EP1196543A2 - Traitement de cellules dendritiques pour l'induction de la tolerance immunitaire - Google Patents

Traitement de cellules dendritiques pour l'induction de la tolerance immunitaire

Info

Publication number
EP1196543A2
EP1196543A2 EP00942249A EP00942249A EP1196543A2 EP 1196543 A2 EP1196543 A2 EP 1196543A2 EP 00942249 A EP00942249 A EP 00942249A EP 00942249 A EP00942249 A EP 00942249A EP 1196543 A2 EP1196543 A2 EP 1196543A2
Authority
EP
European Patent Office
Prior art keywords
cells
human
agonist
molecule
dendritic cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00942249A
Other languages
German (de)
English (en)
Inventor
David J. Nat. Blood Service Oxford Ctr. ROBERTS
Britta C. Inst. of Molecular Medicine URBAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oxford University Innovation Ltd
Original Assignee
Oxford University Innovation Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oxford University Innovation Ltd filed Critical Oxford University Innovation Ltd
Publication of EP1196543A2 publication Critical patent/EP1196543A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46434—Antigens related to induction of tolerance to non-self
    • 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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464414—CD74, Ii, MHC class II invariant chain or MHC class II gamma chain
    • 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
    • A61K39/464839—Allergens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • C07K14/70557—Integrin beta3-subunit-containing molecules, e.g. CD41, CD51, CD61
    • 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
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2848—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta3-subunit-containing molecules, e.g. CD41, CD51, CD61
    • 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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • C12N5/064—Immunosuppressive dendritic 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
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • 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
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood 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
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/25—Tumour necrosing factors [TNF]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of immune suppression and, in particular, to the identification of molecules which act as agonists of the cell surface receptors CD36, CD51 and thrombospondin receptors expressed on mammalian dendritic cells and other antigen-presenting cells, to ex vivo and in vivo uses of such molecules for inducing peripheral immune tolerance in mammals, to identification of molecules which inhibit the state of immune tolerance induced in a human by the binding of red blood cells infected with the malarial parasite to dendritic cells and to in vivo uses of such molecules in treating malaria. Dysfunction of the immune system has been shown to play a role in the initial development and further progression of many human diseases.
  • Impaired immune function can result in inability to fight infection or to destroy malignant cells as they develop within the body.
  • Other diseases are caused because the immune system mounts an inappropriate response to a particular antigen.
  • This inappropriate response might be to an external antigen resulting in atopic disease such as hay fever, asthma, eczema, coeliac disease and the like or to the body's own antigens resulting in auto-immune disease.
  • both the non-organ specific auto-immune diseases such as systemic lupus erythromatosis and rheumatoid arthritis and the organ specific auto-immune diseases such as auto-immune haemolytic anaemia and idiopathic thrombocytopenic purpura are associated with an inappropriate T-cell response to self ⁇ -antigens .
  • auto-immune diseases where the antigen has been defined include auto-immune connective tissue syndromes, insulin dependent diabetes mellitus and auto-immune thyroid disease.
  • Diseases where the antigen is less well defined include auto-immune skin diseases such as eczema, psoriasis, alopecia areata and vitiligo, auto-immune diseases of the gastrointestinal system such as inflammatory bowel disease and auto-immune hepatitis, auto-immune diseases of the nervous system such as multiple sclerosis and myasthenis gravis and auto-immune diseases of the kidney such as glomerulonephritis .
  • a cellular immune response is mediated by T- lymphocytes which are activated by antigen presenting cells, the most important of which are dendritic cells, which present antigen and activate memory T- cells and naive T-cells.
  • Dendritic cells become potent antigen-presenting cells when exposed to an immune stimulus and thereafter are described as "mature”. Maturation confers enhanced ability to stimulate T-cells and a reduction in pinocytosis and phagocytosis compared with immature cells.
  • maturation is accompanied by enhanced cell surface expression of HLA Class I and class II molecules as well as adhesion molecules, including CD54 and co-stimulatory molecules such as CD80, CD86 and the cell-surface marker CD83.
  • Maturation of dendritic cells is also accompanied by the secretion of cytokines such as TNFoc and IL12p70.
  • the secreted cytokines have an autokrine effect on dendritic cell maturation itself and parakrine effects on interacting T-cells.
  • Immature dendritic cells present the cell surface antigens CD36 and CD51 ( ⁇ v ) (part of the vibronectin receptor v ⁇ 3 ) .
  • CD36 and integrin heterodimers ⁇ v ⁇ 3 or v ⁇ 5 can be cross-linked by the soluble bridging molecule thrombospondin (TSP) .
  • TSP soluble bridging molecule thrombospondin
  • LPS lipopolysaccharide
  • Plasmodi um falciparum is one of the most successful human pathogens for which virulence factors remain poorly defined, although adhesion of infected erythrocytes to venular endothelium has been associated with some of the symptoms of severe disease. Immune responses are unable to prevent symptomatic infections throughout life and immunity to severe disease develops only slowly during childhood. Understanding the obstacles to the development of protective immunity is crucial for rational approaches to prevent the disease.
  • the present inventors have now identified a further mechanism by which the malarial parasite prevents the infected host from mounting an effective immune response and preventing recurrence of the disease.
  • human erythrocytes which are infected with Plasmodium falciparum are capable of adhering to human dendritic cells and that immature dendritic cells exposed to infected erythrocytes are no longer able to mature into full antigen-presenting cells or to stimulate T- cell proliferation, when subsequently exposed to an immune stimulus.
  • this state of immune tolerance is not observed when the dendritic cells are exposed to uninfected erythrocytes, uninfected erythrocyte lysate, infected erythrocyte lysate, parasite-conditioned medium or a crude pigment preparation derived from infected erythrocytes.
  • CD36 and CD51 influence the process of dendritic cell maturation and that agonists thereof, including the malarial parasite derived protein pf-EMP-1, antibodies specific for CD36 and CD51, negatively charged phospholipids and apoptotic cells, are able to inhibit dendritic cell maturation in response to an immune stimulus.
  • agonists reduce the ability of the dendritic cells to stimulate T-cell proliferation in response to an antigen to a level which is lower than cells which have not been exposed to an immune stimulus at all.
  • agonists of CD36 and CD51 can induce a state of immune tolerance.
  • dendritic cells may be treated by CD36 and/or CD51 agonists in vi tro together with an antigen specific to the immune- response manifested in the auto-immune disease in question.
  • tolerance may be induced to a specific antigen so that, when the dendritic cells are reintroduced into the host, further auto-immune reaction is avoided or substantially reduced.
  • CD36 and CD51 agonists are useful for inducing a state of immune tolerance in both host and donor dendritic cells where bone marrow transplantation or lymphocyte infusion is contemplated.
  • the feasibility of such treatment is demonstrated herein in vivo in mice.
  • the ability to inhibit maturation of dendritic cells can be demonstrated in vi tro so that molecules which act as CD36 or CD51 agonists can be easily identified in a high throughput screening assay.
  • agonist means a composition, molecule, cell or a component thereof which induces the same response when interacting with a receptor as the naturally-occurring ligand for that receptor.
  • the invention provides a method of identifying a molecule which is an agonist of cell surface receptor CD36 and/or CD51 as expressed by mammalian dendritic cells which method comprises:
  • impaired maturation in response to the immune stimulus is an indication that said molecule under test is a CD36 and/or CD51 agonist.
  • the method is performed using human dendritic cells.
  • dendritic cells means cells that present antigen to and activate lymphocytes and which are distinguished by their ability to activate, not only memory T-cells but also naive T-cells.
  • Dendritic cells for use in the method of the invention may be derived by cultivation of adherent peripheral blood mononuclear cells with the addition of Granulocyte-Macrophage Stimulating Factor and Interleukin-4 for about 6 to 10 days.
  • Such dendritic cells can be characterised by their level of expression of the cell-surface markers HLA Class I and II (high), CDll c (high), CD3 and CD19 (negative), CD14 (low) and CD86 (high) .
  • the level of expression of the HLA Class I and II molecules and/or adhesion molecules and/or co-stimulatory molecules is measured.
  • maturation of dendritic cells is detected by measurement of the level of expression of two or more of the cell-surface antigens HLA DR, CD54, CD40, CD83 and CD86 whose level of expression is particularly enhanced.
  • the level of expression of all of the above in response to an immune stimulus is measured.
  • the expression level of CD80 may also be measured.
  • Suitable labels are well-known to those skilled in the art and include radioactive labels, enzyme labels, fluorescent labels, metallic particles and the like.
  • Antibodies suitable for carrying out the screening method of the present invention, as well as a commercial source, are shown in Table 1 below:
  • T-cell proliferation As an alternative to measuring the level of cell surface antigen to determine whether or not dendritic cell maturation has occurred, it is possible to measure the cell's ability to induce T-cell proliferation. This is inhibited by agonists of CD36 or CD51.
  • Dendritic cells which have been exposed to the molecule to be tested and to an immune stimulus may be exposed to T-cells, for example allogeneic lymphocytes in a mixed lymphocyte reaction (MLR) with the T-cell receptor.
  • MLR mixed lymphocyte reaction
  • the T-cells respond by growing and dividing, something which can easily be measured using methods well-known to one skilled in the art. For example, growth and division can be assessed visually using a light microscope to observe clumps of dividing cells.
  • cell proliferation can be quantified using a suitably labelled metabolite, for example tritiated thymidine, which is incorporated into the cell's DNA.
  • a yet further alternative for determining the degree of dendritic cell maturation is to measure the level of secretion of cytokines such as TNF ⁇ , IL2p70 or IL10.
  • IL12p70 is secreted by mature cells but not by immature cells.
  • the level of TNF ⁇ secretion is reduced in immature as opposed to mature cells. Kits are commercially available for detection and quantitation of all of the above cytokines. (see Examples) .
  • the levels of TNF ⁇ , IL12p70 and IL10 secretion are measured.
  • lipidopolysaccharide available from
  • CD40L which is expressed from plasmids having the ATCC Accession No's 79812,79813,79814 or 79815.
  • the plasmids may be expressed in mouse fibroblasts STO (ATCC-CRL-1503) .
  • TNF ⁇ would not be used as both immune stimulant and indicator of cell maturation in the same assay.
  • immature dendritic cells (about 10 6 ) are exposed in duplicate to various concentrations of the test molecule for about 3 to about 12 hours in a multiwell plate.
  • the test compound is prepared in a suitable diluent which is not toxic to the dendritic cells such as tissue culture medium, PBS, water or a suitable non-toxic organic solvent, if appropriate.
  • the duplicate wells are subsequently exposed to LPS (about 500 ng/ml) or left untreated for about 48 hours.
  • LPS about 500 ng/ml
  • the surface expression of the molecules identified above is compared with the surface expression on immature dendritic cells exposed to the test compound as well as untreated immature dendritic cells.
  • the increase in cell surface expression is evaluated using indirect immunofluorescence and FACScan analysis.
  • a compound is a candidate for further evaluation if the surface expression on dendritic cells of at least two cell- surface antigens is not increased by addition of the immune stimulant, LPS.
  • molecules identified as potential CD36 or CD51 agonists by the method of the invention will be subject to further evaluation.
  • surface expression of lineage-specific molecules has been used to determine the degree of maturation it would be usual to check whether the compound can also prevent immune-stimulated dendritic cells from inducing proliferation of T-cells and visa versa.
  • the ability of the molecule to vary cytokine secretion could also be tested.
  • direct binding of the candidate molecule to CD36, CD51 or TPS should also be confirmed. This latter confirmation may be easily obtained by applying a sample of the candidate molecule to a purified sample of CD36, CD51 or TPS.
  • Purified CD36 may be prepared as described by Tandon et al (1989) The Journal of Biological Chemistry, 264 pp 7570-7575.
  • Purified CD51 may be prepared as described by Smith et al, (1990), Journal of Biological Chemistry, 2.65, 11008-11013 and purified TSP may be prepared as described by Silverstein et al (1985) , Journal of Clinical Investigation, 75., pp 2065-2073.
  • Tests to detect binding of the test molecule are conveniently carried out by immobilizing the CD36, CD51 or TSP to a solid surface, for example the surface of a well of a microtitre plate. Methods of immobilization of protein molecules on such surfaces are well-known to those skilled in the art.
  • the test molecule identified as a CD36 or CD51 agonist is then applied to the immobilized protein. Following removal of unbound test molecule the presence of bound molecule is directly detected. This may be achieved in a number of ways depending on the chemical or biochemical characteristics of the test molecule.
  • test molecule is a protein it would be usual to detect binding with a labelled antibody to that protein. If the test molecule is a non-antigenic small molecular weight compound then the compound itself may be radioactively labelled for detection.
  • the molecule whose activity is to be tested in the method of the invention may have any type of molecular structure. For example, it may be a protein, a peptide, an amino acid, DNA, RNA, PNA, a nucleotide or a nucleoside, or a low molecular weight compound. It may be a molecule having known pharmacological or biochemical activity or a molecule with no such known activity and may be a novel molecule.
  • the method of the invention is suitable for testing entire libraries of molecules, for example libraries such as would be created by combinatorial chemistry. Indeed, all the embodiments of the screening method above may be adapted for an automated high throughput compound screen.
  • Plasmodium falciparum derived protein pf-EMP-1 is an agonist of both CD36 and CD51.
  • a fragment of pf- EMP-1 known as CIDR/A4 which comprises the CD36 binding domain is an agonist of CD36.
  • CIDR/A4 is described by Smith et al (1998) Molecular and Biochemical Parasitology, .97, pp 133-148 and comprises amino acids 402 to 846 of pf-EMP-1 as shown in Figure 2.
  • Antibodies which bind CD36 and CD51 have also been identified as having agonist activity and are capable of inhibiting the maturation of dendritic cells. Thrombospondin is also an agonist of CD51.
  • the present invention is also directed to any individual molecule identified as an agonist of CD36 or CD51 by the methods described herein.
  • the assays of the invention have allowed the inventors to make the further observation that apoptotic cells, the natural ligand of CD36, are also able to inhibit dendritic cell maturation in response to LPS. This is yet further evidence of the role of CD36 in modulating immune response.
  • the invention provides a pharmaceutical composition suitable for inducing immune tolerance in a mammal which comprises an agonist of the cell surface receptor CD36 and a pharmacologically acceptable carrier or diluent.
  • the CD36 agonist may be a molecule identified by the method described above.
  • Agonists which are suitable for incorporation into a pharmaceutical composition in accordance with the invention for the treatment of humans include antibodies with an affinity for an epitope of CD36, in particular an antibody which blocks the binding domain on CD36 for pf-EMP-1.
  • Monoclonal antibodies specific for CD36 which are designated “clone 89” and “clone SM ⁇ ” and which are commercially available from Serotech or Immunocontact (details above) are suitable for use in the pharmaceutical compositions of the invention.
  • Other commercially available CD36 antibodies which may be included in pharmaceutical compositions are listed in Appendix 1. It is contemplated that compositions comprising antibodies bispecific against CD36 and CD51 will be useful for inhibiting dendritic cell maturation.
  • agonists suitable for inclusion in pharmaceutical compositions are all variants of the Plasmodum falciparium pf-EMP-1 or fragments of such proteins which comprise the binding domain for CD36.
  • a particular example is the fragment CIDR/A4 described herein comprising amino acids 402 to 846 of pf-EMP-1. ( Figure 2) .
  • compositions comprising a bispecific CD36 antibody and the CIDR/A4 fragment are also contemplated in accordance with the invention.
  • Yet another agonist suitable for inclusion in a pharmaceutical composition are negatively charged phospholipids such as phosphatidylserine containing liposomes which have also been shown to bind to CD36 and other cellular receptors of immune cells.
  • Yet another agonist suitable for inclusion in a pharmaceutical composition are apoptotic cells.
  • the invention provides a pharmaceutical composition suitable for inducing peripheral immune tolerance in a mammal which comprises an agonist of the cell surface receptor CD51 as expressed by mammalian dendritic cells and a pharmacologically acceptable carrier or diluent.
  • acceptable agonists are antibodies, preferably monoclonal antibodies, directed against an epitope of CD51.
  • Antibodies suitable for incorporation in a pharmaceutical composition in accordance with this aspect of the invention are commercially available and set out in Appendix 2.
  • Thrombospondin (TSP) is also suitable for incorporation into a pharmaceutical composition as a CD51 agonist.
  • such compositions also include the Plasmodi um falciparum protein pf-EMP-1 or a fragment thereof incorporating the thrombospondin binding domain of pf-EMP-1.
  • negatively charged phospholipids such as phosphatidylserine are also suitable for incorporation as CD51 agonists in pharmaceutical compositions of the invention as well as apoptotic cells.
  • compositions in accordance with the second and third aspects of the invention are useful for the treatment of autoimmune diseases associated with inappropriate dendritic cell maturation and T-cell proliferation such as systemic lupus erythromatosis, rheumatoid arthritis, autoimmune haemolytic anaemia or idiopathic thrombocytopenic purpura.
  • Vehicles suitable for delivery of pharmaceutically active substances are known to those skilled in the art, especially those for delivery of pharmaceutically active proteins.
  • a method of treating mammalian dendritic cells in vi tro to induce immune tolerance therein which comprises exposing said cells to an agonist of cell surface receptors CD36 and/or CD51 as expressed on mammalian dendritic cells.
  • the invention also relates to preparations of cells so treated. Suitable agonists are any of those agonists described above or any molecule or substance identified by the screening method described herein. Treatment of dendritic cells ex-vivo with an agonist of CD36 and/or CD51 is beneficial in many therapeutic applications as described hereinafter.
  • dendritic cells of the donor may also be treated with a CD36 and/or CD51 agonist to induce immune tolerance.
  • the donor may be allogeneic or xenogeneic.
  • the present inventors have demonstrated that in mice tolerance to foreign antigens can be achieved by exposure of dendritic cells from one mouse strain, ex- vivo, to a CD51 agonist followed by introduction of the treated cells into another strain of mice. Thus such therapy is expected to be applicable to humans.
  • the present inventors have shown using a fragment of the ⁇ -subunit of the human acetylcholine receptor that immature dendritic cells treated with a CD36 agonist can be "modulated" to induce tolerance against a specific antigen by subsequent ex-vivo exposure to that antigen. Once reintroduced in vivo, immune response to that antigen is reduced or avoided.
  • dendritic cells may be removed from a patient suffering from an auto-immune disease, for example, and exposed to a CD36 and/or CD51 agonist and an antigenic molecule associated with the auto-immune disease in question and the dendritic cell preparation, with or without maturation, reintroduced into the patient.
  • this method may be used to induce tolerance to a particular allo or xeno-antigen or other therapeutic substance which is likely to induce an unwanted immune response, such as a blood product like factor VIII.
  • the invention includes preparations of dendritic cells tailored to the treatment of a particular auto-immune disease by exposure to an agonist of CD36 and/or CD51 and the specific auto- antigen associated with the disease and cell preparations tolerant to other antigens likely to generate an unwanted immune response.
  • the invention provides a method of identifying a molecule which is an agonist of cell surface receptors CD36 and/or CD51 and/or a thrombospondin receptor as expressed on antigen-presenting cells of the mammalian immune system which method comprises:
  • an impaired response compared to the response in the absence of said test molecule is an indication that said molecule under test is a CD36 and/or CD51 agonist or an agonist of a thrombospondin receptor.
  • the response that is measured is maturation of said antigen presenting cell.
  • Such a screening method may be carried out using the general methodology already described herein for dendritic cells.
  • monocytes can be purified from peripheral blood by adherence of PBMC to plastic dishes. Non-adherent cells are removed and the adherent cells can be detached by incubation with EDTA in PBS. Contaminating lymphocytes are depleted with the aid of magnetic heads and antiCD3 and antiCD19 monoclonal antibodies.
  • Macrophages may be generated by culturing monocytes, which have been isolated as described above, in RPMI 1640 supplemented with M-CSF for six days.
  • ⁇ -lyphocytes can be isolated from blood by virtue of their non-adherence to plastic petri dishes.
  • the non-adherent cells are subjected to depletion of contaminating monocytes and T-cells by exposure to magnetic heads and antiCD14 and antiCD3 monoclonal antibodies.
  • the antigen presenting cells are exposed to a substance to be tested for agonist activity against CD36, CD51 or a thrombospondin receptor and the degree of activation of said cells is measured.
  • activation may be determined by measuring the levels of secretion of various cytokines, or by testing ability of said antigen presenting cells to stimulate T-cell proliferation.
  • the increased expression of certain cell surface receptors is used as a measure of activation.
  • monocytes and macrophages activation is accompanied by an increase in surface expression of HLA-DR, CD54 and CD86 which is measured in the manner described above, preferably with the use of a monoclonal antibodies to HLA-DR, CD54 and CD86.
  • B-cell activation is determined by measuring the level of cell surface expression of HLA-DR, CD86 and CD40.
  • the expression may be detected using antibodies to these cell surface receptors.
  • the invention also further relates to uses of an agonist as identified above using said antigen- presenting cells for treatment of any of the autoimmune diseases listed above and for inducing immune tolerance in said antigen presenting cells ex-vivo as well as to antigen-presenting cell preparations which have been treated with a CD36 and/or CD51 agonist and/or thrombospondin receptor agonist and optionally an antigenic material.
  • the invention also relates to pharmaceutical compositions comprising an agonist of a thrombospondin receptor, for example ⁇ v ⁇ 3 or ⁇ v ⁇ 5; with a pharmaceutically acceptable carrier or diluent suitable agonists include antibodies to the thrombospondin binding domain of said receptor, for example any of the antibodies listed in Appendix 3.
  • suitable agonists include negatively charged phospholipids such as phosphatidylserine containing liposomes.
  • the invention in a sixth aspect thus, further relates to methods of identifying ⁇ -integrin agonists by any of the procedures described above and to uses of ⁇ -integrin agonists, as defined above, for any of the medical uses which are described herein.
  • the present invention further relates to uses of apoptotic cells as a medicament for inducing immune tolerance in antigen-presenting cells, preferably dendritic cells and to pharmaceutical compositions comprising those cells in a suitable carrier or diluent.
  • Apoptotic cells are suitable for delivering tissue specific antigens including major and minor histocompatibility antigens to dendritic or other antigen-presenting cells. Delivering antigens in this way allows delivery of unknown antigens or antigens where the class II restricted epitope (s) are not defined.
  • the tissue origin of the apoptotic cell may be varied depending upon the application. For example, it is preferred for the apoptotic cell to be of the same tissue type as any cell bearing an antigen to which tolerance is to be induced.
  • the invention further relates to the use of negatively charged phospholipids for inducing immune tolerance in antigen presenting cells.
  • Said immune tolerance may be induced by treatment of said antigen presenting cells, for example dendritic cells, with said negatively- charged phospholipid either ex-vivo by the methods described herein or by administration of the phospholipid to a patient by any of the conventional administration routes known to those skilled in the art.
  • a preferred form of composition is liposomes comprising the negatively charged phospholipid.
  • a preferred phospholipid is phosphatidylserine.
  • a method comprising the following steps is used to identify a molecule capable of preventing adherence of erythrocytes infected with a malarial parasite to human dendritic cells:
  • a reduction in the level of adherence to CD36 or TSP in the presence of the test molecule compared to the level of adherence in the absence of said test molecule is an indication that said molecule is capable of preventing the adherence of erythrocytes infected with the malarial parasite to human dendritic cells .
  • the erythrocytes may be infected with Plasmodium falciparum or another Plasmodium species.
  • Suitable falciparum strains include ITO/A4 or ITO/C24 which may be derived as described by Roberts et al (1992) Nature 357 pp 689-692 or Malayan Camp (MC) which may be obtained as described by Roberts et al (1985) Nature 318:64-66.
  • a suitable format for carrying out a screening method as described above is to immobilize the purified CD36 or TSP onto a solid surface.
  • immobilization is secured by adsorption of the protein molecules to a plastic surface such as a petri dish.
  • Parasitised erythrocytes suspended in a suitable binding medium are added to the adsorbed CD36 or TSP and incubated for a period sufficient to allow adherence, for example, about 1 hour. Thereafter the binding medium and any non-adhered erythrocytes are removed and a suitable erythrocyte stain for example, Giemsa, added to the petri dish.
  • Adhered erythrocytes may be quantified by counting under a light microscope.
  • erythrocyte adherence may be quantified by spectrometry, fluorescence microcopy and the like.
  • the invention provides a method of identifying a molecule capable of preventing the adherence of red blood cells infected with a malarial parasite to human dendritic cells which comprises:
  • any maturation of said dendritic cells in the presence of the test molecule over and above that manifested in the absence of said molecule is an indication that said molecule is capable of preventing adherence of red blood cells infected with a malarial parasite to human dendritic cells.
  • Maturation of dendritic cells may be measured by any of the methods already described herein.
  • Suitable immune stimulants include LPS, TNF ⁇ , CD40L and monocyte conditioned medium (MCM) .
  • MCM monocyte conditioned medium
  • the pf- EMP-1 preparation for use in the method is that designated in pf-EMP-1 A4var as described by Smith et al (see before) and having the Genbank Accession No. L42244.
  • the fragment CIDR/A4 may also be used.
  • the invention provides for use of molecules identified by the aforementioned methods which inhibit infected erythrocyte adherence to dendritic cells in pharmaceutical compositions for the treatment of malarial infection.
  • a modified CIDR region of the pf-EMP-1 A4 variant protein could be incorporated in a multisubunit vaccine against falciparum malaria. This would induce blocking antibodies against the CD36 binding domain of pf-EMP-1 variant proteins so that the immune responses against other proteins are not inhibited.
  • FIGURE 1 shows schematically the molecular basis for the binding of Plasmodium falciparum infected red blood cells to CD36 and TSP on the surface of dendritic cells;
  • FIGURE 2 shows the amino acid sequence of the pf-EMP-1 fragment CIDR/A4;
  • FIGURE 3 shows the increase in surface expression of dendritic cell marker antigens HLA DR, CD54, CD40,
  • CD80, CD83 and CD86 following immune stimulation after exposure to (a) LPS matured dendritic cells, (b) dendritic cells matured with LPS, with and without prior exposure to RBC, (c) dendritic cells matured with LPS with and without prior exposure to parasite lysate and (d) dendritic cells matured with LPS with and without prior exposure to intact ITO/A4 infected RBC;
  • FIGURE 4 shows the absolute binding of erythrocytes infected with parasite lines IT0/A4, ITO/C24, MC and T9/96 to CD54, CD56, and TSP (a,c,e,g) and (B) shows the increase in surface expression of LPS matured dendritic cells compared with dendritic cells exposed to the respective parasite line prior to maturation (b,d,f,h);
  • FIGURE 5 shows transmission electron micrographs illustrating the interaction of dendritic cells with (a) ITO/A4 infected erythrocytes and (d) non-adherent T9/96 infected erythrocytes;
  • FIGURE 6 shows dendritic cell stimulation of T-cell proliferation (a) induced by immature dendritic cells ( ⁇ ) , LPS-matured dendritic cells (D) and dendritic cells co-cultivated with intact ITO/A4 infected erythrocytes (T) prior to maturation, primary CD4+ T- cell responses to parasite lysate (b) and to keyhole limpit haemocyanin (c) induced by LPS-matured autologous dendritic cells (D,o) and autologous dendritic cells co-cultivated with intact ITO/A4 infected erythrocytes ( ⁇ ,•) prior to maturation;
  • FIGURE 7 shows the effect of monoclonal antibodies against CD36 and CD51 on maturation of dendritic cells represented graphically as relative increase in surface expression of dendritic cells matured with LPS compared with immature dendritic cells;
  • FIGURE 8 shows the effect of monoclonal antibodies against CD36 and CD51 on dendritic cell maturation as a FACscan output
  • FIGURE 9 shows further results of experiments with apoptotic cells (a) output of FACscan, (b) staining with potassium iodide to exclude dead cells, (c) proliferation of allogenic T-cells stimulated by increasing numbers of immature dendritic cells, ( ⁇ ) LPS- matured dendritic cells (A) or dendritic cells exposed to apoptotic dendritic cells and then matured with LPS(" .
  • FIGURE 10 shows the effect of apoptotic neutrophils on the maturation of dendritic cells
  • FIGURE 11 shows results of a T-cell proliferation assay including antigen specific T-cell proliferation, (a) Proliferation of allogeneic T-cells. (b) proliferation of KLH specific CD4+CD45RO- autologous T-cells (c, d) proliferation of the T-cell clone TB-2 specific for the human Acetylcholine Receptor a- subunit in response to polypeptide (c) or peptide (d) .
  • Stimulator dendritic cells were treated as follows: immature DC alone ( x ) or matured with LPS (D) ; dendritic cells exposed to irrelevant antibodies with (O) or without (•) antigen and then matured with LPS; dendritic cells exposed to antiCD36 antibody with (v) or without (T) antigen and then matured with LPS; dendritic cells exposed to antiCD51 antibody with ( ⁇ ) or without (A) antigen and then matured with LPS; dendritic cells exposed to antiCD36 and antiCD51 antibody with (>) or without ( ⁇ ) antigen and then matured with LPS.
  • FIGURE 12 shows secretion of cytokines TNF ⁇ , IL12p70 and IL10 by dendritic cells exposed to an antiCD36 antibody or to apoptotic dendritic cells and respective controls;
  • FIGURE 13 shows in vi tro maturation of mouse dendritic cells following exposure to an antiCD51 antibody
  • FIGURE 14 shows results from mouse popliteal lymph node assay.
  • Immature dendritic cells were derived from peripheral human blood cells using standard procedures as described by Sallusto et al (1995) J. Exp. Med. 182 pp 389-400. Briefly, monocytes were cultivated in RPMI 1640 supplemented with 2mM Glutamine, 50 ⁇ g/ml Kanamycin, 1% nonessential amino acids (GibcoBRL) , 10% human AB serum and 50 ng/ml of each IL-4 (specific activity >2xl0 6 U/mg, PeproTech) and GM-CSF (specific activity > lxlO 7 U/mg, Schering-Plough) for 6 days.
  • RPMI 1640 supplemented with 2mM Glutamine, 50 ⁇ g/ml Kanamycin, 1% nonessential amino acids (GibcoBRL) , 10% human AB serum and 50 ng/ml of each IL-4 (specific activity >2xl0 6 U/mg, PeproTech) and
  • Monocytes were purified from peripheral blood by adherence of PBMC to plastic dishes for 2 hours. Non adherent cells were removed and the adherent cells layer washed 2 times with warm PBS. For further purification, the adherent cells were detached by incubation with 2 mM EDTA in PBS for 20 min and contaminating lymphocytes depleted with the aid of magnetic beads (Dynal or Miltenyi) and anti-CD3 and anti-CD19 monoclonal antibodies (DAKO) .
  • Monocytes isolated as described above were cultured in RPMI 1640 supplemented with 2 mM Glutamine, 50 ⁇ g/ ml Kanamycin, 10% human AB serum and 50 ng/ml of M-CSF (specific activity > 2 x 10 6 U/mg, Peprotech) for 6 days .
  • B-lymphocytes were isolated from human blood according to standard procedures. Briefly, non-adherent PBMC were subjected to depletion of contaminating monocytes and T-cells with the aid of magnetic beads (Dynal or Miltenyi) and anti-CD14 and anti-CD3 monoclonal antibodies (DAKO) . B-cells were cultured in RPMI 1640 supplemented with 2 mM Glutamine, 50 ⁇ g/ ml Kanamycin, 1% to 10% human AB serum.
  • RPMI 1640 supplemented with 2 mM Glutamine, 50 ⁇ g/ ml Kanamycin, 1% to 10% human AB serum.
  • CD34+ cells were isolated from PBMC with the aid of anti-CD34 antibody conjugated magnetic beads (Dynal or Miltenyi) .
  • CD34+ progenitor were then cultured in RPMI 1640 supplemented with 2 mM Glutamine, 50 ⁇ g/ ml Kanamycin, 1% to 10% human AB serum and the following cytokines: 100 ng/ml of GM-CSF (Schering-Plough), 50 ng/ml TNF ⁇ and 50 ng/ml SCF (Peprotech) for 12 days.
  • CD34+ cells could be expanded in the above mentioned medium but supplemented with 25 ng/ml FLT3-L , lOU/ml TPO, SCF 20 ng/ml (Peprotech) for up to 8 weeks and then induced to differentiate to dendritic cell by culture of a further 3 days in medium supplemented with 25 ng/ml GM-CSF and 25 ng/ml IL-4.”
  • Plasmodium falciparum Laboratory strains of Plasmodium falciparum were cultured in human RBC as described by Trager et al (1976) Science. 193 pp673 to 675.
  • the cytoadherent cell lines ITO/A4 and ITO/C24 were clones isolated by manipulation from the IT04 line, which is derived from a parasite isolate from Ituxi in Brazil.
  • the cytoadherent parasite line Malayan Camp (MC) and the non-adherent cell line T9/96 were both adapted to in vitro culture from parasites originally isolated from Thailand.. All cultures were free from mycoplasma contamination.
  • Infected erythrocytes were purified either by differential sedimentation in Plasmagel or through 65% Percoll both of which gave a yield of more than 90% infected erythrocytes. Examination of a thin film revealed that more than 90% of infected erythrocyes were viable.
  • Parasite lysate was obtained by three rounds of freezing and thawing of mature infected RBC. Parasite pigment was prepared as described by Schwarzer et al (1994) BR. J. Haematol. 88, pp740-745.
  • Parasite conditioned medium was the supernatant derived after culturing lxlO 8 purified infected erythrocytes in dendritic cell medium for 24 hours. All materials were from Sigma unless otherwise stated.
  • Binding of parasitised RBCs to purified proteins was measured as previously described by Craig et al (1997) Infect. Immun. 65, pp 4580-4585. Briefly, two microlitres of a solution of TSP (Gibco-BRL) , purified CD36 or purified CD54 (ICAM-Fc) were adsorbed onto bacteriological, plastic plates. Mature erythrocytes parasitised with P. falciparum strains (a) IT0/A4, (c) ITO/C24, (e) MC and (g) T9/96, were suspended in binding medium and added to each dish. The erythrocytes were allowed to settle and then resuspended by gentle rotation every 10 minutes for 1 hour.
  • Non-adherent cells were removed, the remaining cells fixed and stained with Giemsa.
  • Adherent parasitised cells were counted by light microscopy and the number of cells bound per square millimeter were corrected to binding at 2% haematocrit and 5% parasitaemia.
  • the results are shown in Figure 4A and confirm that like IT0/A4, ITO/C24 and MC are able to adhere to CD36 and TSP. However, their adherence to CD54 was much reduced. T6/96 does not adhere to CD54, CD36 or TSP.
  • a maturation assay as described in Example 2 was carried out but exposing immature dendritic cells to erythrocytes infected with (b) ITO/A4, (d) ITO/C24, (f) MC and (h) T9/96.
  • the results are shown in Figure 4B. While parasite lines MC and ITO/C24 inhibited the maturation of dendritic cells in a similar vein to clone IT0/A4, the non-adherent line T9/96 did not inhibit maturation of dendritic cells even at a ratio of infected erythrocytes to dendritic cells of 100:1.
  • ITO/A4 infected erythrocytes were observed to be in intimate contact with immature dendritic cells with cytoplasmic processes partially enclosing the parasites (Fig. 5a).
  • the plasmalemma of the infected erythrocytes was in close apposition to the limiting membrane of the dendritic cell particularly at the site of knobs (Fig. 5b) .
  • a similar apposition between parasitised erythrocytes and host cells is seen between infected red blood cells and endothelial cells (Berendt et al (1994) Parasitology 108 Suppl. 519-28).
  • T-cell responses were purified using a Cellect column (TCS) .
  • TCS Cellect column
  • dendritic cells were added in increasing numbers (156 to 10,000) to 1 x 10 5 T-cells in triplicate and incubated for 5 days. T-cells were pulsed with 0.5 ⁇ Ci 3H-thymidine/well for the last 18 hours of the culture.
  • 1 x 10 6 dendritic cells were incubated with medium alone or with 1 x 10 8 infected erythrocytes for 18 h and then pulsed with 10 ⁇ g/ml parasite-lysate or with 30 ⁇ g/ml keyhole limpet haemocyanin, respectively.
  • the dendritic cells were purified by sedimentation through LymphoprepTM and 1 X 10 5 dendritic cells were culterd with 1.5 x 10 6 CD4+ T-cells from the same donor. From day 4 to day 6 of culture, 50 ⁇ l aliquots were taken in triplicate and pulsed with 0.5 ⁇ Ci 3 H-thymidine/well for 8 hours.
  • Dendritic cells exposed to intact infected erythrocytes are poor stimulators of T-cell proliferation. Allogeneic T-cell proliferation (a) induced by immature dendritic cells ( ⁇ ) , LPS-matured dendritic cells (D) and dendritic cells co-cultivated with intact ITO/A4 infected erythrocytes (T) prior to maturation.
  • Dendritic cells matured after incubation with uninfected RBC, a crude pigment preparation or a lysate of infected erythrocytes induced a similar degree of T-cell proliferation in a mixed leukocyte, reaction to that induced by control mature dendritic cells (data not shown) .
  • dendritic cells incubated with LPS after exposure to intact infected erythrocytes from the parasite line ITO/A4 were strikingly less efficient in their induction of T-cell proliferation compared with the T-cell proliferation induced by mature dendritic cells (Fig 6a) .
  • dendritic cells exposed to intact infected erythrocytes before maturation with LPS did not induce primary CD4+ T-cell responses to lysate of infected erythrocytes or to keyhole limpet haemocyanin (Plebanski et al) (Fig 6, b,c).
  • Maturation assay with monoclonal antibody A maturation assay was carried out as described in Example 2 except that instead of infected erythrocytes the immature dendritic cells were exposed to monoclonal antibodies to CD36, CD51 or both prior to immune stimulation with LPS.
  • dendritic cells were incubated in duplicate wells without or with either 25 ⁇ g irrelevant IgM antibody, 25 ⁇ g irrelevant IgGl antibody, 25 ⁇ g antiCD36 antibody, 25 ⁇ g antiCD51 antibody or a combination thereof for at least 3 hours. Thereafter, dendritic cells were matured with 100 ng/ml LPS ( Salmonella typhimuri um) for 48 hours or left untreated as a control.
  • the monodonal antibodies tested were CD36 clone SMQ (Immunocontact) and clone 89 (Serotec), CD51 clone 13C2
  • a maturation assay was carried out as described in Example 2 except that instead of infected erythrocytes the immature dendritic cells were exposed to apoptotic cells prior to immune stimulation with LPS.
  • Apoptotic or necrotic cells were derived from purified autologous dendritic cells, monocytes or neutrophils.
  • lxlO 6 purified dendritic cells were incubated in duplicate wells without or with either 2 x 10 6 autologous apoptotic or necrotic cells for 12 hours. Maturation was induced by the addition of LPS or TNF ⁇ as stated above.
  • Apoptosis was induced by radiation with a calibrated UV lamp at a dose of 2500 mJ/cm 2 and evaluated by staining with FITC-AnnexinV/Propidium Iodide according to manufacturers recommendations (Roche Diagnostics) 3 hours and 12 hours after UV radiation. Necrosis was induced by at least three cycles of rapid freezing at -70 ° C and thawing at 37 ° C. Thereafter, more then 90% of cells were permeable for trypan blue. The results are shown in Figure 9 as follows:
  • Apoptotic cells but not necrotic cells inhibit the maturation of dendritic cells (a) Immature dendritic cells were left untreated, matured with LPS or exposed autologous apoptotic or necrotic dendritic cells prior to maturation with LPS and subsequently stained with antibodies directed against surface marker and analysed by FACScan as indicated, (b) Dead Cells and especially apoptotic cells were efficiently excluded from analysis by gating on forward scatter and exclusion of cells positive for Propidium Iodide.
  • T-cell proliferation was measured as described in Example 7.
  • 1 x 10 6 dendritic cells were incubated with medium alone or with antibodies as indicated and then pulsed for 6 h with 0.025 mM AChR ⁇ : 3-181 polypeptide before or 1 mM AChR ⁇ : 144-163 peptide after maturation with LPS.
  • IL12 p70 was secreted by control dendritic cells matured with LPS whereas IL10 was secreted by dendritic cells exposed to anti- CD36 antibodies or apoptotic cells.
  • the absolute amount of IL10 varied considerably between dendritic cells treated with anti-CD36 and dendritic cells exposed to apoptotic cells in response to LPS. It is possible that intact cells bind to more than one receptor thus modifying the cytokine secretion induced by CD36 alone.
  • human monocyte derived dendritic cells can be modulated in their maturation and function by a variety of agents including antibodies binding to CD36 and or CD51, in this study we began to investigate whether a similar phenomenon could be observed in mouse dendritic cells .
  • Bone marrow from male Balb/c (H-2 ) mice was harvested and total cells were cultured in RPMI supplemented with 2 mM glutamine, 50 mg Kanamycin, 10 % FCS, 10 ng/ml each murine recombinant GM-CSF and IL-4. On day two of culture half the medium was replaced with fresh medium supplemented with cytokines and on day four of culture non-adherent cells were harvested.
  • bone-marrow derived mouse dendritic cells In vitro maturation of bone-marrow derived mouse dendritic cells: One million of bone marrow derived dendritic cells (approximately 50% total cells) in duplicate were exposed to either medium alone, 25 mg isotpype control antibody or 25 mg antiCD51 antibody for 8 hours. Cells were subsequently exposed to 100 ng LPS for 48 hours or left alone as a control. Maturation of dendritic cells was analysed by double staining with FITC conjugated antibodies against CDllc and PE- conjugated antibodies directed against either CD40, CD54, CD86 or I-A and subsequent FACScan analysis. Analysis was performed on CDllc-FITC positive cells.
  • Popliteal lymph node assay Dendritic cells were exposed to medium alone or to antiCD51 antibodies and then matured with LPS as described above. The cells were then harvested and washed four times in PBS in order to remove LPS. Cells were resuspended in 10% FCS/PBS at a concentration of 6 x 10 5 total cells/20 ml.
  • mice Groups of six male C3H3/HE (H-2 d ) mice were injected with 20 ml of PBS into the right footpad and LPS matured dendric cells into the left footpad, with 20 ml of PBS into the right footpath and dendritic cells exposed to antiCD51 antibody prior to LPS maturation into the left footpad or with 20 ml of PBS into the right and the left footpad. After one week mice were sacrificed and the popliteal lymphnodes were removed. The weight of the left and the right lymphnode of each mouse in all three groups were determined and the ratio of the weight of the left lymphnode to the weight of the right lymphnode was calculated. The mean and SE of the ratio was determined for each group.
  • Dendritic cells matured with LPS increased the surface expression of the molecules CD40, CD54 and CD86 as compared to immature dendritic cells (DC) .
  • DC LPS dendritic cells matured with LPS
  • DC CD51 lps dendritic cells were treated with antiCD51 antibodies prior to exposure to LPS
  • the results are shown in Figure 13.
  • Agonists of CD36, CD51, thrombospondin receptors or ⁇ -integrin may be used to modulate human immune response in patients with unwanted and/or harmful allo- or auto-immune responses.
  • immature human dendritic cells which are defined and identifiable as described herein and with the ability to phagocytose are derived from preparations of human peripheral blood.
  • the dendritic cells are derived from CD34+ stem cells or monocytes isolated from human peripheral blood by the method described in Example 1.
  • an agonist of C36, CD51, thrombospondin receptor or ⁇ -integrin is added.
  • the relative concentration of agonist to cells is adjusted depending on the nature of the agonist used. For example, if the agonist is a monoclonal antibody, about 25 ⁇ g antibody to about 10 6 dendritic cells is appropriate at a concentration of 25 ⁇ g Ml "1- The cells are treated for between 3 and 24 hours.
  • Adminitration may be by intravenous infusion, by inhalation or by sub-cutaneous or intramuscular injection.
  • dendritic cells to a human following the method described above will give rise to a generalized immune suppressive effect which will be useful, in a number of situations, for example in the prevention of rejection of allografts and xenografts or for treatment of disease suspected of having an auto-immune basis but for which the auto- antigen is not known.
  • the method described above may be modified to produce dendritic cells which are tolerant to a specific antigen.
  • the dendritic cell preparation is exposed to an antigen against which tolerance is to be induced as well as to the CD36, CD51, thrombospondrin receptor or ⁇ - integrin agonist.
  • the cells may be exposed to antigenic material, before, after or simultaneously with the aforesaid agonist molecule.
  • the antigenic material may be linked to, fused to or otherwise associated with said agonist molecule. Exposure to the antigenic material is for about 6 to about 24 hours with or without an immune stimulant, then the cells are reintroduced to the patient as described above .
  • tolerance can be induced in respect of the following antigens:
  • auto-immune diseases for example: components of autologous red blood cells to modulate the immune response in patients with auto-immune hemolytic anaemia components of autologous platelets in patients with auto-immune thrombocytopeniacomponents of beta islet cells of the pancreas in patients with insulin dependent diabetes mellitus components of other endocrine organs in patients with other organ specific auto-immune diseases components of the acetylcholine receptor in patients with myasthenia gravis other antigens or apoptotic cells containing antigens causing harmful or pathological immune responses in other auto-immune diseases
  • antigens to which there is pathological immune response causing atopic or allergic diseases for example antigens eliciting an immune response in hay fever, asthma, eczema or coeliac disease
  • pathological immune response may be defined in other diseases for example in non-organ specific immune diseases (systemic lupus erythematosis or rheumatoid arthritis) or other immune mediated arthritis or other connective tissue diseases in inflammatory bowel disease in auto-immune hepatitis in multiple sclerosis or in other auto-immune disease
  • allo-antigens to which there is a harmful or pathological immune response for example components of red blood cells in haemolytic disease of the newborn or in previously transfused patients components of platelets for example in neo-natal allo immune thrombocytopenia or in conditions where there is an allo-immune response to transfused platelets other blood products or substitutes for example Factor VIII in haemophilia patients other synthesized or manufactured or naturally occurring products or substances
  • dendritic cells such as macrophages, monocytes or ⁇ -lymphocytes could be used for treatment with an agonist of CD36, CD51, thrombospondin receptor or ⁇ -integrin and optionally an antigenic material. Specific tolerance can be introduced in such cells for use in any of the applications listed above.
  • phosphatidylserine liposome compositions Preparation of phosphatidylserine liposome compositions and their therapeutic uses.
  • Liposomes encapsulating antigens and phospholipids with or without additional targeting molecules induce antigen specific immune unresponsiveness .
  • Liposomes are prepared as described by Coradini et al, Anticancer Research 1998 18 177-182. In brief clean glass tubes are coated with 2 micromolar of mixtures of phosphatidylcholine and phosphatidylserine, other negatively charged phospholipids or other phospholipids including cholesterol and/or cholesterol ester dissolved in chloroform. The solvent is evaporated under nitrogen gas and the tubes incubated in a vacuum for 45 minutes.
  • Sterilised phosphate buffered saline (unmodified liposomes) or containing the antigen (s) to which unresponsiveness will be induced with or without molecules allowing targeting of the liposomes to CD36 and/or CD51 and/or beta-integrins and/or other receptors of apoptotic cells or other molecules expressed on the surface of antigen presenting cells, is added to the lipid shell.
  • Suitable targeting molecules are monoclonal antibodies to the respective receptors or fragments of the P. falciparum erythrocyte membrane protein-1 that bound to CD36 and/or thrombospondin.
  • the tubes are shaken at high' speed for 5 minutes and separated from free fatty acid by ultracentrifugation at 100,000g for 60 minutes.
  • Targeting molecules may be covalently or non-covalently attached to the surface of liposomes.
  • the liposomes are filtered through a 0.22 micrometer filter. Encapsulation of antigens and targeting molecules can also be achieved by freeze-thawing or dehydration/rehydration or by reverse phase evaporation (Monnard PA et al, Biochem. Biophys. Acta 1997 1329 39-50) or by other published methods of preparing liposomes.
  • Liposomes prepared as described above would be added to 1 x 10 6 isolated immature dendritic cells or to other antigen presenting cells at a concentration of 25 micrograms per ml. The maturation and function of the dendritic cells or other antigen presenting cells is assessed as previously described.
  • the liposomes containing phosphatidylserine (with or without targeting molecules) is used to treat dendritic cells or other antigen presenting cells ex vivo or for systemic treatment.
  • SD is standard designation
  • the selected databases contain 18 documents matching your query:
  • AB CD36 is also known as platelet GPIV, GPIV, platelet GPIIIb,
  • AB CD36 is also known as GPIIIb, GPIV
  • SD is standard designation
  • AB Reactant#l: CD51/61 complex is also known as integrin alpha V beta 3.
  • AV / R-phycoerythrin conjugate / f luorescein conjugate AB CD51 is also known as integrin alpha V subunit and vitronectin receptor AB alpha subunit.
  • SD 13C2 SD 21255108 SD 21255114 SD 21255119 LD USA MCM El DA>9811 CI /catalog
  • AB Reactant is also known as vitronectin receptor alpha subunit and CD51.
  • AB Reactant is also known as CD51 and vitronectin receptor alpha subunit .

Abstract

On décrit des procédés et des compositions qui permettent d'induire une tolérance immunitaire dans des cellules de présentation d'antigène de mammifère telles que des cellules dendritiques, des macrophages, des monocytes et des lymphocytes B. Ces procédés et ces compostions impliquent l'utilisation d'agonistes des récepteurs de surface cellulaire CD36, CD51, des récepteurs de thrombospondine et/ou des Bêta intégrines qui, une fois exposés à une cellule présentant l'antigène telle qu'une cellule dendritique sont capables d'inhiber la maturation dans ces derniers. Par conséquent l'aptitude des cellules à favoriser la réponse immunitaire est inhibée. La tolérance à un antigène spécifique peut être induite dans des cellules présentant l'antigène au moyen de l'exposition à un ou plusieurs agonistes précités et à l'antigène. Des préparations de cellules peuvent ainsi être préparées pour être administrées à des humains chez qui la tolérance à un ou plusieurs antigènes spécifiques doit être induite, par exemple en cas de transplants d'allogreffes ou de xénogreffes ou en cas de maladie auto-immune.
EP00942249A 1999-06-30 2000-06-30 Traitement de cellules dendritiques pour l'induction de la tolerance immunitaire Withdrawn EP1196543A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB9915311 1999-06-30
GBGB9915311.6A GB9915311D0 (en) 1999-06-30 1999-06-30 Modulation of dendritic cell maturation
PCT/GB2000/002546 WO2001002005A2 (fr) 1999-06-30 2000-06-30 Induction de la tolerance immunitaire

Publications (1)

Publication Number Publication Date
EP1196543A2 true EP1196543A2 (fr) 2002-04-17

Family

ID=10856356

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00942249A Withdrawn EP1196543A2 (fr) 1999-06-30 2000-06-30 Traitement de cellules dendritiques pour l'induction de la tolerance immunitaire

Country Status (5)

Country Link
EP (1) EP1196543A2 (fr)
AU (1) AU5695200A (fr)
CA (1) CA2378477A1 (fr)
GB (1) GB9915311D0 (fr)
WO (1) WO2001002005A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012135550A1 (fr) * 2011-03-30 2012-10-04 Luna Innovations Incorporated Agents de contraste ciblant un biomarqueur et leur utilisation en imagerie par résonance magnétique pour détection de plaque d'athérosclérose

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0477270A1 (fr) * 1989-06-15 1992-04-01 Med-Tal, Inc. Prophylaxie et traitement d'infections microbiennes avec des phosphoglycerides
WO1993006848A1 (fr) * 1991-10-03 1993-04-15 The Center For Blood Research IMMUNOADHESINES DE CD36 ET LEUR UTILISATION POUR TUER DE MANIERE SELECTIVE LES ERYTHROCYTES INFECTES PAR $i(PLASMODIUM FALCIPARUM)
AU697624B2 (en) * 1993-08-13 1998-10-15 Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The Methods and compositions for stimulating and inhibiting TGF-beta activity
WO1996033736A1 (fr) * 1995-04-27 1996-10-31 Affymax Technologies N.V. Peptides du paludisme et vaccins associes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0102005A2 *

Also Published As

Publication number Publication date
WO2001002005A3 (fr) 2001-07-05
CA2378477A1 (fr) 2001-01-11
WO2001002005A2 (fr) 2001-01-11
GB9915311D0 (en) 1999-09-01
AU5695200A (en) 2001-01-22

Similar Documents

Publication Publication Date Title
ES2346877T3 (es) Composicion y procedimiento para modular la interaccion de las celulas t y las celulas dendriticas.
Carrera et al. Involvement of the CD4 molecule in a post‐activation event on T cell proliferation
Li et al. Mechanism and localization of CD8 regulatory T cells in a heart transplant model of tolerance
JP2020018315A (ja) 免疫抑制樹状細胞を得るための方法
JP7447388B2 (ja) 感染性疾患の治療のための共受容体システム
JP2016504912A (ja) 免疫賦活樹状細胞を得るための方法
JP2000508226A (ja) Ｌａｇ−３タンパク質によるｔｈ１リンパ球を検出、同定、単離並びに選択的に標識およびターゲッティングする方法
WO1997003695A9 (fr) Procedes pour detecter, identifier, isoler, marquer et cibler selectivement les lymphocytes th1, au moyen de la proteine lag-3
US20220064255A1 (en) Anti-tcr antibody molecules and uses thereof
CA2131299A1 (fr) Regulation de l'activite lymphocitaire par les peptides hla
KR20180054663A (ko) CD8+CD45RClow TREGS의 신규한 아집단 및 이들의 용도
AU2018262788A1 (en) Mesenchymal lineage precursor or stem cells with enhanced immunosuppression
CN102112147B (zh) Xbp1、cd138、和cs1肽
US20230048244A1 (en) Anti-tcr antibody molecules and uses thereof
US20220411478A1 (en) Cd70 targeted chimeric antigen receptor (car) t cells and uses thereof
Iannone et al. T-lymphocyte immunophenotyping in polymyositis and dermatomyositis
KR20200083990A (ko) 종양 관련 골수 세포의 조절 방법 및 면역 체크포인트 차단을 증강시키는 방법
US20080248011A1 (en) Methods for Isolating Monocytes
JPH10501791A (ja) クラスｉｍｈｃペプチドによる細胞傷害性ｔ細胞リンパ球（「ｃｔｌ」）活性の調節
JP2021511041A (ja) キメラ抗原受容体を発現する制御性ｔ細胞
EP1196543A2 (fr) Traitement de cellules dendritiques pour l'induction de la tolerance immunitaire
CN112470007A (zh) 治疗性物质，其制备和诊断方法
AU2021323388B2 (en) Binding proteins recognizing HA-1 antigen and uses thereof
US20230190798A1 (en) Cd19-directed chimeric antigen receptor t cell compositions and methods and uses thereof
Schroeder et al. FTY720 prevents anti-CD4 mAb-induced tolerance but cannot reverse established tolerance in a rat kidney transplantation model

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20020129

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

17Q First examination report despatched

Effective date: 20040630

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20060103