WO1999038530A1 - Agent capable de moduler l'activite liee a un glycosphingolipide, agissant ainsi sur un trouble immunitaire - Google Patents

Agent capable de moduler l'activite liee a un glycosphingolipide, agissant ainsi sur un trouble immunitaire Download PDF

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Publication number
WO1999038530A1
WO1999038530A1 PCT/GB1999/000290 GB9900290W WO9938530A1 WO 1999038530 A1 WO1999038530 A1 WO 1999038530A1 GB 9900290 W GB9900290 W GB 9900290W WO 9938530 A1 WO9938530 A1 WO 9938530A1
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WIPO (PCT)
Prior art keywords
agent
glycosphingolipid
associated activity
modulating
immune disorder
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PCT/GB1999/000290
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English (en)
Inventor
Neil Andrew Williams
Timothy Raymond Hirst
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University Of Bristol
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Publication date
Application filed by University Of Bristol filed Critical University Of Bristol
Priority to EP99902688A priority Critical patent/EP1049489A1/fr
Priority to CA002318190A priority patent/CA2318190A1/fr
Priority to AU22900/99A priority patent/AU2290099A/en
Publication of WO1999038530A1 publication Critical patent/WO1999038530A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55544—Bacterial toxins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
    • G01N2405/08—Sphingolipids
    • G01N2405/10—Glycosphingolipids, e.g. cerebrosides, gangliosides

Definitions

  • the present invention relates to a medicament.
  • the present invention relates to a medicament useful in the treatment of immune disorders.
  • these disorders include inflammatory conditions, autoimmune diseases, allergic conditions, the treatment of cancers, including human leukaemias of a T cell origin and as agents in the prevention of human transplantation rejection and graft versus host disease (GNHD).
  • GNHD graft versus host disease
  • the present invention relates to an immunomodulatory agent.
  • the present invention relates to such an agent optionally co-
  • a specific antigen for use in the treatment of mammalian particularly human immune disorders include inflammatory conditions, autoimmune diseases, allergic conditions, the treatment of cancers, including human leukaemias of a T cell origin and as agents in the prevention of human transplantation rejection and graft versus host disease (GNHD).
  • GNHD graft versus host disease
  • Autoimmumty is the term used to describe the mechanism by which the body generates an immune response to self-antigens. It has been disclosed in the art that agents which have GM-1 binding activity, or which have an effect on GM-1 mediated intracellular signalling events but no GM-1 binding activity, are useful as therapeutic
  • WO95/10301 disclosed agents which consisted of a mucosa-binding molecule linked to a specific tolerogen.
  • GM-1 binding agents provided in WO97/02045 were cholera toxin (Ctx), the B subunit of cholera toxin (CtxB), the
  • E.coli heat labile toxin Etx
  • EtxB E.coli heat labile toxin
  • allergen a normally harmless environmental stimulus, called an "allergen”
  • Allergies or hypersensitivities are distinguished into four types of reactions. The first three are antibody-mediated, the fourth is mediated mainly by T cells and macrophages.
  • IgE In Type I Immediate Hypersensitivity/ Atopic Allergy, the principal immune response to the allergen involves the production of IgE antibodies. Such disorders are by far the most prevalent in humans and are seen as principal targets for new therapeutic approaches. Although these diseases are not exclusively IgE mediated, IgE binds to cells within the tissues such as mast cells and basophils and the cross-linking of IgE on the cells surfaces by allergen invokes the release of many inflammatory mediators.
  • Typical examples of such diseases include asthma, allergic cough, allergic rriinitis and conjunctivitis, atopic eczema and dermatitis, urticaria, hives, insect bite allergy, dietary and certain drug allergies.
  • the particular allergens are known.
  • the principal allergen in asthma is DerPl from house dust mite but other triggers of asthma such as pet dander antigens also exist.
  • Type II or antibody dependent cytotoxic hypersensitivity occurs when antibodies of a different type, usually IgG and IgM, binds to either self antigen or foreign antigen on cells and leads to phagocytosis, killer cell activity or complement mediated lysis. These types of allergies are relatively unusual but can include some allergies to drugs.
  • Type III hypersensitivity develops when immune complexes are formed in large quantites or cannot be cleared adequately by the reticuloendothelial system. The immune complexes usually result from the deposition of antibody, usually IgM or IgG, allergen complexes at these sites. In normal circumstances, antibody binds to allergen and is cleared by a variety of tissue cells.
  • Type IN or delayed type hypersensitivity does not involve antibody but instead the prolonged activation of T lymphocytes. These T cells are capable of secreting soluble factors causing tissue damage and enhancing the recruitment and activation of other cell types to the tissues. Incoming cells themselves contribute to the inflammation and tissue damage. DTH is most seriously manifested when antigens (for example those associated with mycobacteria tuberculosis) are trapped in a macrophage and cannot be cleared. T cells are then stimulated to elaborate cytokines which mediate a range of inflammatory responses. DTH reactions are less common than Type I reactions but are seen in graft rejection and allergic contact dermatitis which is generally manifested as a contact sensitivity (allergy usually involving skin rash) to environmental "contact allergens" such as heavy metals.
  • WO 95/01301 discloses an immunological tolerance-inducing agent comprising a mucosa-binding agent linked to a specific tolerogen.
  • WO95/10301 also includes mention of the treatment of allergy using a mucosa binding agent coupled to an allergen.
  • Other researchers such as Tamura et al (1997 Vaccine 15: 225-229) have taken directly the protocol of WO 95/10301 and tested its efficacy in preventing allergy in a murine model of Type I allergy. They reported a significant lowering of IgE levels which are a strong predictor of efficacy and they cite data, following administration of EtxB coupled to ovalbumin (the results were not included), which shows that EtxB was not effective once IgE levels are established.
  • immunological tolerance may include a number of different mechanisms which may be summarised as follows:
  • GM1 is a member of family of ganglioside receptors comprising sialic acid containing glycolipids (also called glycosphingolipids) which are formed by a hydrophobic portion, the ceramide and a hydrophilic part, that is the oligosaccharide chain.
  • glycolipids also called glycosphingolipids
  • gangliosides are usually associated with plasma membranes, where they can act as receptors for a variety of molecules and have been shown to take part in cell-to-cell interaction and in signal transduction.
  • Glycosphingolipids other than GM1 gangliosides, are also capable of acting as plasma membrane receptors.
  • One member of this glycosphingolipid family which acts as a plasma membrane receptor is globotriaosylceramide, (Gb3), which is optionally represented as
  • Gb3 mediates the internalisation of a family of E. coli derived toxins called verotoxins (NT), into susceptible cells by capping and receptor mediated endocytosis (RM ⁇ ) (Khine and Lingwood 1994 J Cell Physiol 161: 319-332).
  • the action of the verotoxins (NT) in causing human vascular disease is dependent on the specific recognition of receptors on target endothelial cells ( ⁇ yholm et al 1996 Chem Biol 3: 263-275).
  • Gb3 has been shown to be the most effective receptor for VT-1 in vitro and to be a functional plasma membrane receptor which mediates cytopathology for most sensitive cells (Boyd et al 1994 ⁇ ur J Biochem 223: 873-878).
  • E. coli verotoxin has been characterized as having an "A" subunit of approximately 31,000 daltons and five "B" subunits each having an approximate molecular weight of 5,500 daltons.
  • the A subunit possesses the biological activity of the toxin which is involved in inhibiting protein synthesis, whereas the B subunits are presumed to mediate specific binding and receptor-mediated uptake of the toxin.
  • the verotoxins are closely related to Shiga toxin and have been widely referred to as Shiga-like toxins (SLTs) as they mediate the same enterotoxic, cytotoxic, and neuro toxic effects of Shiga toxin, produced by the Shigella dysenteriae type 1. It has been shown that the antigenically distinct Shiga like toxins (SLTs) SLT-1 and SLT-II use Gb3 as functional receptors (Samuel et al 1990 Infect Immun 58: 611-618) whereas the SLT II variants may use different receptors.
  • the B subunits of verotoxins, VT1 and VT2 demonstrate terminal Gal 1-4 Gal- dependent binding.
  • the binding B oligomers of SLTI, SLTII and SLTIIvh recognize host cell glycolipid receptors containing at minimum the disaccharide subunit ⁇ Gal(l-4) ⁇ Gal at the non-reducing terminus.
  • SLTII vp has been shown to bind to the receptors containing this subunit but not necessarily at the non-reducing end (Samuel, J.
  • the carbohydrate recognition domain also known as the P (k) trisaccharide, of the Gb3 receptor for SLT-1 (St Hilaire et al 1994 Biochem 33: 14452-14463) is represented as
  • VTEC VT producing E coli
  • SLT-EC Shiga-like toxin producing E coli
  • the present invention now seeks to provide new ways of treating disorders such as allergic conditions or hypersensitivity conditions, autoimmune and inflammatory diseases, cancers such as human leukaemias of a T cell origin, transplantation rejection and graft versus host disease (GVHD), all collectively called "immune disorders”.
  • disorders such as allergic conditions or hypersensitivity conditions, autoimmune and inflammatory diseases, cancers such as human leukaemias of a T cell origin, transplantation rejection and graft versus host disease (GVHD), all collectively called "immune disorders”.
  • an agent in the manufacture of a medicament to affect an immune disorder; wherein the agent is capable of modulating a glycosphingolipid associated activity; and wherein the modulation of the glycosphingolipid associated activity affects an immune disorder.
  • an agent in the manufacture of a medicament to affect an immune disorder; wherein the agent is capable of modulating a globotriaosylceramide (Gb3) associated activity; and wherein the modulation of the Gb3 associated activity affects an immune disorder.
  • Gb3 globotriaosylceramide
  • the agent is capable in vivo of modulating lymphocyte populations.
  • the agent is capable of acting as a vaccine adjuvant.
  • the agent is capable of promoting the antigenicity of a protein.
  • an assay method for identifying an agent according to the present invention capable of affecting an immune disorder comprising: (a) contacting an agent with a glycosphingolipid; (b) determining whether the agent modulates a glycosphingolipid associated activity; such that the modulation of the glycosphingolipid associated activity is indicative that the agent may be capable of affecting an immune disorder.
  • the assay is an assay to screen for an agent useful in the prevention and/or treatment of an immune disorder.
  • a process comprising the steps of: (a) performing the assay according to the present invention; (b) identifying one or more agents capable of modulating a glycosphingolipid associated activity; and (c) preparing a quantity of those one or more agents.
  • a process comprising the steps of: (a) performing the assay according to the present invention; (b) identifying one or more agents capable of modulating a glycosphingolipid associated activity; and (c) preparing a pharmaceutical composition comprising one or more identified agents.
  • a process comprising the steps of: (a) performing the assay according to the present invention; (b) identifying one or more agents capable of modulating a glycosphingolipid associated activity; and (c) modifying one or more identified agents capable of modulating a glycosphingolipid associated activity; and (d) preparing a pharmaceutical composition comprising those one or more modified agents.
  • the agent identified had not previously been known to affect an immune disorder through modulation of a glycosphingolipid associated activity.
  • a method of affecting an immune disorder with one or more agents wherein the agent is capable of modulating a glycosphingolipid associated activity in an in vitro assay method; and wherein the in vitro assay method is the assay method defined in the present invention.
  • a method of affecting in vivo an immune disorder with one or more agents wherein the agent is capable of modulating a glycosphingolipid associated activity in an in vitro assay method; and wherein the in vitro assay method is the assay method defined in the present invention.
  • an agent according to the present invention for use as a pharmaceutical.
  • an agent according to the present invention in the manufacture of a medicament to affect an immune disorder.
  • a pharmaceutical composition comprising or prepared from an agent according to the present invention.
  • the agent capable of modulating a glycosphingolipid associated activity is a Gb3 binding agent.
  • the agent is a verotoxin.
  • the agent is VtxB.
  • the agent is the wild type VtxB.
  • the agent has a pentameric structure.
  • the agent(s) is/are non-toxic.
  • the agent is either a mutant of VtxB which is capable of modulating a glycosphingolipid associated activity or other equivalent proteins thereof.
  • the agent is VtxB and mutants thereof which are capable of modulating a glycosphingolipid associated activity.
  • glycosphingolipid is Gb3.
  • the agent capable of modulating a glycosphingolipid associated activity is capable of cross-linking Gb3 receptors.
  • VtxB is a suitable example of an agent which is capable of cross-linking Gb3.
  • the medicament is used for the treatment or prophylaxis of an immune disorder.
  • the medicament includes one or more antigens which are optionally co- administered with the agent.
  • the agent is coupled to an antigen.
  • the agent is uncoupled to an antigen.
  • the agent may be administered to a mammal with or without TO- administration of an antigen.
  • the mammal is a human - e.g. a human patient.
  • VtxB E.coli verotoxin
  • VtxB can serve as a vaccine adjuvant which is capable of promoting the antigenicity of a protein.
  • immune disorders may be treated with an agent, such as VtxB, capable of modulating a glycosphingolipid associated activity (for example, a Gb3 associated activity) for instance, which is not coupled with an antigen.
  • the linkage of the components was not found to be necessary. Furthermore, our findings indicate that the mechanisms of protection against immune disorders may include, though not be limited to an immunomodulation of the immune system.
  • the present invention is advantageous because immune disorders can be treated with an agent capable of modulating a glycosphingolipid associated activity.
  • the agent may be coupled or uncoupled to an antigen.
  • glycosphingolipid as used with respect to the present invention include glycosphingolipids capable of acting as receptors for VtxB or VtxB-like agents as well as active fragments thereof.
  • the glycosphingolipid is not GM1.
  • glycosphingolipid associated activity includes any one or more of modulating or immunomodulating a glycosphingolipid receptor, modulating any signalling event prior to, during or subsequent to glycosphingolipid binding.
  • Vtx refers to the verotoxin and VtxB refers to the B subunit of the vero toxin. In other texts, these may sometimes be identified as VT or VTB respectively.
  • the toxin can be referred to as a Shiga-like toxin (SLT) or Stx.
  • SLT Shiga-like toxin
  • Stx Stx.
  • the verotoxin can be made synthetically or isolated from natural sources. Alternatively, it can be obtained from commercial sources.
  • shiga-like toxin or “SLT” refers to group of toxins produced by enterohemorrhagic E. coli that resemble the S ⁇ tge/V ⁇ -produced shiga toxins as is commonly understood in the art. These toxins comprise an enzymatically active A subunit and a multimeric receptor binding B subunit.
  • SLTs include SLTI and the various grouped toxins designated in the art SLTII.
  • GM1 binding agent includes any agent which acts as an immunomodulator through interacting with a GM1 ganglioside receptor.
  • Etx herein means the E. coli heat labile enterotoxin and EtxB is the B subunit of Etx. In other texts, these may sometimes be identified as LT or Lt and LTB or LtB respectively.
  • adjuvant includes a substance that enhances an immune response to an antigen.
  • vaccine adjuvant includes an agent which is delivered with an unrelated antigen, such that the agent is capable of facilitating an immune response to the unrelated antigen. In this way, the agent acts as a so-called vaccine adjuvant.
  • vaccine adjuvant includes the term "mucosal adjuvant”.
  • mucosal adjuvant includes an agent which is delivered mucosally with an unrelated antigen, such that the agent is capable of facilitating a mucosal immune response to the unrelated antigen. In this way, the agent acts as a so-called mucosal adjuvant.
  • a “vaccine carrier” includes a carrier of relevant antigens (Szostak et al 1996 J Biotechnol 44: 161-170)
  • macosal surfaces includes but is not limited to oral, sublingual, nasal, vaginal, rectal, salivary, intestinal and conjunctival surfaces.
  • macosal membrane and/or “mucosal tissue” includes but is not limited to the intestine, the lung, the mouth, the genital tract, the nose and the eye.
  • macosal immunogen includes an agent administerable by a mucosal route that has the capability to evoke cell mediated immune reactions and/or delayed type hypersensitivity reactions.
  • immunological or oral tolerance means a reduction in immunological reactivity of a host towards a specific antigen(s). Immunological or oral tolerance may not mean a complete suppression of the immune response to a particular antigen but it is a form or tolerance also known as “immune deviation” or “split tolerance”.
  • immune deviation or “split tolerance” can be used to describe the likely preservation of local IgA responses with the retention of some IgG responses but with the down regulation of delayed hypersensitivity and IgE responses.
  • tolerance means a state of specific immunological unresponsiveness.
  • immune disorder includes conditions such as allergic conditions or hypersensitivity conditions, autoimmune and inflammatory diseases, cancers such as human leukaemias of a T cell origin, transplantation rejection and graft versus host disease (GVHD).
  • autoimmune and inflammatory diseases include conditions such as allergic conditions or hypersensitivity conditions, autoimmune and inflammatory diseases, cancers such as human leukaemias of a T cell origin, transplantation rejection and graft versus host disease (GVHD).
  • GVHD graft versus host disease
  • autoimmunity is used to describe the mechanism by which the body generates an immune response to self-antigens.
  • agent capable of modulating a glycosphingolipid associated activity can be used to describe any agent which acts as an immunomodulator through interacting with a glycosphingolipid.
  • immunomodulator includes any agent that alters the extent of the immune response to an antigen, by altering the antigenicity of the antigen or by altering in a nonspecific manner the specific reactivity or the nonspecific effector associated mechanisms of the host.
  • Viral delivery mechanisms include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectos, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors.
  • Non-viral delivery mechanisms include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof.
  • the routes for such delivery mechanisms include but are not limited to mucosal, nasal, oral, parenteral (such as intravenous (iv), intramuscular (im), or subcutaneous (sc) route), gastrointestinal, topical, or sublingual routes.
  • co-administered means that the site and time of administration of each of the agent and the antigen are such that the necessary modulation of the immune system is achieved.
  • agent and the antigen may be administered at the same moment in time and at the same site, there may be advantages in administering the agent at a different time and to a different site from the antigen.
  • the agent and antigen may even be delivered in the same delivery vehicle (such as a MacrosolTM-see WO95/13795 and WO96/14871).
  • administered includes but is not limited to delivery by a mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestable solution; a parenteral route where delivery is by an injectable form, such as, for example, an intravenous, intramuscular or subcutaneous route.
  • systemic immunisation means the introduction of an antigen into a non- mucosal tissue such as the skin or the blood.
  • self antigens means components derived from host tissues.
  • target interaction components includes but is not limited to an agent capable of modulating a glycosphingolipid associated activity, a glycosphingolipid and/or an antigen.
  • uncoupled which is synonymous with the term “unlinked” - means that the agent is not coupled to the antigen.
  • Coupled which is synonymous with the term “linked” - means the linkage of the agent with the antigen - which includes but is not limited to direct linkage (such as by a covalent bond) or indirect linkage such as by the provision of suitable spacer groups.
  • the agent and/or antigen can be coupled to another entity.
  • fect includes modulation, such as treatment, prevention, suppression, alleviation, restoration or other alteration of pre-existing condition and/or to potentially affect a future condition, as well as any combination thereof.
  • an “antigen” means an agent which, when introduced into an immunocompetent animal, stimulates the production of a specific antibody or antibodies that can combine with the agent.
  • the antigen may be a pure substance, a mixture of substance or soluble or particulate material (including cells or cell fragments).
  • the term includes any suitable antigenic determinant, auto-antigen, self-antigen, tolerogen, allergen, hapten, and immunogen, or parts thereof, as well as any combination thereof, and these terms are used interchangeably throughout the text.
  • a "tolerogen” means a tolerated antigen.
  • a "hapten” means a small molecule which can act as an epitope but is incapable by itself of eliciting an antibody response.
  • antigens include but are not limited to: insulin; myelin basic protein; antibody or antibody fragments; gamma globulins; transplantation antigens or cells expressing said transplantation antigens, such as red blood cells, platelets and lymphocytes; bacterial toxins; and/or synthetic peptides and/or corresponding variants, homologues, derivatives or fragments thereof of the above mentioned antigens.
  • An "allergen” includes any antigen that stimulates an allergic reaction, inducing a Type I hypersensitivity reaction.
  • allergic condition includes but is not limited to asthma, allergic cough, allergic rhinitis and conjunctivitis, atopic eczema and dermatitis, uticaria, hives, insect bite allergy, dietary allergy (peanut, fish milk, wheat etc) and drug allergies.
  • hypersensitivity condition includes but is not limited to conditions such as contact hypersensitivity induced by plant poison ivy.
  • agent includes entities capable of modulating a glycosphingolipid associated activity.
  • the agent can be one or more of an inorganic or organic chemical, as well as combinations thereof.
  • the agent can be a polypeptide as well as a variant/homologue/derivative/fragment thereof so long as they retain the required immunomodulatory activity. It also includes mimics and equivalents and mutants thereof.
  • Other agents for the prevention and/or treatment of immune disorders include antibodies to the target interaction components. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, fragments produced by a Fab expression library and specifically designed humanised monoclonal antibodies.
  • Agents capable of modulating a glycosphingolipid associated activity may be designed and produced as outlined above, by methods which are known in the art.
  • the agent of the invention is a protein such as the VtxB subunit it may be produced, for use in all aspects of this invention by a method in which the gene or genes coding for the specific polypeptide chain (or chains) from which the protein is formed, is inserted into a suitable vector and then used to transfect a suitable host.
  • the gene coding for the polypeptide chain from which the VtxB assemble may be inserted into suitable expression vectors (such as plasmids) which are then used to transfect host cells, such as Vibrio sp.60.
  • the protein is purified and isolated in a manner known per se. Mutant genes expressing active mutant VtxB protein may then be produced by known methods from the wild type gene.
  • verotoxins are encoded by one or more bacteriophages and, furthermore, individual strains may produce either one or both VT1 and VT2. Both the natural and the recombinant forms of E. coli verotoxin have been isolated.
  • One such recombinant cloned toxin is pJLB28 which expresses both the A and B subunits (Huang, A. et al (1986) J. Bacteriol. 166, 375-379).
  • a target interaction component is a protein
  • procedures well known in the art may be used for the production of antibodies to that component.
  • various hosts including goats, rabbits, rats, mice, etc. may be immunized by injection with the target interaction component or any derivative or homologue thereof or oligopeptide which retains immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not limited to, Freund's, mineral gels such as aluminium hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are potentially useful human adjuvants.
  • a target interaction component is a protein
  • monoclonal antibodies to that component may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, pp 77-96).
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991; Nature 349:293-299).
  • Antibody fragments which contain specific binding sites for a target interaction components may also be generated.
  • fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al (1989) Science 256:1275-128 1).
  • the target interaction components of the present invention or a derivative or homologue thereof and/or a cell line that expresses the target interaction components of the present invention or a derivative or homologue thereof may be used to screen for antibodies, peptides, or other agent, such as organic or inorganic molecules, that act as modulators of the target interaction, thereby identifying a therapeutic agent capable of modulating the target interaction. For example, antibodies capable of modulating the target interaction may be identified.
  • screening of peptide libraries or organic libraries made by combinatorial chemistry with recombinantly expressed target interaction components or a derivative or homologue thereof or cell lines expressing the target interaction components or a derivative or homologue thereof may be useful for identification of therapeutic agents that function by modulating the target interaction.
  • Synthetic compounds, natural products, and other sources of potentially biologically active materials can be screened in a number of ways deemed to be routine to those of skill in the art.
  • a target interaction component polypeptide, its immunogenic fragments or oligopeptides thereof can be used for screening therapeutic compounds in any of a variety of drug screening techniques.
  • the polypeptide employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The abolition of activity or the formation of binding complexes between the target interaction component and the agent being tested may be measured.
  • phage display can be employed in the identification of candidate agents which affect the target interaction components.
  • Phage display is a protocol of molecular screening which utilises recombinant bacteriophage.
  • the technology involves transforming bacteriophage with a gene that encodes an appropriate ligand (in this case a candidate agent) capable of reacting with a target interaction component (or a derivative or homologue thereof) or the nucleotide sequence (or a derivative or homologue thereof) encoding same.
  • the transformed bacteriophage (which preferably is tethered to a solid support) expresses the appropriate ligand (such as the candidate agent) and displays it on their phage coat.
  • the entity or entities (such as cells) bearing the target molecules which recognises the candidate agent are isolated and amplified.
  • the successful candidate agents are then characterised.
  • Phage display has advantages over standard affinity ligand screening technologies.
  • the phage surface displays the candidate agent in a three dimensional configuration, more closely resembling its naturally occuring conformation. This allows for more specific and higher affinity binding for screening purposes.
  • the present invention provides a method for screening a plurality of agents for specific binding affinity with the target interaction component or a derivative or homologue thereof comprising providing a plurality of agents; combining the target interaction components or a derivative or homologue thereof with each of a plurality of agents for a time sufficient to allow binding under suitable conditions; and detecting binding of the target interaction components, or a derivative or homologue thereof to each of the plurality of agents, thereby identifying the agent or agents which specifically bind the target interaction components.
  • the plurality of agents may be produced by combinatorial chemistry techniques known to those of skill in the art.
  • Another technique for screening provides for high throughput screening of agents having suitable binding affinity to the target interaction component polypeptides and is based upon the method described in detail in WO 84/03564.
  • large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the peptide test agents are reacted with the target interaction component fragments and washed.
  • a bound target interaction component is then detected - such as by appropriately adapting methods well known in the art.
  • a purified target interaction component can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non- neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising administering a therapeutically effective amount of an agent capable of modulating a glycosphingolipid associated activity and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
  • the pharmaceutical compositions may be for human or animal usage and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, excipient or adjuvant.
  • a pharmaceutically acceptable diluent carrier, excipient or adjuvant.
  • the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • the pharmaceutical composition may be formulated together with an appropriate antigen.
  • kits comprising separate compositions for each of the therapeutic agent and the antigen.
  • the pharmaceutical compositions will comprise one or more of: an agent that has been screened by an assay of the present invention; wherein the agent is capable of modulating a glycosphingolipid associated activity.
  • the present invention also relates to pharmaceutical compositions comprising effective amounts of antigen in admixture with a pharmaceutically acceptable diluent, carrier, excipient or adjuvant (including combinations thereof)-
  • the present invention also provides a method of treating a mammal, such as a human patient, comprising administering to said mammal an effective amount of the pharmaceutical composition of the present invention.
  • the present invention relates to pharmaceutical compositions which may comprise all or portions of the target interaction components alone or in combination with at least one other agent, such as a stabilizing compound, and may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • composition formulation requirements may be different composition formulation requirements dependent on the different delivery systems.
  • the pharmaceutical composition of the present invention may be formulated to be delivered by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.
  • the formulation may be designed to be delivered by both routes.
  • the agent is to be delivered mucosally through the gastrointestinal mucosa, it is preferably stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.
  • a physician will determine the actual dosage which will be most suitable for a subject and it will vary with the age, weight and response of the particular subject. While a single dose of the agent and the antigenic determinant may be satisfactory, multiple doses are contemplated within the scope of the invention.
  • compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously.
  • compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
  • compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
  • Expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of the agent to the targeted tissue and/or cell population. Methods which are well known to those skilled in the art can be used to construct recombinant vectors containing the agent. Alternatively, the agent can be delivered to target cells in liposomes.
  • the controlled release of vaccine antigens on mucosal surfaces using biodegradable polymer microspheres may help to target antigens and reduce the numbers of doses required for primary immunisation (Gupta and Siber 1995 Vaccine 13: 1263-1276)
  • rNV Norwalk Virus-like particles
  • VLPs Viral Like Particles
  • One preferred method of oral delivery uses formations as described in WO95/13795, WO96/17593 and WO96/17594. These formulations allow macromolecules such as proteins to be solubilised in “oils” for oral delivery. Such formulations therefore allow delivery of the macromolecules to mucosal surfaces in the gut.
  • the therapeutic agent is a protein
  • This system utilises the bacterium Lactococcus lactis to deliver proteins, for instance orally or indeed by other mucosol routes such as nasally.
  • the present invention provides the use of an agent in the manufacture of a medicament to affect an immune disorder; wherein the agent is capable of modulating a glycosphingolipid associated activity.
  • the present invention provides an agent capable of modulating a glycosphingolipid associated activity and which is potently immunogenic.
  • Gb3 binding agent or an agent having an effect on Gb3 intracellular signalling events, but no Gb3 binding activity, in the preparation of a medicament for the treatment or prophylaxis of inflammatory conditions, autoimmune diseases, transplant rejection, graft versus host disease, immune disorders or cancer.
  • the agent is VtxB.
  • a pharmaceutical formulation comprising a Gb3 binding agent, or an agent having an effect on Gb3 intracellular signalling events, but no Gb3 binding activity, optionally together with one or more pharmaceutically acceptable carriers, diluents or excipients.
  • a pharmaceutical formulation as claimed in paragraph 5 which also comprises one or more allergens/antigens.
  • a pharmaceutical formulation as claimed in paragraph 5 or paragraph 6 which is a vaccine formulation.
  • a method for the treatment or prophylaxis of an inflammatory condition, an autoimmune disease, transplant rejection, GVHD, allergic, or other hypersensitive conditions or cancer which comprises administering to a subject an effective amount of a Gb3 binding agent, or an agent having an effect on Gb3 intracellular signalling events, but no Gb3 binding activity.
  • a method of vaccinating a subject against, for example an immune disorder which comprises administering to a subject an effective amount of a Gb3 binding agent, or an agent having an effect on Gb3 intracellular signalling events, but no Gb3 binding activity.
  • a product comprising an agent having Gb3 binding activity, or an agent having an effect on Gb3 mediated intracellular signalling events, but no Gb3 binding activity, in the preparation of a medicament to treat an inflammatory condition, an autoimmune disease, transplant rejection, CVHD, an immune disorder or cancer, and at least one antigen allergen as a combined preparation for simultaneous, separate or sequential use.
  • FIG 1 shows the serum anti-VtxB antibody responses in NTH mice following subcutaneous immunisation with VtxB.
  • Figure 2 shows the upregulation in IL-2R (CD25) expression induced by binding of EtxB (A), CtxB (B) and VtxB (C) to lymphocyte populations.
  • the serum anti-VtxB antibody titre in column 1 of Figure 1 was obtained in NTH mice immunised with lO ⁇ g VtxB in complete Freund's adjuvant on day 1 and lO ⁇ g VtxB in incomplete Freund's adjuvant on Days 8 and 15 whereas the serum anti-VtxB antibody titre in column 2 was obtained in NTH mice immunised with lO ⁇ g of VtxB without complete or incomplete Freund's adjuvant on days 1, 8 and 15. Mice were bled on day 28. Antibody titres to VtxB were determined on VtxB-coated microtitre plates.
  • Example 1 Serum anti-VtxB antibody responses in NIH mice following subcutaneous immunisation with VtxB.
  • NTH mice were immunised (s.c.) on days 1, 8 and 15 with lO ⁇ g of VtxB in Freund's adjuvant, lO ⁇ g of VtxB alone or PBS (control). Mice were bled on day 28. Antibody titres to VtxB were determined on VtxB-coated microtitre plates.
  • mice ranging between the ages of 6 to 8 weeks were sacrificed and Mesenteric lymph node tissue was subsequently removed into Hanks balanced salt solution (HBSS without Calcium and Magnesium). Lymphocytes were then dispersed into the solution and away from fibrous tissue through gently pressing the tissue through a wire mesh.
  • Hanks balanced salt solution HBSS without Calcium and Magnesium
  • CD8 + T cells were purified by separation in a magnetic MACS column. Briefly, 100 million cells were resuspended in 300 ⁇ l MACS buffer (PBS/0.5% BSA/0.5mM EDTA). For positive selection 20 ⁇ l of MACS anti-CD8 antibody was added to cell suspension and the mixture was incubated on ice for a period of 30 minutes to allow time for effective antibody interaction. Following incubations cells were subsequently washed in cold MACS buffer and passed through a pre-washed VS+ selection column (MACS).
  • MACS buffer PBS/0.5% BSA/0.5mM EDTA
  • VtxB B subunit of E.coli verotoxin
  • Gb3 glycolipid receptor
  • Agents capable of modulating glycosphingolipid associated activity are tested by any one of a variety of methods. Examples of such methods include, but are not limited to the following methods:
  • Binding to a glycosphingolipid is determined by using purified Gb3 to coat microtiter plates. Following blocking of further non-specific protein binding to the plate, the agent under investigation is applied to the plate and allowed to interact prior to washing and detection with specific antibodies to said agent. Conjugation of the antibodies either directly or indirectly to an enzyme or radiolabel allows subsequent quantification of binding either using colormetric or radioactivity based methods (ELISA or RIA respectively).
  • the pentasaccharide moiety of a glycosphingolipid such as Gb3 is bound to a suitable column matrix in order to allow standard affinity chromatography to be performed. Removal of known compounds applied to the column from the diluent are used as evidence for binding activity. Alternatively, where mixtures of compounds are applied to the column, elution and subsequent analysis allows the properties of the agent capable of modulating glycosphingolipid associated activity to be determined.
  • Protein analysis includes peptide sequencing and tryptic digest mapping followed by comparisons with available databases. If eluted proteins cannot be identified in this way, then standard biochemical analysis, such as, for example, mass determination by laser desorption mass spectrometry is used to further characterise the compound. Non- proteins eluted from Gb3-affinity columns are analysed by HPLC and mass spectrometry of single homogenous peaks.
  • glycosphingolipid such as Gb3
  • affinity of the interaction may be determined using plasmon surface resonance as previously reported by (Kuziemko et al (1996) Biochem 35:6375-6384).
  • the identification of agents capable of modulating a glycosphingolipid associated activity such that the modulation of the glycosphingolipid associated activity affects an autoimmune condition is determined as follows:
  • Animal models of an autoimmune disease can be used to assess the efficacy of an agent capable of modulating a glycosphingolipid associated activity to prevent autoimmune disease.
  • An example of the use of an animal model of autoimmune disease is the induction of arthritis in DBA/1 mice. Evaluation of agents is conducted by injecting groups of DBA/1 mice with bovine collagen in Complete Freund's Adjuvant (CFA) by intradermal (id) injection in the flank in the presence or absence of the agent capable of modulating a glycosphingolipid associated activity in order to assess prevention of disease development.
  • CFA Complete Freund's Adjuvant
  • mice are given a second injection of collagen in Incomplete Freund's Adjuvant (IF A) in the presence/absence of the agent capable of modulating a glycosphingolipid associated activity. All animals are assessed for severity of disease on day 45 by measuring hind limb ankle thickness or scoring each hind limb digit for swelling.
  • IF A Incomplete Freund's Adjuvant
  • the identification of agents capable of modulating a glycosphingolipid associated activity such that the modulation of the glycosphingolipid associated activity affects an allergic condition and/or a hypersensitivity condition is determined as follows: Laboratory animals are stimulated to produce antigen-specific IgE by methods well known in the art. By way of example, mice are challenged with alum precipitated soluble protein antigen (e.g. ovalbumin or allergens known to be involved in human allergic diseases such as ragweed or house dust mite antigens) either subcutaneously or intraperitoneally.
  • alum precipitated soluble protein antigen e.g. ovalbumin or allergens known to be involved in human allergic diseases such as ragweed or house dust mite antigens
  • agents capable of modulating glycosphingolipid associated activity are admimstered to mice either in the presence or absence of the challenge antigen at a range of doses, and by a variety of routes.
  • oral route is the preferred method of administration, delivery can be by other mucosal surfaces or parenterally.
  • the frequency of such administration as well as the timing of repetitive dosing is also investigated.
  • intervention strategies are utilised either prior to the IgE inducing antigen challenge (prophylaxis) or after the
  • Antigen challenge can be either with (i) the antigen used as part of the prophylactic or treatment protocol; (ii) an unrelated antigen or (iii) a mixture of the challenge and unrelated antigen in order to test the specificity of the response and the induction of bystander suppression respectively.
  • Efficacy is determined in a variety of ways and is manifested as a number of different outcomes.
  • Antigen-specific IgE levels Measurement of serum IgE by specific ELISA (as described) is used to determine whether prophylactic or treatment protocols are capable of reducing levels of serum antigen-specific IgE. Other methods known in the art for the determination of IgE response are used either as alternatives to ELISA or in order to provide complementary data. Such methods include the so-called “Ussing Chamber test” or "passive cutaneous anaphylaxis” assay. A reduction in specific IgE, as determined by any of these assays, is a strong marker of potential clinical efficacy.
  • T-cell reactivity The responses of T-cells, derived from secondary lymphoid organs of the treated animals to the challenge antigen, is investigated using established methodology. Cell suspensions are prepared and cultured, in the presence or absence of the challenge antigen. At appropriate time intervals after the initiation of the cultures, samples are assessed for cell proliferation and cytokine production.
  • Cytokines are measured by specific capture ELISA, by intracellular staining followed by cytometric analysis, by RT-PCR or by other established procedures. Comparison of cell proliferation and cytokine production, in the presence of antigen as opposed to its absence, provides in each case a measure of that part of the response which is specific to the challenge antigen.
  • Evidence of efficacy of prophylactic or treatment protocols is demonstrated by a reduction in the production of Th2 associated cytokines (in particular IL-4) or by an increased expression of cytokines which are involved in down-regulating the allergic response (for example, IL-10 or TGF ⁇ ).
  • IgG and IgA levels Protocols which do not reduce the levels of antigen specific IgE can still be considered as potentially effective in the event that they are also able to enhance the production of other non-allergy associated antibody isotopes.
  • Standard antigen specific ELISA assays utilising detecting antibodies specific for IgG and specific subclass thereof, and IgA are used for this purpose.
  • Enhanced production of secreted or serum IgG or IgA antibodies indicate efficacy since such antibodies can be expected to prevent an allergen from cross-linking IgE bound to mast cells, basopbils and eosinophils or limit the uptake of antigen across the mucosal epithelium and hence prevent the subsequent allergic inflammatory response.
  • glycosphingolipid binding assay for the detection of glycosphingolipid binding agents such as verotoxins has been developed according to the teachings as set out in US5164298. This assay is based on the immobilization of deacylated globotriaosyl ceramide in microtitre wells.
  • deacylated Gb3 is bound to a microtitre plate for use in an ELISA for the detection of a Gb3 binding agent such as verotoxin.
  • a Gb3 binding agent such as verotoxin.
  • the verotoxin present in verotoxin containing samples, or verotoxin positive controls will bind to the deacylated Gb3 which has been bound to the plate.
  • glycolipid-bound toxin is visualized by use of a polyclonal rabbit antiserum and an immunoperoxidase indicator system.
  • Other indicator systems well known to those skilled in the art of ELISA would also be suitable.
  • a deacylated Gb3, having a free amino group could also be covalently bound to another protein either directly or through the incorporation of a spacer arm. This second protein could then be used in the primary binding step in the assay.
  • the glycosphingolipid instead of attaching the glycosphingolipid to a protein as an assay component, it could be covalently bound directly to a solid phase support as an assay component or alternatively, the assay component may be a liposome which contains the glycosphingolipid receptor.
  • solid phase supports include microtitre plates, test tubes, glass beads, nitrocellulose and latex particles.
  • the plates or test tubes may be of glass or a plastic such as polyvinyl chloride, polystyrene or latex.
  • the principle of using a glycosphingolipid in a receptor-based assay for verotoxin could be applied to any of the well known assay technologies, including radioimmunoassay, cell-binding cytotoxicity assays, thin layer chromatography assays and agglutination assays.
  • the principle could also be used in a fluorescence based receptor assay for verotoxin using toxin sensitive target cells as the receptor bearing vehicle.
  • Binding of VtxB to Gb3 or deacylated Gb3 can also be examined using an adaptation of the GM1 ELISA reported by Amin, T., & Hirst, T.R (1994 Prot. Express, and Purif. 5, 198-204) where GM1 is replaced by Gb3 or deacylated Gb3 respectively.
  • Sera and gut secretions are examined for the presence of anti-B subunit IgG and IgA antibodies by ELISAs in which samples are applied to microtitre plates (Immulon I, Dynateck, USA) coated with 5 ⁇ g/ml of VtxB in PBS.
  • Anti-B subunits IgA antibodies in gut secretion supernatants are extrapolated from a standard curve made by coating 2 rows of wells on each plate with l ⁇ g/ml rabbit anti-mouse IgA ( ⁇ chain specific; Zymed Lab, USA) in PBS followed by addition of l ⁇ g/ml of mouse myeloma IgA (MOPC 315, Sigma, USA).
  • LFN- ⁇ LFN- ⁇ is used. Briefly, microtiter plates are coated with rat antibodies to mouse IL-2, IL-4, IL-5, IL-10 and IFN- ⁇ . Plates are blocked with 2% (w/v) bovine serum albumin. Supernatants from culture medium are added to wells and diluted down. One row on each plate for each cytokine contains a standard amount of recombinant cytokines. Plates are then incubated with 0.5 ⁇ g/ml of biotinylated anti-cytokine monoclonal antibodies followed by addition of avidine-peroxidase and 3,3', 5,5' - Tetramethylbenzidene (TMB) substrate and read at A 450nm .
  • TMB Tetramethylbenzidene

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Abstract

L'invention concerne l'utilisation d'un agent dans la fabrication d'un médicament destiné à agir sur un trouble immunitaire. L'agent est capable de moduler une activité liée à un glycosphingolipide. La modulation de l'activité liée au glycosphingolipide agit sur le trouble immunitaire.
PCT/GB1999/000290 1998-01-28 1999-01-28 Agent capable de moduler l'activite liee a un glycosphingolipide, agissant ainsi sur un trouble immunitaire WO1999038530A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP99902688A EP1049489A1 (fr) 1998-01-28 1999-01-28 Agent capable de moduler l'activite liee a un glycosphingolipide, agissant ainsi sur un trouble immunitaire
CA002318190A CA2318190A1 (fr) 1998-01-28 1999-01-28 Agent capable de moduler l'activite liee a un glycosphingolipide, agissant ainsi sur un trouble immunitaire
AU22900/99A AU2290099A (en) 1998-01-28 1999-01-28 Agent capable of modulating a glycosphingolipid-associated activity thus affecting an immune disorder

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GB9801871.6 1998-01-28
GBGB9801871.6A GB9801871D0 (en) 1998-01-28 1998-01-28 Therapies

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997002045A1 (fr) * 1995-07-05 1997-01-23 Oratol Limited Agents therapeutiques pour maladies auto-immunes
CA2163716A1 (fr) * 1995-11-24 1997-05-25 Clifford A. Lingwood Compositions pharmaceutiques a base de verotoxine et traitements medicaux qui l'utilisent
WO1997029181A1 (fr) * 1996-02-09 1997-08-14 The Ontario Cancer Institute Procede d'elimination selective des cellules cd77+ de la moelle osseuse
WO1998051326A1 (fr) * 1997-05-16 1998-11-19 Hsc Research And Development Limited Partnership Inhibition de l'angiogenese au moyen de verotoxines

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997002045A1 (fr) * 1995-07-05 1997-01-23 Oratol Limited Agents therapeutiques pour maladies auto-immunes
CA2163716A1 (fr) * 1995-11-24 1997-05-25 Clifford A. Lingwood Compositions pharmaceutiques a base de verotoxine et traitements medicaux qui l'utilisent
WO1997029181A1 (fr) * 1996-02-09 1997-08-14 The Ontario Cancer Institute Procede d'elimination selective des cellules cd77+ de la moelle osseuse
WO1998051326A1 (fr) * 1997-05-16 1998-11-19 Hsc Research And Development Limited Partnership Inhibition de l'angiogenese au moyen de verotoxines

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GORDON V M ET AL: "AN ENZYMATIC MUTANT OF SHIGA-LIKE TOXIN II VARIANT IS A VACCINE CANDIDATE FOR EDEMA DISEASE OF SWINE", INFECTION AND IMMUNITY, vol. 60, no. 2, 1 February 1992 (1992-02-01), pages 485 - 490, XP002054488 *
RAMEGOWDA B. ET AL: "Differentiation-associated toxin receptor modulation, cytokine production, and sensitivity to shiga-like toxins in human monocytes and monocytic cell-lines", INFECTION AND IMMUNITY, vol. 64, no. 4, April 1996 (1996-04-01), pages 1173 - 1180, XP002106550 *

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