WO1999051247A1 - Use of human mesenchymal stem cells to induce t-cell apoptosis - Google Patents

Use of human mesenchymal stem cells to induce t-cell apoptosis Download PDF

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Publication number
WO1999051247A1
WO1999051247A1 PCT/US1999/005349 US9905349W WO9951247A1 WO 1999051247 A1 WO1999051247 A1 WO 1999051247A1 US 9905349 W US9905349 W US 9905349W WO 9951247 A1 WO9951247 A1 WO 9951247A1
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cells
mesenchymal stem
stem cells
cell
antigen
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PCT/US1999/005349
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French (fr)
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Joseph D. Mosca
Kevin R. Mcintosh
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Osiris Therapeutics, Inc.
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Priority to AU29041/99A priority Critical patent/AU2904199A/en
Publication of WO1999051247A1 publication Critical patent/WO1999051247A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0081—Purging biological preparations of unwanted cells
    • C12N5/0087—Purging against subsets of blood cells, e.g. purging alloreactive T cells
    • 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
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • 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
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/001—Preparations to induce tolerance to non-self, e.g. prior to transplantation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4637—Other peptides or polypeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46443—Growth factors
    • A61K39/464433—Platelet-derived growth factor [PDGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to the field of inducing death of specific T- lymphocyte cells which are deleterious to an organism.
  • the present invention relates particularly to the area of autoimmune disease in humans.
  • T cell tolerance is achieved 1) in the thymus where thymocytes reactive for self-peptides are eliminated by clonal deletion (central tolerance), and 2) in the periphery by exposure to self-antigens under tolerogenic conditions (peripheral tolerance). Peripheral tolerance is manifested by clonal anergy, and by clonal deletion where autoreactive cells are elirninated.
  • Clonal deletion can also result from expression of cell death molecules on the antigen presenting cells.
  • Classic examples of death molecules are Fas ligand
  • FasL FasL
  • TRAIL ligand which ligate their receptors, Fas and DR4, respectively, on activated T cells, inducing apoptosis of the T cells.
  • CD27 a member of the TNFR superfamily
  • CD27-ligand CD70
  • the immune system may generate a response against self- constituents, as happens in autoimmune disease.
  • Autoimmune disease wherein antibodies or T cells attack self proteins, may be caused by abnormal immune response.
  • the cause may be an autoreactive T cell component, the T cells may themselves be pathogenic, or T cells may help trigger autoreactive B cells to produce antibodies to self antigens.
  • Patients with autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease and myasthenia gravis, are either inadequately treated with existing non-selective drug therapies, or experience deleterious side effects from long-term immunosuppressive treatment.
  • human mesenchymal stem cells can be used to deliver antigens to the immune system for interaction with T cells.
  • Mesenchymal stem cells can further be used to present to the immune system molecules that induce apoptotic death in cells of the immune system that express receptors for the molecules.
  • the methods of the present invention are particularly useful for eliminating, reducing or ameliorating unwanted or activated T cell responses and can be used as a method to treat or inhibit specific unwanted or abnormal immune responses such as occurs in autoimmune disease.
  • the method involves reducing, ameliorating or eliminating T cells that have been activated against an antigen by ad ⁇ unistering to a subject autologous human mesenchymal stem cells which have been modified to present such antigen, and to express a molecule that induces apoptosis of activated T cells.
  • the mesenchymal stem cells can be used to deliver to the immune system a molecule that induces apoptosis of activated T cells since activated T cells carry a receptor for the molecule. This results in the deletion of activated T lymphocytes and in the suppression of an unwanted immune response.
  • autologous human mesenchymal stem cells are modified to express a cell death molecule.
  • the mesenchymal stem cells express the cell death molecule Fas ligand which will interact with the Fas receptor found on activated T cells.
  • the method of the present invention provides administering to a host a human mesenchymal stem cell that (i) has been modified to have at least one exogenous antigen fragment bound to a primary surface molecule of the cell such that the antigen fragment is presented to the immune system, and (ii) has been modified to express a cell death molecule.
  • the mesenchymal stem cell presents the antigen and thereby interacts with T cells that have previously been activated.
  • the mesenchymal stem cells of the invention further contain exogenous genetic material that codes for a molecule that induces activated T cell apoptosis.
  • the exogenous genetic materials are in one or more expression vectors.
  • the mesenchymal stem cells are modified to deliver to the immune system a molecule that induces activated T cell elimination.
  • the mesenchymal stem cells which may be allogeneic to the host, are modified to express a cell death molecule such as Fas ligand or TRAIL.
  • a cell death molecule such as Fas ligand or TRAIL.
  • the invention relates to methods for reducing, inhibiting or elin inating an immune response to an antigen, in vivo, by employing human mesenchymal stem cells to present antigen and to simultaneously present "a cell death molecule", a molecule that induces immune cell apoptosis.
  • the administration of these modified mesenchymal stem cells results in the deletion of activated T cells and thus a reduction of the T cell response.
  • the human mesenchymal stem cells are preferably autologous to the recipient of the human mesenchymal stem cells.
  • the invention relates to a method of eliniinating activated T cells by administering, in vivo, mesenchymal stem cells which deliver a specific antigen to T cells, and in addition are modified to express a cell death molecule.
  • the present invention is based in part on the discovery mat human mesenchymal stem cells do not provide costimulatory signals to fully stimulate T cells. Therefore, when antigen bearing mesenchymal stem cells are present in the immune system, the mesenchymal stem cells present the antigen without providing the costimulatory signal required for T cell activation.
  • the mesenchymal stem cells are modified to present antigen to T cells by contacting the mesenchymal cells with antigen, in vitro, prior to contact with the T cells.
  • the antigen can be a protein, a polypeptide, lipid or glycoprotein bound to a primary surface molecule of the cell.
  • the mesenchymal stem cell is contacted with at least one antigen (antigen-pulsing) which the mesenchymal stem cell processes into an antigen fragment.
  • the mesenchymal stem cells can alternatively be genetically manipulated to express an antigenic molecule.
  • the mesenchymal stem cell contains exogenous genetic material that codes for at least one exogenous antigenic polypeptide, which the mesenchymal stem cell expresses, processes into an antigen fragment and presents to the T cells.
  • the mesenchymal stem cells are modified to present an autoantigen, for example, an autoantigen that mediates the immune response in an autoirnmune disease.
  • the mesenchymal stem cells are preferably autologous to the recipient of the mesenchymal stem cells. This method can be used to reduce or inhibit a T cell immune response involved in autoimmune disease, for example, rheumatoid arthritis, systemic lupus erythematosus, iriflammatory bowel disease and myastiienia gravis.
  • autoimmune disease for example, rheumatoid arthritis, systemic lupus erythematosus, iriflammatory bowel disease and myastiariaa gravis.
  • the mesenchymal stem cells are also modified to express a molecule that will induce T cell apoptosis, i.e., a cell death molecule.
  • a cell death molecule is a molecule that interacts or binds with its cognate receptor on an activated T cell, the interaction inducing T cell death or apoptosis.
  • the Fas system has been shown to be involved in a number of cell functions in vivo including negative selection of thymocytes, mamtaining immune privilege sites within the body, and cytotoxic T-lymphocyte (CTL)-mediated cytotoxicity (Green and Ware, PNAS 1997).
  • CTL cytotoxic T-lymphocyte
  • TRAIL receptor interacts with TRAIL ligand which can induce apoptosis in a variety of transformed cell lines (G. Pan SCIENCE, 1997); and the interaction of CD27 and its ligand CD70 (Prasad et al, PNAS 1997) also induces apoptosis.
  • FasL expression is restricted to stimulated T cells and cites of immune privileges, TRAIL is detected in many normal tissues.
  • Both TRAIL-ligand and CD70, but not Fas-ligand are expressed on unmanipulated human mesenchymal stem cells. Activated, but not resting, T cells express the TRAIL receptor and CD27.
  • the mesenchymal stem cells can be induced to express an endogenous cell death molecule or can be genetically engineered to express exogenous molecules that cause cell death.
  • the mesenchymal stem cells which present an antigen to which T cells have been previously activated cause the T cells to be drawn to such mesenchymal stem cells.
  • the activated T cells express either TRALL-receptor, Fas or CD27 on the T cell.
  • TRALL-receptor TRALL-receptor
  • Fas Fas
  • CD27 CD27 on the T cell.
  • Other ligands either present within the mesenchymal stem cell or introduced into the mesenchymal stem cell can bind to their cognate receptors on the activated T cells to induce apoptosis.
  • mesenchymal stem cells administered to an individual can delete autoreactive cells, reducing the severity or incidence of autoimmune disease.
  • An advantage of the method of the present invention over current treatment for autoimmune disease is specificity; mesenchymal stem cells can be targeted to reduce a specific immune response while reducing or eliminating the effect on other segments of the immune system.
  • the elimination of an antigen specific immune response enables the treatment of or prevention of an unwanted or abnormal hnmune response to a specific antigen.
  • the methods of the present invention are particularly applicable to therapy of autoimmune disease and preferably el minate the response to autoantigen specifically, while reducing or el ⁇ riinating the effect on other aspects of the immune system.
  • the invention can be utilized for treatment of autoimmune diseases where the autoantigen mediating the disease is known.
  • the method involves genetically engineering mesenchymal stem cells, to express an autoantigen in order to induce specific immunotherapy to inactivate or elirninate abnormal immune responses.
  • the invention encompasses administering the mesenchymal stem cells to a host as a method for the treatment of autoimmune diseases such as myasthenia gravis or rheumatoid arthritis.
  • the mesenchymal stem cells are modified to express a molecule that will induce T cell apoptosis, without modification to present an antigen against which the T cells have been activated.
  • the mesenchymal stem cells thus modified will have a nonspecific effect on the immune system, i.e., will eliminate activated T cells at or near the site of administration of the mesenchymal stem cells. Upon contact with the mesenchymal stem cells, apoptosis of the activated T cells will be induced.
  • Mesenchymal stem cells are the formative pluripotential blast cells found, inter alia, in bone marrow, blood, dermis and periosteum. These cells can be expanded in culture, for example by methods described for isolating, purifying, and greatly replicating these cells in culture, i.e., in vitro, in Caplan and Haynesworth, U.S. Patent No. 5,486,359.
  • the human mesenchymal stem cells of the invention can be engineered
  • the engineered human mesenchymal stem cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying exogenous genes therein.
  • the culture conditions such as temperature, pH and the like, can be those previously used with engineered human mesenchymal stem cells. See, for example, Gerson et al, U.S. Patent No. 5,591,625.
  • the mesenchymal stem cells and method of the invention can be appropriately applied to treatment strategies requiring immunosuppressive reagents. Accordingly, the present invention provides for the modification of and expansion of mesenchymal stem cells in vitro for use in cellular immunotherapy, and the in vivo adrninistration of the immunosuppressive mesenchymal stem cells for treating or ameliorating unwanted immune responses.
  • One aspect of the invention is the development of the mesenchymal stem cells into a vehicle for presenting antigen and delivering a cell death molecule for eliminating a specific cellular response.
  • the dosage of the active ingredient varies within wide limits and will, of course be fitted to the individual requirements in each particular case.
  • it is customary to adniinister from about 0.5 to about 5 million cells per kilogram of recipient body weight.
  • the number of cells used will depend on the weight and condition of the recipient and other variables known to those of skill in the art.
  • the cells can be adrninistered by a route which is suitable for the particular disease state to be treated.
  • the antigen-modified mesenchymal stem cells can be targeted to a particular tissue or organ such as bone marrow.
  • the cells can be suspended in an appropriate diluent, at a concentration of from about 5 x 10 6 to about 50 x 10 6 cells/ ml.
  • Suitable excipients for injection solutions are those that are biologically and physiologically compatible with the recipient, such as buffered saline solution.
  • the composition for administration should be sterile, stable and physiological acceptable. It is contemplated that the mesenchymal stem cells of the present invention can be used in conjunction with current modes of treating autoimmune disease. By ameliorating the severity of the immune response in autoirnmune disease, the amount of drug used in treatment and/or the frequency of adrninistration of drug therapy can be reduced, resulting in alleviation of general irnmune suppression and unwanted side effects.

Abstract

The present invention provides a method for inducing antigen-specific T-cell lymphocyte elimination using human mesenchymal stem cells as antigen presenting cells which additionally express a molecule that induces T-cell apoptosis.

Description

USE OF HUMAN MESENCHYMAL STEM CELLS TO INDUCE T-CELL APOPTOSIS
This application is based on and claims priority of U.S. provisional application serial no. 60/080,533 filed April 3, 1998.
The present invention relates to the field of inducing death of specific T- lymphocyte cells which are deleterious to an organism. The present invention relates particularly to the area of autoimmune disease in humans.
Background of the Invention The function of the immune system is to eliminate foreign cells which may contain pathogens, while mamtaining unresponsiveness or "tolerance" against self- antigens. In a normal immune response, activation of naive T-cells requires recognition of a foreign antigenic fragment bound to a self MHC molecule and the simultaneous delivery of a co-stimulatory signal by a specialized antigen-presenting cell. T cell tolerance is achieved 1) in the thymus where thymocytes reactive for self-peptides are eliminated by clonal deletion (central tolerance), and 2) in the periphery by exposure to self-antigens under tolerogenic conditions (peripheral tolerance). Peripheral tolerance is manifested by clonal anergy, and by clonal deletion where autoreactive cells are elirninated.
Clonal deletion can also result from expression of cell death molecules on the antigen presenting cells. Classic examples of death molecules are Fas ligand
(FasL) and TRAIL ligand, which ligate their receptors, Fas and DR4, respectively, on activated T cells, inducing apoptosis of the T cells. The interaction of CD27, a member of the TNFR superfamily, and the CD27-ligand (CD70) also induces T cell apoptosis.
However, the immune system may generate a response against self- constituents, as happens in autoimmune disease. Autoimmune disease, wherein antibodies or T cells attack self proteins, may be caused by abnormal immune response. The cause may be an autoreactive T cell component, the T cells may themselves be pathogenic, or T cells may help trigger autoreactive B cells to produce antibodies to self antigens. Patients with autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease and myasthenia gravis, are either inadequately treated with existing non-selective drug therapies, or experience deleterious side effects from long-term immunosuppressive treatment.
Infusion of individuals with drugs that prevent T-cell activation can inhibit immune cell response, but these treatments result in general immune suppression, toxicity and sometimes death due to opportunistic infections. Because of the toxicity and incomplete response rate of conventional treatment of autoimmune diseases, alternative approaches are needed for patients who cannot withstand or do not respond to drug therapy.
Summary of the Invention
It has been discovered that human mesenchymal stem cells can be used to deliver antigens to the immune system for interaction with T cells. Mesenchymal stem cells can further be used to present to the immune system molecules that induce apoptotic death in cells of the immune system that express receptors for the molecules.
Accordingly, the methods of the present invention are particularly useful for eliminating, reducing or ameliorating unwanted or activated T cell responses and can be used as a method to treat or inhibit specific unwanted or abnormal immune responses such as occurs in autoimmune disease.
In one aspect the method involves reducing, ameliorating or eliminating T cells that have been activated against an antigen by adπunistering to a subject autologous human mesenchymal stem cells which have been modified to present such antigen, and to express a molecule that induces apoptosis of activated T cells. The mesenchymal stem cells can be used to deliver to the immune system a molecule that induces apoptosis of activated T cells since activated T cells carry a receptor for the molecule. This results in the deletion of activated T lymphocytes and in the suppression of an unwanted immune response. In accordance with an aspect of the invention, autologous human mesenchymal stem cells are modified to express a cell death molecule. In a preferred embodiment, the mesenchymal stem cells express the cell death molecule Fas ligand which will interact with the Fas receptor found on activated T cells.
Thus, the method of the present invention provides administering to a host a human mesenchymal stem cell that (i) has been modified to have at least one exogenous antigen fragment bound to a primary surface molecule of the cell such that the antigen fragment is presented to the immune system, and (ii) has been modified to express a cell death molecule. The mesenchymal stem cell presents the antigen and thereby interacts with T cells that have previously been activated. The mesenchymal stem cells of the invention further contain exogenous genetic material that codes for a molecule that induces activated T cell apoptosis. Preferably, the exogenous genetic materials are in one or more expression vectors.
In another aspect, the mesenchymal stem cells are modified to deliver to the immune system a molecule that induces activated T cell elimination. The mesenchymal stem cells, which may be allogeneic to the host, are modified to express a cell death molecule such as Fas ligand or TRAIL. When the mesenchymal stem cell comes into contact with an activated T cell, apoptosis of the activated T cell will be induced. The mesenchymal stem cell-antigen presentation system described herein has a wide range of applications, including but not limited to, deletion of large numbers of antigen-specific T cells for use in immunotherapy against, inter alia, autoirnmune disease.
Detailed Description of Preferred Embodiments The invention relates to methods for reducing, inhibiting or elin inating an immune response to an antigen, in vivo, by employing human mesenchymal stem cells to present antigen and to simultaneously present "a cell death molecule", a molecule that induces immune cell apoptosis. The administration of these modified mesenchymal stem cells results in the deletion of activated T cells and thus a reduction of the T cell response. The human mesenchymal stem cells are preferably autologous to the recipient of the human mesenchymal stem cells.
Accordingly, the invention relates to a method of eliniinating activated T cells by administering, in vivo, mesenchymal stem cells which deliver a specific antigen to T cells, and in addition are modified to express a cell death molecule. The present invention is based in part on the discovery mat human mesenchymal stem cells do not provide costimulatory signals to fully stimulate T cells. Therefore, when antigen bearing mesenchymal stem cells are present in the immune system, the mesenchymal stem cells present the antigen without providing the costimulatory signal required for T cell activation.
In one embodiment of the invention, the mesenchymal stem cells are modified to present antigen to T cells by contacting the mesenchymal cells with antigen, in vitro, prior to contact with the T cells. For human mesenchymal stem cells modified to have at least one exogenous antigen fragment, the antigen can be a protein, a polypeptide, lipid or glycoprotein bound to a primary surface molecule of the cell. Thus, in this embodiment, the mesenchymal stem cell is contacted with at least one antigen (antigen-pulsing) which the mesenchymal stem cell processes into an antigen fragment. The mesenchymal stem cells can alternatively be genetically manipulated to express an antigenic molecule. Thus, in another embodiment, the mesenchymal stem cell contains exogenous genetic material that codes for at least one exogenous antigenic polypeptide, which the mesenchymal stem cell expresses, processes into an antigen fragment and presents to the T cells.
In a preferred embodiment of this aspect of the invention, the mesenchymal stem cells are modified to present an autoantigen, for example, an autoantigen that mediates the immune response in an autoirnmune disease. In accordance with this aspect, the mesenchymal stem cells are preferably autologous to the recipient of the mesenchymal stem cells. This method can be used to reduce or inhibit a T cell immune response involved in autoimmune disease, for example, rheumatoid arthritis, systemic lupus erythematosus, iriflammatory bowel disease and myastiienia gravis. By using mesenchymal stem cells that present the autoantigen against which the T cells have been activated, such activated T cells will recognize the presented antigen.
In accordance with this embodiment of the invention, the mesenchymal stem cells are also modified to express a molecule that will induce T cell apoptosis, i.e., a cell death molecule. As defined herein a "cell death molecule" is a molecule that interacts or binds with its cognate receptor on an activated T cell, the interaction inducing T cell death or apoptosis.
Fas mediates apoptosis of recently activated T cells which are again exposed to stimulation (Parijs, et al, 1996). Fas is a type I membrane receptor that, when crosslinked by its cognate ligand, induces apoptosis in a wide variety of cells. The interaction between the Fas molecule (CD95) on target cells and its ligand Fas L on activated T cells results in receptor aggregation, which transduces signals leading to apoptosis of the target cell. The Fas system has been shown to be involved in a number of cell functions in vivo including negative selection of thymocytes, mamtaining immune privilege sites within the body, and cytotoxic T-lymphocyte (CTL)-mediated cytotoxicity (Green and Ware, PNAS 1997).
Other members of the tumor necrosis factor receptor (TNFR) family have roles in programmed cell death: DR4 TRAIL receptor interacts with TRAIL ligand which can induce apoptosis in a variety of transformed cell lines (G. Pan SCIENCE, 1997); and the interaction of CD27 and its ligand CD70 (Prasad et al, PNAS 1997) also induces apoptosis. Whereas FasL expression is restricted to stimulated T cells and cites of immune privileges, TRAIL is detected in many normal tissues. Both TRAIL-ligand and CD70, but not Fas-ligand, are expressed on unmanipulated human mesenchymal stem cells. Activated, but not resting, T cells express the TRAIL receptor and CD27. Thus, in accordance with the present invention, the mesenchymal stem cells can be induced to express an endogenous cell death molecule or can be genetically engineered to express exogenous molecules that cause cell death.
It is believed that the mesenchymal stem cells which present an antigen to which T cells have been previously activated cause the T cells to be drawn to such mesenchymal stem cells. The activated T cells express either TRALL-receptor, Fas or CD27 on the T cell. The engagement of these receptors with their ligands on the mesenchymal stem cells results in T cell death via apoptosis. Other ligands either present within the mesenchymal stem cell or introduced into the mesenchymal stem cell can bind to their cognate receptors on the activated T cells to induce apoptosis. In this manner, mesenchymal stem cells administered to an individual can delete autoreactive cells, reducing the severity or incidence of autoimmune disease.
An advantage of the method of the present invention over current treatment for autoimmune disease is specificity; mesenchymal stem cells can be targeted to reduce a specific immune response while reducing or eliminating the effect on other segments of the immune system. The elimination of an antigen specific immune response enables the treatment of or prevention of an unwanted or abnormal hnmune response to a specific antigen. The methods of the present invention are particularly applicable to therapy of autoimmune disease and preferably el minate the response to autoantigen specifically, while reducing or elύriinating the effect on other aspects of the immune system.
The invention can be utilized for treatment of autoimmune diseases where the autoantigen mediating the disease is known. The method involves genetically engineering mesenchymal stem cells, to express an autoantigen in order to induce specific immunotherapy to inactivate or elirninate abnormal immune responses. Accordingly, the invention encompasses administering the mesenchymal stem cells to a host as a method for the treatment of autoimmune diseases such as myasthenia gravis or rheumatoid arthritis.
In another aspect of the invention, the mesenchymal stem cells are modified to express a molecule that will induce T cell apoptosis, without modification to present an antigen against which the T cells have been activated. The mesenchymal stem cells thus modified will have a nonspecific effect on the immune system, i.e., will eliminate activated T cells at or near the site of administration of the mesenchymal stem cells. Upon contact with the mesenchymal stem cells, apoptosis of the activated T cells will be induced.
Mesenchymal stem cells are the formative pluripotential blast cells found, inter alia, in bone marrow, blood, dermis and periosteum. These cells can be expanded in culture, for example by methods described for isolating, purifying, and greatly replicating these cells in culture, i.e., in vitro, in Caplan and Haynesworth, U.S. Patent No. 5,486,359.
The human mesenchymal stem cells of the invention can be engineered
(transduced or transformed or transfected) with genetic material of interest. The engineered human mesenchymal stem cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying exogenous genes therein. The culture conditions, such as temperature, pH and the like, can be those previously used with engineered human mesenchymal stem cells. See, for example, Gerson et al, U.S. Patent No. 5,591,625.
Unless otherwise stated, genetic manipulations are performed as described in Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL, 2nd
Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).
The mesenchymal stem cells and method of the invention can be appropriately applied to treatment strategies requiring immunosuppressive reagents. Accordingly, the present invention provides for the modification of and expansion of mesenchymal stem cells in vitro for use in cellular immunotherapy, and the in vivo adrninistration of the immunosuppressive mesenchymal stem cells for treating or ameliorating unwanted immune responses. One aspect of the invention is the development of the mesenchymal stem cells into a vehicle for presenting antigen and delivering a cell death molecule for eliminating a specific cellular response.
The dosage of the active ingredient varies within wide limits and will, of course be fitted to the individual requirements in each particular case. In general, in the case of parenteral adniinistration, it is customary to adniinister from about 0.5 to about 5 million cells per kilogram of recipient body weight. The number of cells used will depend on the weight and condition of the recipient and other variables known to those of skill in the art. The cells can be adrninistered by a route which is suitable for the particular disease state to be treated. The antigen-modified mesenchymal stem cells can be targeted to a particular tissue or organ such as bone marrow.
The cells can be suspended in an appropriate diluent, at a concentration of from about 5 x 106 to about 50 x 106 cells/ ml. Suitable excipients for injection solutions are those that are biologically and physiologically compatible with the recipient, such as buffered saline solution. The composition for administration should be sterile, stable and physiological acceptable. It is contemplated that the mesenchymal stem cells of the present invention can be used in conjunction with current modes of treating autoimmune disease. By ameliorating the severity of the immune response in autoirnmune disease, the amount of drug used in treatment and/or the frequency of adrninistration of drug therapy can be reduced, resulting in alleviation of general irnmune suppression and unwanted side effects.

Claims

What Is Claimed Is:
1. A method of eliminating T cells comprising administering to a host a human mesenchymal stem cell which expresses a cell death molecule.
2. The method of claim 1 wherein the cell death molecule is selected from the group consisting of Fas Ligand, TRAIL ligand and CD27 ligand.
3. A method of reducing T cells activated against an antigen, comprising administering to a host human mesenchymal stem cells which present the antigen against which the T cells have been activated, and which express a cell death molecule.
4. The method of claim 3 wherein the antigen is an autoantigen.
5. The method of claim 3 wherein the mesenchymal stem cells are autologous to the host.
6. The method of claim 3 wherein the cell death molecule is selected from the group consisting of Fas Ligand, TRAIL ligand and CD27 ligand.
7. Use of mesenchymal stem cells which express a cell death molecule for the preparation of a composition for eliminating T cells.
8. Use of mesenchymal stem cells which present an antigen and which express a cell death molecule for the preparation of composition for reducing T cells activated against the antigen.
10
PCT/US1999/005349 1998-04-03 1999-03-12 Use of human mesenchymal stem cells to induce t-cell apoptosis WO1999051247A1 (en)

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US9528088B2 (en) 2002-06-28 2016-12-27 Life Technologies Corporation Methods for eliminating at least a substantial portion of a clonal antigen-specific memory T cell subpopulation

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Publication number Priority date Publication date Assignee Title
US9528088B2 (en) 2002-06-28 2016-12-27 Life Technologies Corporation Methods for eliminating at least a substantial portion of a clonal antigen-specific memory T cell subpopulation
EP2824175A3 (en) * 2004-03-22 2015-05-06 Mesoblast International Sàrl Mesenchymal stem cells and uses therefor
US9694035B2 (en) 2004-03-22 2017-07-04 Mesoblast International Sarl Mesenchymal stem cells and uses therefor
EP2298862B1 (en) 2004-03-22 2017-08-30 Mesoblast International Sàrl Mesenchymal stem cells and uses therefor
US9943547B2 (en) 2004-03-22 2018-04-17 Mesoblast International Sàrl Mesenchymal stem cells and uses therefor
EP3461884A1 (en) * 2004-03-22 2019-04-03 Mesoblast International Sàrl Mesenchymal stem cells and uses therefor
US10668101B2 (en) 2004-03-22 2020-06-02 Mesoblast International Sárl Mesenchymal stem cells and uses therefor
US10716814B2 (en) 2004-03-22 2020-07-21 Mesoblast International Sàrl Mesenchymal stem cells and uses therefor
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US11389484B2 (en) 2004-03-22 2022-07-19 Mesoblast International Sárl Mesenchymal stem cells and uses therefor

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