CA2318190A1 - Agent capable of modulating a glycosphingolipid-associated activity thus affecting an immune disorder - Google Patents

Agent capable of modulating a glycosphingolipid-associated activity thus affecting an immune disorder Download PDF

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CA2318190A1
CA2318190A1 CA002318190A CA2318190A CA2318190A1 CA 2318190 A1 CA2318190 A1 CA 2318190A1 CA 002318190 A CA002318190 A CA 002318190A CA 2318190 A CA2318190 A CA 2318190A CA 2318190 A1 CA2318190 A1 CA 2318190A1
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agent
glycosphingolipid
associated activity
modulating
immune disorder
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Timothy Raymond Hirst
Neil Andrew Williams
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University of Bristol
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
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Abstract

The use of an agent in the manufacture of a medicament to affect an immune disorder is described. The agent is capable of modulating a glycosphingolipid associated activity. The modulation of the glycosphingolipid associated activity affects an immune disorder.

Description

AGENT CAPABLE OF MODULATING A GLYCOSPHINGOLIPID-ASSOCIATED ACTIVITY THUS
AFFECTING AN
IMMUNE DISORDER
The present invention relates to a medicament.
In particular, the present invention relates to a medicament useful in the treatment of immune disorders. In particular, 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 (GVHD).
More in particular, in one aspect the present invention relates to an immunomodulatory agent.
More in particular, the present invention relates to such an agent' optionally co-administered with a specific antigen for use in the treatment.of mammalian particularly human 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 (GVHD).
Autoimmunity 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-I binding activity, or which have an effect on GM-I
mediated intracellular signalling events but no GM-1 binding activity, are useful as therapeutic agents for, inter alia, autoimmune diseases. W095/10301 disclosed agents which consisted of a mucosa-binding molecule linked to a specific tolerogen.
However, in W097/02045 we disclosed that in fact linkage between a GM-1 binding agent and the tolerogen was not required. Particular examples of GM-1 binding agents provided in W097/02045 were cholera toxin (Ctx), the B subunit of cholera toxin (CtxB), the E.coli heat labile toxin (Etx) and the B subunit of the E.coli heat labile toxin (EtxB).
The term "allergy or hypersensitivity" is used to describe an adaptive immune response which occurs in an exaggerated or inappropriate form. Allergic or hypersensitivity reactions are the result of normally beneficial immune responses acting inappropriately to foreign antigens (usually environmental macromolecules) and sometimes cause inflammatory reactions and tissue damage. In these situations, a normally harmless environmental stimulus, called an "allergen", triggers an immune response which upon re-exposure, is re-activated to generate pathological damage.
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.
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 rhinitis and 2o conjunctivitis, atopic eczema and dermatitis, urticaria, hives, insect bite allergy, dietary and certain drug allergies. In many cases, the particular allergens are known.
By way of example, 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. However, a number of factors may influence the persistence of the immune complexes and where they remain in the blood for prolonged periods, they can lodge and establish inflarnmation in the kidneys, skin (where they cause rashes) and joints (where they can cause a type of arthritis other than rheumatoid arthritis).
Type IV or delayed type hypersensitivity (DTH) 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.
Researchers have shown that a state of immunological unresponsiveness, also known as "immunological or oral tolerance", can be induced by the oral administration of dietary protein antigens (Weiner, HL, Immunol Today 1997 18: 335-343; Sun et al 1994 ibic~. The inhalation of antigens can also induce a state of specific immunological unresponsiveness or "nasal tolerance". Thus, systemic immunological tolerance can be induced when antigen is administered at mucosal sites (such as orally or nasally). WO 95/01301 discloses an immunological tolerance-inducing agent comprising a mucosa-binding agent linked to a specific tolerogen. W095/10301 also includes mention of the treatment of allergy using a mucosa binding agent coupled to an allergen. Other researchers such as Tamara 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 marine 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. It has also been shown that orally administered Ctx and Etx can act on several humoral and cellular immune responses not only at the gastrointestinal tract, but also in distant mucosal 1o effector sites such as the respiratory tract. These data suggest that these mucosal adjuvants have a potential use in oral immunisation strategies to improve the local immune responses in remote mucosal tissues, in accordance with the concept of a corlmon mucosal immune system (Bienenstock J 1974 The physiology of the local immune system and the gastrointestinal tract. In: Progress in Immunology II, vol 4:
clinical aspects, LL. Brent, J.Holborrow, Eds. Amsterdam, North Holland, pp197-207;
Ruedl et al 1996 ibid; Umesaki 1992 ibid; Czerkinsky and Holmgren (1994 Cell Mol Biol 40: 37-44).
The induction of immunological tolerance may include a number of different mechanisms which may be summarised as follows:
(i) a process whereby antigen reactive cells are removed through triggering them to commit suicide (apoptosis);
(ii) an induction of anergy or the long term inactivation of the antigen reactive cells;
(iii) immune deviation of the antigen reactive cells away from the production of pathological responses;

(iv) suppression of the antigen reactive cells or their regulation by specific factors or regulatory cells In the treatment of allergy, it is possible that the treatment of any of these mechanisms 5 may be useful. However, while the deletion of antigen reactive cells and/or the induction of anergy are useful strategies once the precise allergens are known, invoking these mechanims will usually silence only those cells which respond to the allergen which was given in the treatment regime. On the other hand, the implementation of immune deviation or suppression strategies has the advantage of l0 potential regulation of responses to antigens which are involved in the condition but were not part of the treatment. This phenomenon, known as "bystander suppression"
allows the "spread" of tolerance to other antigens (such as allergens) in the target tissues through the possible secretion of non-antigen specific suppressor molecules in that tissue as a result of the interaction between the antigen specific cells and the specific immunising antigen. In this way, as Iong as at least one of the antigens involved in the disorder is known, the condition may be treated even if there are other antigens implicated as well. Thus, the goal of a good treatment is the induction of a specific immune deviation or suppression.
Oral administration of antigens - such as allergens and autoantigens - has long been recognised as a method to prevent peripheral T cell responses and, in the case of autoantigens, has also been shown to prevent or delay the onset of several experimental autoimmune diseases including experimental allergic encephalomyelitis (EAE). Major problems recognised with such strategies are that it usually requires feeding of large, if not massive, doses of autoantigens and it is generally less efficient in an immune as opposed to a naive host. The latter problem has limited the therapeutic potential of this strategy. However, it has now been shown by Sun et al (1994 Proc Natl Acad Sci 91: 10795-10799) that oral administration of minute amounts of prototype particulate and soluble protein antigens conjugated to cholera toxin B subunit (CtxB), the nontoxic receptor-binding moiety of cholera toxin, can readily induce tolerance in the peripheral T-cell compartment and is effective not only in naive but also in systemically sensitised animals. In addition, oral administration of minute amounts of an autoantigen, myelin basic protein (MBP) coupled to CtxB
can prevent EAE in Lewis rats (Sun et al 1996 Proc Natl Acad Sci 93: 7196-7201).
Other researchers have also shown that feeding even a single dose of minute amounts (microgram) of antigens conjugated to the receptor binding nontoxic B subunit moiety of cholera toxin (CtxB) can markedly suppress systemic T cell mediated inflammatory reactions in naive as well as in experimental animals (Bergerot et al 1997 Proc Natl Acad Sci 94: 4610-4614).
to Nashar and co-workers (Proc Natl Acad Sci 1996 93: 223-226; Int Immunol 1996 8:
731-736; Immunol 1997 91: 572-578) have demonstrated that the administration of EtxB and other homologues can modulate the immune response away from the production of Thl cytokines such as IFNy and interleukin 2 (IL-2) and towards the secretion of Th2 cytokines such as IL-4, IL-10 and IL-13. IFNy is the classical Thl cytokine, IL-4 is the classical Th2 cytokine. This "immune deviation" is the basis of the disclosure in WO 97/02045.
Earlier, we surprisingly found that agents having GM1 binding activity exert immunomodulatory effects useful in the treatment of allergic conditions and/or 2o hypersensitivity conditions (UK patent application No. 9800487.2).
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. Within cells, 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 3o membrane receptors.
One member of this glycosphingolipid family which acts as a plasma membrane receptor is globotriaosylceramide, (Gb3), which is optionally represented as Gal al-4Gal (31-4Glc ail-1 Cer or Gal al-4Ga1 (3 1-4Glc-Cer io Gb3 mediates the internalisation of a family of E. coli derived toxins called verotoxins (VT), into susceptible cells by capping and receptor mediated endocytosis (RNIE) (Khine and Lingwood 1994 J Cell Physiol 161: 319-332). The action of the verotoxins (VT) in causing human vascular disease is dependent on the specific recognition of receptors on target endothelial cells (Nyholin 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 Eur 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 neurotoxic 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, VTI and VT2 demonstrate terminal Gal a 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 aGal(1-4)(3Ga1 at the non-reducing terminus. SLTIIvp has been shown to bind to the receptors containing this subunit but not necessarily at the non-reducing end (Samuel, J. E., et al Infect Immun (1990) 58:611-618; Boyd, B., et al Nephron (1989) 51:207-210; DeGrandis, S., et al J Biol Chem (1989) 264:12520-12525; Waddell, T., et al Biochem Biophys Res Chem (1988) 152:674-679; Lingwood, C. A., et al J
Biol Chem (1987) 262:8834-8839; Waddell, T., et al Proc Natl Acad Sci USA (1990) 87:7898-7901; Cohen, A., et al J Biol Chem (1987) 262:17088-17091; Jacewicz, M., et al J. Exp Med (1986) 163:1391-1404; Lindberg, A. A., et al J Biol Chem (1987) 262:1779-1785).
The carbohydrate recognition domain, also known as the P (k) trisaccharide, of the Gb3 receptor for SLT-I (St Hilaire et al 1994 Biochem 33: 14452-14463) is represented as methyl 4-O-(4-O-alpha-D-galactopyranosyl)-4-O-beta-D-glucopyranoside}
2o The terms "VT producing E coli (VTEC)" (Lingwood 1996 Trends Microbiol 4:

153) or "Shiga like toxin producing E coli (SLT-EC)" (Newburg et al 1993 J
Infect Dis 168: 476-479) are widely used and interchangeable. Recently, a new unified nomenclature for the verotoxin, Shiga-like toxin and Shiga toxin family was proposed with the designation Stx being used for the entire family. The nomenclature Vtx shall be used throughout the text of the present invention.
Although workers know that the Gb3 receptor acts as a functional receptor for verotoxins, the possibility that receptor binding modulates the immune response has not yet been explored.
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".
According to a first aspect of the present invention, there is provided 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; and wherein the modulation of the glycosphingolipid associated activity affects an immune i0 disorder.
According to a second aspect of the present invention, there is provided the use of 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.
Preferably, the agent is capable in vivo of modulating lymphocyte populations.
Preferably, the agent is capable of acting as a vaccine adjuvant.
Preferably, the agent is capable of promoting the antigenicity of a protein.
According to a fourth aspect of the present invention, there is provided an assay method for identifying an agent according to the present invention capable of affecting an immune disorder; wherein the assay method comprises: (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.

According to a fifth aspect of the present invention, there is provided an assay method according to the present invention wherein the assay is an assay to screen for an agent useful in the prevention and/or treatment of an immune disorder.
5 According to a sixth aspect of the present invention, there is provided a process comprising the steps of (a) perfornling 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.
l0 According to a seventh aspect of the present invention, there is provided 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.
According to an eighth aspect of the present invention, there is provided 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.
According to a ninth aspect of the present invention, there is provided an agent identified by the process of the present invention.
Preferably the agent identified had not previously been known to affect an immune disorder through modulation of a glycosphingolipid associated activity.
According to a tenth aspect of the present invention, there is provided a method of 3o 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.
Preferably there is provided 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.
According to a eleventh aspect of the present invention, there is provided an agent according to the present invention for use as a pharmaceutical.
According to a twelfth aspect of the present invention, there is provided the use of an agent according to the present invention in the manufacture of a medicament to affect an immune disorder.
According to a thirteenth aspect of the present invention, there is provided a pharmaceutical composition comprising or prepared from an agent according to the present invention.
Preferably, the agent capable of modulating a glycosphingolipid associated activity is a Gb3 binding agent.
Preferably the agent is a verotoxin.
Preferably the agent is VtxB.
In a particularly preferred embodiment the agent is the wild type VtxB.
In one embodiment, the agent has a pentameric structure.

Prefefably the agents) is/are non-toxic.
Alternatively, preferably the agent is either a mutant of VtxB which is capable of modulating a glycosphingolipid associated activity or other equivalent proteins thereof.
Preferably the agent is VtxB and mutants thereof which are capable of modulating a glycosphingolipid associated activity.
Preferably the glycosphingolipid is Gb3.
In one embodiment, the agent capable of modulating a glycosphingolipid associated activity is capable of cross-linking Gb3 receptors.
Preferably VtxB is a suitable example of an agent which is capable of cross-linking Gb3.
Preferably good cross-linking is achieved by virtue of its pentameric form.
Preferably the medicament is used for the treatment or prophylaxis of an immune 2o disorder.
Preferably the medicament includes one or more antigens which are optionally co-administered with the agent.
In one preferred embodiment, the agent is coupled to an antigen.
In one preferred embodiment, the agent is uncoupled to an antigen.

WO 99/3$530 PCT/GB99/00290 Preferably the agent may be administered to a mammal with or without ~6-administration of an antigen.
Preferably the mammal is a human - e.g. a human patient.
In accordance with the present invention we have surprisingly found that the B
subunit of E.coli verotoxin (VtxB) induces a similiar modulatory effect on CD8+ T
cells to that of CtxB and EtxB. This effect is very surprising as VtxB binds to an alternative receptor (the glycolipid receptor Gb3) which is different from that utilised by EtxB and io CtxB and is was not expected that VtxB would induce similiar effects to that of CtxB
and EtxB.
In particular, we have surprisingly found that the use of agents having Gb3 associated activity, when given alone or when co-administered with suitable antigens, exert immunomodulatory effects useful in the treatment of immune disorders.
We have also surprisingly found that a verotoxin administered on its own and without adjuvant can serve to immunomodulate the immune system. Thus, VtxB can serve as a vaccine adjuvant which is capable of promoting the antigenicity of a protein. In 2o addition, 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.
Significantly, 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.

Thus, 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.
The term "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. For the purposes of this invention; the glycosphingolipid is not GM1.
1o The term "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.
The term "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.
Alternatively, the toxin can be referred to as a Shiga-like toxin (SLT) or Stx. The verotoxin can be made synthetically or isolated from natural sources.
Alternatively, it can be obtained from commercial sources.
2o As used herein, "shiga-like toxin" or "SLT" refers to group of toxins produced by enterohemorrhagic E. coli that resemble the Shigella-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. Such SLTs include SLTI
and the various grouped toxins designated in the art SLTII.
The term "GM1 binding agent" includes any agent which acts as an immunomodulator through interacting with a GM 1 ganglioside receptor.

WO 99/38530 PC"T/GB99/00290 The term "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.
5 The term "adjuvant" includes a substance that enhances an immune response to an antigen.
The term "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 1o unrelated antigen. In this way, the agent acts as a so-called vaccine adjuvant. The term "vaccine adjuvant" includes the term "mucosal adjuvant".
The term "mucosal adjuvant" includes an agent which is delivered mucosally with an unrelated antigen, such that the agent is capable of facilitating a mucosal immune 15 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 Biotechnol 44: 161-170) The term "mucosal surfaces" includes but is not limited to oral, sublingual, nasal, vaginal, rectal, salivary, intestinal and conjunctiva) surfaces.
The term "mucosal 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.
The term "mucosal 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.

The term "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".
The term "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.
The term "tolerance" means a state of specific immunoIogical unresponsiveness.
The term "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).
The term "autoimmunity" is used to describe the mechanism by which the body generates an immune response to self antigens.
The term "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.
The term "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.
The term "administered" includes delivery by viral or non-viral techniques.
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 hut are not limited to mucosal, nasal, oral, parenteral (such as intravenous (iv), intramuscular (im), or subcutaneous (sc) route), gastrointestinal, topical, or sublingual routes.
The term "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 1o is achieved. Thus, whilst the 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 W095/13795 and W096/14871).
The term "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.
The term "systemic immunisation" means the introduction of an antigen into a non-mucosal tissue such as the skin or the blood.
The term "self antigens" means components derived from host tissues.
The term "target interaction components" includes but is not limited to an agent capable of modulating a glycosphingolipid associated activity, a glycosphingolipid and/or an antigen.

The team "uncoupled" - which is synonymous with the term "unlinked" - means that the agent is not coupled to the antigen.
The term "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.
However, in accordance with the present invention, the agent and/or antigen can be 1 o coupled to another entity.
The term "affect" 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 specif c 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). In this sense, 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.
Examples of antigens include but are not limited to: insulin; myelin basic protein;
3o 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.
Examples of common allergen sources are outlined in the Table below.
Group E:ampler of Allergens Airborne grass pollens ragweed, rye, couch, wild oat, timothy, Bermuda, Kentucky blue, mugwort tree pollens alder, birch, hazel, beech, Cupressae, oak,olive moulds Aspergillus spp., Cladosporium spp., Alternaria spp., Basidospores, Ascomycetes cereal grains wheat, rye, oat animal dander and cat, dog, horse, rabbit, guinea urine pig, hamster bird feathers budgerigar, parrot, pigeon, duck, chicken house dust mite Dermatophagoides pteronyssinus, D.farinae, Euroglyphus maynei insects cocla~oach, fly, locust, midge Oral seafood, legumes, peanuts, nuts, cereals, dairy foods products, eggs, fruits, tomatoes, mushrooms, alcoholic beverages, coffee, chocolate Wigs penicillins, sulphonamides and other antibiotics, sulphasalazine, carbamazepine Injected bee and wasp stings, ant and mosquito bites insects blood products, sera, vaccines, contrast media, drugs (including anti-asthma drugs and Wigs antibiotics) The term "allergic condition" includes but is not limited to asthma, allergic cough, allergic rhinitis and conjunctivitis, atopic eczema and dermatitis, uticaria, hives, insect 5 bite allergy, dietary allergy (peanut, fish milk, wheat etc) and drug allergies.
The term "hypersensitivity condition" includes but is not limited to conditions such as contact hypersensitivity induced by plant poison ivy.
to The teen "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. By way of example the agent can be a polypeptide as well as a variantlhomologue/derivative/fragment thereof so long as they retain the required immunomodulatory activity. It also includes mimics and equivalents and 15 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. By way of example, when 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. For example, 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 io 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.
In E. coli, 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).
Where a target interaction component is a protein, procedures well known in the art 2o may be used for the production of antibodies to that component.
For the production of antibodies, 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.
Depending on the host species, various adjuvants may be used to increase immunological response. Such 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) and Corynebacterium parvum are potentially useful human adjuvants.

Where 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). In addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human 1o antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison et al (1984) Proc Natl Acad Sci 81:6851-6855;
Neuberger et al (1984) Nature 312:604-608; Takeda et al (1985) Nature 314:452-4.54).
Alternatively, techniques described for the production of single chain antibodies (US
Patent No. 4,946,779) can be adapted to pmduce target interaction component specific single chain antibodies.
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. For example, such 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. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Ruse 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.
Alternatively, screening of peptide libraries or organic libraries made by combinatorial to 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 2o 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.
Alternatively, 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 Iigand (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.
Accordingly, 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. In such an assay, 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. In summary, 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. Alternatively, 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 comprising 5 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 1o comprise any one or more of 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), 15 coating agent(s), solubilising agent(s).
The pharmaceutical composition may be formulated together with an appropriate antigen.
2o Alternatively, a kit may be provided comprising separate compositions for each of the therapeutic agent and the antigen.
In some embodiments of the present invention, the pharmaceutical compositions will comprise one or more of an agent that has been screened by an assay of the present 25 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, 3o excipient or adjuvant (including combinations thereofj.

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.
There 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. Alternatively, the formulation may be designed to be delivered by both routes.
Where 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.
Typically, 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.

Where appropriate, the pharmaceutical 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. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example 1o enough salts or monosaccharides to make the solution isotonic with blood.
For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
There may be different delivery requirements dependent on the different composition/formulation systems.
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.
By way of example, 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) Encapsulation of vaccines in biodegradable microspheres provides excellent mucosal immunogens. Recombinant Norwallc Virus-like (rNV) particles may also be used for the oral delivery of rnucosal immunogens (Ball et al 1996 Arch Virol Suppl 12:

249) Viral Like Particles (VLPs) have been utilised as vaccine delivery system for multiple immunogens including B and T cell epitopes (Roy 1996 Intervirology 39: 62-71).
One preferred method of oral delivery uses formations as described in W095/13795, W096/17593 and W096/17594. These formulations allow macromolecules such as proteins to be solubilised in "oils" for oral delivery. Such formulations therefore allow to delivery of the macromolecules to mucosal surfaces in the gut.
In a further approach, again when the therapeutic agent is a protein, it is possible to deliver such proteins by means of a bacterial delivery system such as that described in WO 93/17117. This system utilises the bacterium Lactococcus lactis to deliver proteins, for instance orally or indeed by other mucosol routes such as nasally.
In summary, 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.
In another broad aspect, the present invention provides an agent capable of modulating a glycosphingolipid associated activity and which is potently immunogenic.
Other aspects of the present invention are now presented below by way of numbered paragraphs, which include:
1. The use of a 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 3o diseases, transplant rejection, graft versus host disease, immune disorders or cancer.

2. The use as claimed in paragraph 1 wherein the agent is VtxB.
3. The use as claimed in paragraph 1 or paragraph 2 wherein the medicament also comprises one or more antigens/allergens.
4. 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.
5. A pharmaceutical formulation as claimed in paragraph 4 wherein the agent is VtxB.
6. A pharmaceutical formulation as claimed in paragraph 5 which also comprises one or more allergens/antigens.
7. A pharmaceutical formulation as claimed in paragraph 5 or paragraph 6 which is a vaccine formulation.
8. 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.
9. 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.
10. 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 5 antigen/allergen as a combined preparation for simultaneous, separate or sequential use.
The present invention will now be further described by example in which reference is made to the following Figures:
Figure 1 shows the serum anti-VtxB antibody responses in 1VIH mice following subcutaneous immunisation with VtxB; and Figure 2 shows the upregulation in IL-2R (CD25) expression induced by binding of 15 EtxB (A), CtxB (B) and VtxB (C) to lymphocyte populations.
In slightly more detail, the serum anti-VtxB antibody titre in column 1 of Figure 1 was obtained in 1VIH mice immunised with lOp,g VtxB in complete Freund's adjuvant on day 1 and lOpg VtxB in incomplete Freund's adjuvant on Days 8 and 15 whereas the 20 serum anti-VtxB antibody titre in column 2 was obtained in N»i mice immunised with lOp,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.
25 The upregulation of CD25 expression on CD8+ T cells induced by CtxB, EtxB
and VtxB in Figure 2 is represented by increased binding of CD25 antibodies labelled with FITC and detected as a shift in the intensity of FITC fluorescence by FACs analysis The dotted lines represent levels of CD25 expression in unstimulated cells.

EXAMPLES
Example 1. Serum anti-VtxB antibody responses in NI~I mice following subcutaneous immunisation with VtxB.
NIH mice were immunised (s.c.) on days 1, 8 and 15 with 10~g of VtxB in Freund's adjuvant, lOp.g of VtxB alone or PBS (control). Mice were bled on day 28.
Antibody titres to VtxB were determined on VtxB-coated microtitre plates.
The results in Figure 1 demonstrate that the serum anti-VtxB antibody titre in NIH
mice treated with VtxB without adjuvant were almost twice that seen in NIH
mice treated with VtxB with adjuvant. These results indicate that (i) in the absence of adjuvant, VtxB is potently immunogenic (ii) VtxB administered on its own and without adjuvant can serve to immunomodulate the immune system and (iii) that (i) and (ii) above indicate that VtxB is capable of acting as a vaccine adjuvant.
Example 2. Examination of CD25 (IL-2R) cell surface expression on VtxB
treated lymphocyte populations by FRCS analysis.
2o NIH female 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.
Following 3 washes in HBSS, CD8+T cells were purified by separation in a magnetic MACS column. Briefly, 100 million cells were resuspended in 300~t1 MACS buffer {PBS/0.5% BSA/O.SmM EDTA). For positive selection 201 of MACS anti-CD8 antibody was added to cell suspension and the mixture was incubated on ice for a 3o 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). The CD8+ T cells bound to the column and then eluted by the force of a plunger, washed and cultured for 20 hours at a concentration of 2 million cell/ml in the presence of either no antigen or 40~,g/ml EtxB, VtxB or CtxB.
After the period of stimulation, cells were washed and resuspended in.I-IBSS/2%
sodium azide at a concentration of 2 million/ml. Phycoerythrin (PE) conjugated anti-CD8 and biotin labelled anti CD25 were incubated on ice with the cellular suspensions l0 at dilutions of 1 in 250 and 1 in 125 respectively for 45 minutes.
Streptavidin conjugated FITC was then added to cell samples following a brief spin and incubated for a further 45 minutes.
Following antibody incubations, cell samples were washed twice in cold HBSS
followed by once in ISOTON solution. Levels of FITC and PE fluorescence as a representation of CD25 and CD8 expression respectively were analysed by a FACS
machine (Hewlettt Packard).
The results demonstrate the surprising finding that the B subunit of E.coli verotoxin (VtxB) induces a similiar modulatory effect on CD8+ T cells, in terms of upregulation of IL-2R expression, to that of CtxB and EtxB. This effect is very surprising as VtxB
binds to an alternative receptor (the glycolipid receptor Gb3) which is different from that utilised by EtxB and CtxB and is was not expected that VtxB would induce similiar effects to that of CtxB and EtxB.
Screens for Agents capable of modulating glycosphingolipid associated activity 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:
1. Binding to a glycosphingolipid, such as Gb3, 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).
to 2. 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 2o 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.
3. The ability to bind to glycosphingolipid, such as Gb3, and the precise affinity of the interaction may be determined using plasmon surface resonance as previously reported by (Kuziemko et al (1996) Biochem 35:6375-6384).
Other screens for agents capable of activating B cells, CD4+ T cells, depleting CD8+ T
3o cells and inducing alterations in lymphocyte nuclear morphology characteristics of cells undergoing apoptosis are outlined in W097/02045 and are incorporated herein by reference.
Evaluation of identified agents 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:
1o 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 dank in the presence or absence of the agent capable of modulating a glycosphingolipid associated activity in order to assess prevention of disease development.
Such mice are given a second injection of collagen in Incomplete Freund's Adjuvant (IFA) 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.
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 5 intraperitoneally.
In the unmanipulated animal, this procedure routinely leads to the production of antigen-specific IgE which is easily detected in the serum, by standard ELISAs, using the antigen to coat suitable microtiter plates. Serum from the immunised mice is 1o applied to the plates after non-specific protein binding has been blocked and the presence of IgE is determined using widely available labelled antibodies specific for marine IgE.
In order to screen agents for their capability to prevent or treat allergy, agents capable ~5 of modulating glycosphingolipid associated activity are administered to mice either in the presence or absence of the challenge antigen at a range of doses, and by a variety of routes. Although the 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. Such intervention strategies 20 are utilised either prior to the IgE inducing antigen challenge (prophylaxis) or after the IgE inducing antigen challenge (treahnent). 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.
1. Antigen-specific IgE levels. Measurement of serum IgE by specific ELISA (as 3o 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 "IJssing 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.
2. Antigen specific 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 2o 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(3).
3. 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. Thus investigation of serum and mucosal secretions from animals which have been either untreated or given agents under investigation as part of prophylactic or treatment protocols for the presence of IgG and IgA are also carried out. Standard antigen specific ELISA assays (as described) 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, basophils and eosinophils or limit the uptake of antigen across the mucosal epithelium and hence prevent the subsequent allergic inflammatory response.
In one aspect of the present invention, a 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 rnicrotitre wells.
In one preferred embodiment of this invention, deacylated Gb3 is bound to a microtitre plate for use in an ELISA for the detection of 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.
i5 The 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.
2o Those skilled in the art of ELISA would also know that 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. Similarly, instead of attaching the glycosphingolipid to a protein as an assay component, it could be covalently bound 25 directly to a solid phase support as an assay component or alternatively, the assay component may be a liposome which contains the glycosphingolipid receptor.
Such 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.

WO 99!38530 PGT/GB99/00290 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.
Enzyme Linked Immunosorbent Assays (ELISAs) Binding of VtxB to Gb3 or deacylated Gb3 can also be examined using an adaptation of the GMI ELISA reported by Amin, T., & Hirst, T.R (1994 Prot. Express. and Purif.
5, I98-204) where GMI 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 (lmmulon I, Dynateck, USA) coated with S~,g/ml of VtxB in PBS. Anti-B subunits IgA
antibodies in gut secretion supernatants are extrapolated finm a standard curve made by coating 2 rows of wells on each plate with 1 ~g/ml rabbit anti-mouse IgA (a chain specific;
Zymed Lab, USA) in PBS followed by addition of 1 ~,g/ml of mouse myeloma IgA
2o (MOPC 315, Sigma, USA). To measure total IgA, wells are coated with rabbit anti-mouse IgA followed by addition of gut secretion supernatants. All samples are serially diluted. Goat anti-mouse IgG (Fc fragment specific; Jackson Lab., USA) or goat anti-mouse IgA (a chain specific; Sigma) peroxidase conjugate are diluted and added to all wells. The anti-B subunit IgG titer, giving an A4so"m >_ 0.2, is determined.
The IgA
anti-B subunit response for the VtxB subunit in gut secretions is calculated as "IgA
specific activity" [mean IgA anti-B subunit (pg/ml) /total IgA (~,g/ml)].
A known ELISA method for measuring cytokine levels of IL-2, IL-4, IL-5, IL-10 and IFN-y is used. Briefly, microtiter plates are coated with rat antibodies to mouse IL-2, 3o IL-4, IL-5, IL-10 and IFI~-y. 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 O.S~g/ml of biotinylated anti-cytokine monoclonal antibodies followed by addition of avidine-peroxidase and 3,3', S,5' -Tetramethylbenzidene (TMB) substrate and read at A~spnm~
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the 1o scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.
Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims (23)

1. 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; and wherein the modulation of the glycosphingolipid associated activity affects an immune disorder.
2. The use of 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.
3. The use of an agent according to claim 1 or claim 2 wherein the agent is capable of in vivo modulating lymphocyte populations.
4. The use of an agent according to claim 1 or claim 2 wherein the agent is also capable of acting as a vaccine adjuvant.
5. The use of an agent according to claim 1 or claim 2 wherein the agent is capable of promoting the antigenicity of a protein.
6. An assay method for identifying an agent according to any one of claims 1 to 5 that is capable of affecting an immune disorder;
wherein the assay method comprises:
(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.
7. An assay method according to claim 6 wherein the assay is to screen for an agent useful in the prevention and/or treatment of an immune disorder.
8. A process comprising the steps of:
(a) performing the assay according to claim 6 or claim 7;
(b) identifying one or more agents capable of modulating a glycosphingolipid associated activity; and (c) preparing a quantity of those one or more agents.
9. A process comprising the steps of:
(a) performing the assay according to claim 6 or claim 7;
(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.
10. A process comprising the steps of:
(a) performing the assay according to claim 6 or claim 7;
(b) identifying one or more agents capable of modulating a glycosphingolipid associated activity; and (c) modifying one or more identified agents that modulates a glycosphingolipid associated activity; and (d) preparing a pharmaceutical composition comprising those one or more modified agents.
11. An agent identified by the process of claim 8 or claim 9 or claim 10.
12. An agent according to claim 11 wherein the agent is a glycosphingolipid binding agent.
13. An agent according to claim 11 wherein the agent is a Gb3 binding agent.
14. An agent according to claim 13 wherein the agent is VtxB.
15. An agent according to claim 11 wherein the agent had not previously been known to affect an immune disorder through modulation of a glycosphingolipid associated activity.
16. An agent according to claim 11 wherein the agent had not previously been known to affect an immune disorder through modulation of a Gb3 associated activity.
17. The invention according to claims 6 to 10 wherein the glycosphingolipid is a Gb3 glycosphingolipid.
18. 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 claim 6 or claim 7.
19. Use of an agent as defined in any one of claims 6 to 18 in the manufacture of a medicament to affect an immune disorder
20. An agent according to claim 11 or claim 12 or claim 13 or claim 14 or an agent prepared by a process according to claim 8 or claim 9 or claim 10 for use as a pharmaceutical.
21. A pharmaceutical composition comprising or prepared from an agent according to any one of claims 11 or claim 12 or claim 13 or claim 14 or an agent prepared by a process according to claim 8 or claim 9 or claim 10 and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant or any combination thereof.
22. Use of an agent in the preparation of a pharmaceutical composition wherein the agent is that defined in any one of claims 11 or claim 12 or claim 13 or claim 14 or an agent prepared by a process according to claim 8 or claim 9 or claim 10 for the treatment of an immune disorder.
23. An agent capable of modulating a glycosphingolipid associated activity substantially as described herein.
CA002318190A 1998-01-28 1999-01-28 Agent capable of modulating a glycosphingolipid-associated activity thus affecting an immune disorder Abandoned CA2318190A1 (en)

Applications Claiming Priority (3)

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GBGB9801871.6A GB9801871D0 (en) 1998-01-28 1998-01-28 Therapies
GB9801871.6 1998-01-28
PCT/GB1999/000290 WO1999038530A1 (en) 1998-01-28 1999-01-28 Agent capable of modulating a glycosphingolipid-associated activity thus affecting an immune disorder

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GB9513733D0 (en) * 1995-07-05 1995-09-06 Univ Bristol Therapeutic agents
CA2163716C (en) * 1995-11-24 2009-05-19 Clifford A. Lingwood Verotoxin pharmaceutical compositions and medical treatments therewith
US5801145A (en) * 1996-02-09 1998-09-01 Ontario Cancer Institute Method for selectively purging CD77+ cells from bone marrow
AU7348998A (en) * 1997-05-16 1998-12-08 Hospital For Sick Children, The Inhibition of angiogenesis by verotoxins

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GB9801871D0 (en) 1998-03-25

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