
  • UCP levels were increased in staurosporin (approximately 175 Geo MFI over background) and PMA (approximately 175 Geo MFI over background) treated cells.
  • XIA approximately 190 Geo MFI over background
  • XIE approximately 240 Geo MFI over background
  • UCP levels in L1210/DDP cells were also significantly higher than L1210 cells (approximately 300 Geo MFI over background
  • Example 4 Involvement of IA versus IE in resistance and susceptibility to immune-mediated cardiovascular disease
  • transgenic mice were graciously provided by Dr. Chella David of Mayo Clinic. Dr. David supplied the following strains: 1) AB° mice lack MHC class II I A and IE molecules (class II knockout (KO) mice); 2) AB° Ea b are MHC class II KO mice which have a functional transgenic IE chain, so that the animal express IE but not IA; 3) Bl.Tg.Ea b mice express the wild type IA molecules as well as the IE molecules; and 4) wild type C57B16 express IA only.
  • KO class II knockout mice
  • AB° Ea b are MHC class II KO mice which have a functional transgenic IE chain, so that the animal express IE but not IA
  • Bl.Tg.Ea b mice express the wild type IA molecules as well as the IE molecules
  • wild type C57B16 express IA only.
  • mice Male mice, 4-5 weeks of age were injected ip with 100 ⁇ g GL3-3A (anti- ⁇ ) monoclonal antibody in 0.5 ml PBS, or PBS alone on days -2 and +2 relative to virus. Animals received 10 4 PFU CVB3 on day 0 and surviving animals were euthanized on day 7. Hearts were removed from animals between days 5 and 7 for analysis. Hearts were divided and the apex was formalin fixed, sectioned and evaluated by image analysis for percent of the myocardium affected. The remaining tissue was titered by plaque forming assay for virus. Groups consisted of 4 mice each. The results are summarized in Table 6 below.
  • mice expressing either no class II MHC antigen or IA only were myocarditis resistant having little or no cardiac inflammation and no animal mortality.
  • IE-bearing mice showed increased mortality accompanied by substantial myocardial necrosis.
  • AB° E ⁇ mice, expressing IE only began dying earlier (day 3 post-infection) and had more extensive coaggulative myocardial necrosis with limited cardiac inflammation compared to Bl.Tg.E ⁇ mice (both IA+ and IE+).
  • Cardiac lesions in Bl.Tg.E ⁇ mice were confined to regions of mononuclear cell infiltration and were characterized by extensive myocyte dropout.
  • Viral titers also differed between mouse strains with the highest titers occurring in AB° and AB° E ⁇ mice. This suggests that IA expression is important in virus clearance. Also, the elevated viral titers in AB° E ⁇ mice must not be directly responsible for the necrotic heart lesions in this strain since AB° mice also have elevated virus concentrations but no histological evidence of cardiac injury. Thus, by either animal mortality or histology, the presence of IE in C57BL/6 mice aggravated CVB3-induced disease. Treating the BL tg E ⁇ k strain with antibodies to deplete ⁇ T cells conferred resistance to myocarditis. We measured cytokine profiles from total splenocytes of the animals before and after infection.
  • Example 5 MHC class II IE molecules confer protection from early atherosclerotic fatty lesions Several studies suggest that CD4 + T lymphocytes contribute to the pathogenesis of fat-induced atherosclerotic lesions (Emeson, EE, et al., Am J Path, 1996, 149:675-685). We addressed the possibility that expression of IA and/or IE impacted the development of lesions which result from a high fat diet.
  • C57BL/6 transgenic mice differing in MHC class II antigen expression were kindly supplied by Dr. Chella David (Mayo Clinic). Between 4 and 10 mice of each strain were placed on high-fat, high-cholesterol diet (Teklad #96354;20% total fat, 1.5% cholesterol, 0.5 % sodium cholate) at three weeks of age and were killed 15 weeks later for evaluation of the aorta and splenocytes. An additional group of 7 C57BL/6 mice were placed on high-fat diet as above, but were injected ip every two weeks with 100 ⁇ g monoclonal rat anti-CD4 antibody (clone GK1.5; American Type Tissue Collection, Bethesda, MD).
  • MHC class II IE molecules confer protection from early atherosclerotic fatty lesions
  • Example 6 NGF and EGF-dependent changes in Fas (CD95), B7.1 (CD80) and B7.2 (CD86) expression on PC 12, TrkA, and v-Crk neuronal cell surface.
  • Rat pheochromocytoma cell lines including PC 12, Trk and v-Crk cells were a kind gift from Dr. Raymond Birge. They were maintained in complete RPMI 1640 (GIBCO) supplemented with 7% heat inactivated fetal calf serum and 3% heat inactivated horse serum at 37° C in a humidified incubator with 5% CO2.
  • the PC12 transfectants were generated as described previously (Glassman et al., 1997, Hempstead et al., 1994). Cells were plated in 6 well culture plates at a concentration ranging from 2-8 x 106 cells/well, according to the cell type's growth kinetics, and 5 ml complete medium. NGF-7S from mouse submaxillary glands (Sigma Chemical Co.) or EGF, kindly provided by Dr. Raymond Birge, was added at a concentration of 50 ng/ml culture medium and the cells were incubated for 24 or 48 hours.
  • FITC fluorescein isothiocyanate
  • FITC anti-mouse Fas FITC anti-mouse CD80
  • PE phycoerythrin
  • B7.2 PE anti-mouse CD86
  • Fas cell surface expression after NGF stimulation The data showed that Fas is constitutively expressed on the surface of PC 12 and TrkA cells. NGF stimulation abrogates Fas expression on PC 12 cells and paradoxically increases its expression on Trk cells at 24 hours. Fas levels tend to return to basal values on PC 12 cells and are maintained constant on TrkA cells after 48 hours of NGF stimulation. V-Crk constitutive cell surface Fas expression is minimal but statistically significant, and it totally abrogated after NGF stimulation at 48 hours. Fas cell surface expression after EGF stimulation: EGF stimulation at 24 and 48 hours also downregulates Fas expression on PC 12 and Trk cells; EGF stimulation at 48 hours totally abrogates Fas expression on both cell types. On the other hand, EGF stimulation at 24 hours significantly up-regulates Fas expression on v-Crk cells, but it is again down-regulated and abrogated by EGF stimulation at 48 hours.
  • B7.1 cell surface expression after NGF stimulation B7.1 is constitutively highly expressed on the surface of unstimulated PC 12 and Trk cells. Its expression is minimal on unstimulated v-Crk cells. NGF stimulation initially downregulates B7.1 expression on PC 12 cells at 24 hours, but it tends to return to basal values at 48 hours. NGF stimulation has no effect on B7.1 expression on the surface of Trk cells and v-crk cells.
  • EGF stimulation at 24 hours significantly lowers the detection of high B7.1 levels on the surface of PC 12 cells; EGF stimulation at 48 hours reestablishes the basal values. As in PC 12 cells, B7.1 expression is lowered after EGF stimulation of Trk cells at 24 hours and reestablished at the 48 hour time point. EGF stimulation significantly increases B7.1 expression on v-Crk cells after 24 and 48 hours of culture.
  • B7.2 cell surface expression after NGF stimulation B7.2 cell surface expression is minimal on unstimulated PC 12 cells. NGF stimulation at 24 hours has no effect on its expression but stimulation at 48 hours completely abrogates its expression. Trk B7.2 cell surface expression is also minimal on unstimulated cells, it is slightly down-regulated by NGF stimulation at 24 hours and abrogated by NGF withdrawal. There is no B7.2 cell surface expression on v-Crk cells nor is it induced by NGF stimulation.
  • B7.2 cell surface expression after EGF stimulation EGF stimulation at 48 hours up- regulates B7.2 expression on PC 12 but this is most significant on the surface of Trk and v-crk cells.
  • NGF and EGF receptor pathways appear to be very similar since they both activate the receptor- type tyrosine kineses, the Erk2/MAPK pathway and involve the Ras proteins (Chao, 1992; Ray , Id Menendez Iglesias et al., 1997) It has been found, according to the invention, however, that the effects of these molecules are quite different on the induction and abrogation of Fas, B7.1 and B7.2 expression on the neuronal cell surface suggesting that the induction of these molecules by growth factors NGF and EGF is mediated by a different intra-cellular signaling pathways albeit dependent on tyrosine kinase activation.
  • Fas B7.1 and B7.2 are constitutively expressed on PC 12 and TrkA cells maintained in culture. Fas expression on PC 12 cells is significantly decreased by NGF stimulation (NGFS). Early NGFS of TrkA cells induces an increase in Fas expression over basal levels.
  • Microglia constitutively express Fas ligand (Bonetti and Raine, 1997; Menendez Iglesias et al., 1997) and perhaps direct cell-cell contact between the neurons and microglia is required for the interaction of Fas and Fas ligand and the development of apoptosis or a co-stimulatory mitotic signal.
  • NGF-induced Fas expression can promote cell division as NGF stimulates mitosis at that time period and synchronously play a role in the differentiation process and the development of filopodia.
  • the higher induction of Fas expression correlates with the increased numbers of tyrosine kinase A surface receptors and thus, the development of a sustained increased stimulus for the mRNA translation of the Fas protein and its secondary expression on the cell surface.
  • EGF stimulation significantly diminishes and even abrogates Fas expression on PC 12 and TrkA cells.
  • its expression increases three-fold in v-Crk cells transiently after EGFS at 24 hours and disappears after EGFS at 48 hours.
  • EGFS induces the development of neurite processes on the PC 12 v-crk mutant not on native PC 12 cells (Hempstead et al., 1994) and this clearly correlates with the induction of Fas on the v-Crk membrane cell surface, but does not explain the down-regulation of Fas observed in Trk and PC 12 cells.
  • EGF receptors are also expressed on cortical neurons, the cerebellum and hippocampus, and appear to act on mitotic cells and postmitotic neurons (Yamada et al., 1997).
  • EGFS at 48 hours significantly increases B7.1 expression on all cell variants but its expression clearly decreases after EGFW.
  • EGFS at 48 hours also significantly increase B7.2 expression on PC 12 and Trk cells and induces its expression on v-Crk cells.
  • EGFW at 24 and 48 hours correlates with B7.2 down-regulation in PC 12 and TrkA cells and paradoxically increases B7.2 expression no v-Crk cells at 24 hours; it is immediately down-regulated after EGFW at 48 hours. Therefore, Fas expression on v-Crk cells and B7.1-B7.2 expression on all cell types, but particularly on v-Crk cells, appear to be EGF-dependent.
  • V-Crk cells are characterized by the presence of a fusion protein with viral gag sequences fused to the cellular sequences of the Src homology regions 2 and 3 (SH2 and SH3) (Hempstead et al., 1994).
  • C-src also possesses tyrosine kinase activity (Vaingankar and Martins-Green, 1998) and perhaps these modifications in its sequence allow it to act similarly to the EGF receptor per se and increase the signal intensity for the expression of these cell surface molecules. Withdrawal of the stimulus (EGFW) reverts the expression of these molecules towards basal levels.
  • Ganglia are removed from Po (one-day old mice) brain and plated into cultures. The sensory neurons do not have to be separatedaway from Schwann cells. Isolated ganglia are cultured for at least 72 hours under the following conditions: 1) No Treatment
  • the cells are cultured as described above but in the presence of CTLA-4-HuIg to inhibit cell interactions (synapses) which will protect Group I from death. This shows that B7-bearing cells cause the CD28+ or CTLA4+ cell to release NGF and promote innervation. Additionally histological sections are stained by immunofluorescence (using the anti B7 and TrkA antibodies) immediately ex vivo intact mouse brain.
  • Example 7 is present in a panel of Tumor Cells
  • the L929 cells are fibroblasts and the PC 12 Trk cells which are derived from pheochromocytoma cell lines, respectively.
  • the EL4 cells are a mouse thymoma cell line and Jurkat are human T cell tumor cells.
  • Example 8 Rates of Glucose Utilization, oxidation and Cell surface and IntracellularFas levels in Melanoma Cells.
  • B 16 cells were cultured in the in the presence of different concentrations of sodium acetate and Fas expression was measured. The data shows the level of cell surface Fas expression on non-permeabilized and intracellular Fas expression in permeabilized B16 melanoma cells. With increasing concentrations of sodium acetate, the levels of intracellular Fas declined and the levels of cell surface Fas increased, demonstrating a translocation of Fas from intracellular stores to the surface.
  • CD4 + T cells 5xl0 6 /ml
  • biotinylated antibodies for CD4 GK1.5
  • CD28 CD3
  • 145.2C11 CD4 and CD28
  • CD3 and CD28 or no treatment.
  • Experimental setup included wells of purified T cells and percoll isolated B cells added to control for potential MHC Class IP contaminants.
  • B cells, 5x10 5 cells, which is 5% of purified T cells (far greater than the 1.5% contaminants seen following Cellect Column purification) were added to T cell wells. Cells were then washed, collected and total RNA was isolated using an RNA isolation kit, RNEasy (Qiagen, Chatsworth CA).
  • Example 12 Sodium Acetate as a mitochondrial modifying agent.
  • LI 210 or L1210DDP cells were cultured in the presence of graded concentrations of sodium acetate in the medium. Cells were stained with Jo2.2, a fluorescein conjugated anti-Fas antibody, or an isotype control. Cell surface staining was measured flow cytometrically. The Percentage of mean fluorescence intensity over the isotype control was plotted. The data indicate that the presence of acetate increases cell surface Fas expression in both cell lines.

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Abstract

La présente invention concerne des méthodes de régulation de la croissance et de la division cellulaires qui permettent de maîtriser des processus morbides par manipulation du métabolisme mitochondrial et par expression des protéines immunitaires de la surface cellulaire. L'invention concerne également des compositions et des essais de criblage connexes.
PCT/US1999/006874 1998-04-17 1999-03-30 Methodes et produits concernant les interactions metaboliques dans les maladies WO1999053953A2 (fr)

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JP2002512203A (ja) 2002-04-23
AU768656B2 (en) 2003-12-18
WO1999053953A3 (fr) 2000-01-13

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